Multispecific antibodies that bind to BCMA

Abstract

This provides a multispecific antibody that binds to BCMA. [Solution] A multispecific human heavy chain antibody (e.g., UniAb) that binds to B cell maturation antigen (BCMA). TM Disclosed are methods for producing such antibodies, compositions comprising pharmaceutical compositions containing such antibodies, and their use for treating disorders characterized by BCMA expression. In one embodiment, a multispecific antibody that binds to BCMA is provided, comprising a first binding unit comprising a variable region comprising a CDR3 sequence having at least 85% sequence identity with a specific sequence.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority as of the filing date of U.S. Provisional Patent Application No. 63 / 046,477, filed on 30 June 2020, the disclosure of which is incorporated herein by reference in whole.

[0002] This invention relates to a multispecific human heavy chain antibody (e.g., UniAb) that binds to BCMA. TM The present invention relates to methods for producing such antibodies, compositions comprising pharmaceutical compositions containing such antibodies, and the use thereof for treating disorders characterized by BCMA expression. [Background technology]

[0003] B cell maturation antigen (BCMA) BCMA, also known as Tumor Necrosis Factor Superfamily Member 17 (TNFRSF17) (UniProt Q02223), is a cell surface receptor expressed only in plasma cells and plasmablasts. BCMA is a receptor for two ligands in the Tumor Necrosis Factor (TNF) superfamily: APRIL (TNFSF13; also known as TALL-2 and TRDL-1; a growth-inducing ligand; a high-affinity ligand for BCMA) and B-cell activator (BAFF) (also known as BlyS; TALL-1; THANK; zTNF4; TNFSF20; and D8Ertd387e; a low-affinity ligand for BCMA). APRIL and BAFF are growth factors that bind to BCMA and promote plasma cell survival. BCMA is also highly expressed in malignant plasma cells of human multiple myeloma (MM). Antibodies that bind to BCMA are described, for example, in Gras et al., 1995, Int.Immunol. 7:1093-1106, and in International Publication Nos. 200124811 and 200124812. Anti-BCMA antibodies that cross-react with TACI are described in International Publication No. 2002 / 066516. Bispecific antibodies against BCMA and CD3 are described, for example, in U.S. Patent Application Publication Nos. 2013 / 0156769A1 and 2015 / 0376287A1. Anti-BCMA antibody-MMAE conjugates or anti-BCMA antibody-MMAF conjugates have been reported to selectively induce the death of multiple myeloma cells (Tai et al., Blood 2014, 123(20):3128-38). Ali et al., Blood 2016, 128(13):1688-700 reported that in a clinical trial (NCT02215967), chimeric antigen receptor (CAR) T cells targeting BCMA resulted in remission of multiple myeloma in human patients.

[0004] Heavy chain antibodies In conventional IgG antibodies, the association of the heavy chain and light chain is partly due to hydrophobic interactions between the constant region of the light chain and the CH1 constant domain of the heavy chain. The framework 2 (FR2) and framework 4 (FR4) regions of the heavy chain contain additional residues that contribute to this hydrophobic interaction between the heavy chain and light chain.

[0005] However, the serum of camelids (including camels, dromedaries, and llamas, belonging to the Tylopoda suborder) contains the major type of antibody (heavy chain only antibody or UniAb) that consists only of paired heavy chains. TM It is known that the following are included in the UniAb of the Camelidae family (Camelus dromedarius, Camelus tribacanus, Lama glama, Lama guanaco, Lama alpaca, and Lama vicuña). TM It has a unique structure consisting of a single variable domain (VHH), a hinge region, and two constant domains (CH2 and CH3) that are highly homologous to the CH2 and CH3 domains of classical antibodies. These UniAbs TM It lacks the first domain (CH1) of the constant region present in the genome but spliced ​​out during mRNA processing. The absence of the CH1 domain is because this domain is at the anchor position of the constant domain of the light chain, thus preventing UniAb TM This explains the absence of light chains in such UniAb. TMIt has evolved naturally to confer antigen-binding specificity and high affinity by the three CDRs of conventional antibodies or their fragments (Muyldermans, 2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opin Biol Ther 5:111-124). Cartilaginous fish such as sharks have also evolved a unique type of immunoglobulin called IgNAR that lacks light chain polypeptides and is composed entirely of heavy chains. IgNAR molecules can be engineered to generate variable domains of single-chain polypeptides (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)).

[0006] The ability of antibodies consisting only of heavy chains lacking light chains to bind antigens was established in the 1960s (Jaton et al. (1968) Biochemistry, 7, 4185-4195). Heavy chain immunoglobulins physically separated from light chains retained 80% of the antigen-binding activity of tetrameric antibodies. Sitia et al. (1990) Cell, 60, 781-790 showed that removal of the CH1 domain from the rearranged mouse μ gene resulted in the production of antibodies consisting only of heavy chains lacking light chains in mammalian cell culture. The antibodies produced retained VH binding specificity and effector functions.

[0007] Highly specific and affinity heavy chain antibodies can be generated against various antigens by immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH portion can be easily cloned and expressed in yeast (Frenken, LGJ, et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility, and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, MA et al. FEBS Lett. 414: 521-526 (1997)).

[0008] Mice in which the λ (lambda) light chain (L) locus and / or the λ and κ (kappa) L chain loci are functionally silenced, and antibodies produced by such mice, are described in U.S. Patent Nos. 7,541,513 and 8,367,888. The recombinant production of only heavy chain antibodies in mice and rats is reported, for example, in International Publication No. 2006 / 008548 pamphlet, U.S. Patent Application Publication No. 2010 / 0122358, Nguyen et al., 2003, Immunology; 109(1), 93 - 101; Brueggemann et al., Crit. Rev. Immunol.; 2006, 26(5):377 - 90; and Zou et al., 2007, J Exp Med; 204(13):3271 - 3283. The generation of knockout rats by microinjection of zinc finger nucleases into embryos is 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 constructs containing single domain antibodies as binding (targeting) domains are described, for example, in Iri - Sofla et al., 2011, Experimental Cell Research 317:2630 - 2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378 - 386.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

[0010] [Non-licensed Document 1] Gras et al.,1995,Int.Immunol.7:1093-1106 [Non-licensed Document 2] Tai et al.,Blood 2014,123(20):3128-38 [Non-licensed Document 3] Ali et al.,Blood 2016,128(13):1688-700 [Non-licensed Document 4] Muyldermans,2001;J Biotechnol 74:277-302 [Non-licensed Document 5] Revets et al.,2005;Expert Opin Biol Ther 5:111-124 [Non-licensed Document 6] Nuttall et al.Eur.J.Biochem.270,3543-3554(2003) [Non-licensed Document 7] Nuttall et al. Function and Bioinformatics 55,187-197(2004)

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Non-licensed literature 9

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[0011] Aspects of the present invention include antibodies that bind to BCMA, for example, UniAb, but are not limited to the following. TM This invention relates to heavy chain antibodies. Further aspects of the present invention relate to methods for producing such antibodies, compositions comprising such antibodies, and their use in the treatment of B cell disorders characterized by BCMA expression.

[0012] An aspect of the present invention includes a multispecific antibody that binds to BCMA, comprising a first binding unit that includes a variable region containing a CDR3 sequence having at least 85% sequence identity with SEQ ID NO: 3.

[0013] An aspect of the present invention includes a multispecific antibody that binds to BCMA, comprising a first binding unit including a variable region comprising CDR1, CDR2, and CDR3 sequences, wherein the combined CDR1, CDR2, and CDR3 sequences have at least 85% sequence identity with combined SEQ ID NOs: 1-3.

[0014] One aspect of the present invention includes a multispecific antibody that binds to BCMA, comprising a first binding unit comprising a variable region including a CDR1 sequence containing the sequence of SEQ ID NO: 1; a CDR2 sequence containing the sequence of SEQ ID NO: 2; and a CDR3 sequence containing the sequence of SEQ ID NO: 3.

[0015] In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in the human VH framework. In some embodiments, the variable region is a heavy chain-only variable region. In some embodiments, the variable region includes a sequence having at least 95% sequence identity with sequence number 12. In some embodiments, the variable region includes a sequence containing sequence number 12. In some embodiments, the variable region is a monovalent or divalent structure.

[0016] In some embodiments, the multispecific antibody further comprises a heavy chain constant region sequence in the absence of the CH1 sequence. In some embodiments, the heavy chain constant region sequence comprises the CH2 domain and the CH3 domain, but does not include the CH1 domain. In some embodiments, the CH2 domain comprises the sequence of the wild-type human IgG4 CH2 domain (SEQ ID NO: 36).

[0017] In some embodiments, the CH2 domain includes a variant human IgG4 CH2 domain containing the F234A mutation, the L235A mutation, or both the F234A and L235A mutations. In some embodiments, the CH3 domain includes the sequence of the wild-type human IgG4 CH3 domain (SEQ ID NO: 38). In some embodiments, the CH3 domain includes a variant human IgG4 CH3 domain (SEQ ID NO: 39) containing the T366W mutation. In some embodiments, the CH3 domain includes a variant human IgG4 CH3 domain (SEQ ID NO: 40) containing the T366S mutation, the L368A mutation, and the Y407V mutation.

[0018] In some embodiments, the multispecific antibody further includes a hinge region sequence located between the variable region of the heavy chain only and the CH2 domain. In some embodiments, the hinge region sequence includes the sequence of the wild-type human IgG4 hinge region (SEQ ID NO: 32). In some embodiments, the hinge region sequence includes a variant human IgG4 hinge region sequence containing the S228P mutation (SEQ ID NO: 33).

[0019] In some embodiments, the multispecific antibody further comprises a variant human IgG4 CH2 domain containing the F234A and L235A mutations. In some embodiments, the multispecific antibody further comprises a variant human IgG4 CH3 domain containing the T366W mutation. In some embodiments, the multispecific antibody further comprises a variant human IgG4 CH3 domain containing the T366S, L368A, and Y407V mutations.

[0020] In some embodiments, the multispecific antibody further comprises a second binding unit that binds to the La protein. In some embodiments, the second binding unit comprises (a) a heavy chain variable region comprising a CDR1 sequence comprising (i) either sequence of SEQ ID NO: 4 or 7; and (ii) a CDR2 sequence comprising the sequence of SEQ ID NO: 5; and (iii) a CDR3 sequence comprising either sequence of SEQ ID NO: 6 or 8; and (b) a light chain variable region comprising a CDR1 sequence comprising (ii) the sequence of SEQ ID NO: 9; and (ii) a CDR2 sequence comprising the sequence of SEQ ID NO: 10; and (iii) a CDR3 sequence comprising the sequence of SEQ ID NO: 11.

[0021] In some embodiments, the heavy chain variable region of the second binding unit includes (i) a CDR1 sequence containing the sequence of SEQ ID NO: 4, a CDR2 sequence containing the sequence of SEQ ID NO: 5, and a CDR3 sequence containing the sequence of SEQ ID NO: 6; or (i) a CDR1 sequence containing the sequence of SEQ ID NO: 7, a CDR2 sequence containing the sequence of SEQ ID NO: 5, and a CDR3 sequence containing the sequence of SEQ ID NO: 8.

[0022] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region of the second binding unit are present in the human VH framework. In some embodiments, the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region of the second binding unit are present in the human VL framework. In some embodiments, the human VL framework is the human V-kappa framework. In some embodiments, the human VL framework is the human V-lambda framework.

[0023] In some embodiments, the second binding unit includes a heavy chain variable region containing a sequence having at least 95% sequence identity with sequence number 14 and a light chain variable region containing a sequence having at least 95% sequence identity with sequence number 16. In some embodiments, the second binding unit includes a heavy chain variable region containing the sequence of sequence number 14 and a light chain variable region containing the sequence of sequence number 16. In some embodiments, the second binding unit includes a heavy chain variable region containing a sequence having at least 95% sequence identity with sequence number 15 and a light chain variable region containing a sequence having at least 95% sequence identity with sequence number 17. In some embodiments, the second binding unit includes a heavy chain variable region containing the sequence of sequence number 15 and a light chain variable region containing the sequence of sequence number 17.

[0024] In some embodiments, the heavy chain variable region and light chain variable region of the second binding unit are located on a common polypeptide subunit of the multispecific antibody and linked by a linker sequence. In some embodiments, the heavy chain variable region and light chain variable region of the second binding unit are located on different polypeptide subunits of the multispecific antibody.

[0025] In some embodiments, the second binding unit further includes a heavy chain constant region. In some embodiments, the heavy chain constant region includes a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the CH2 domain includes the sequence of the wild-type human IgG4 CH2 domain (SEQ ID NO: 36). In some embodiments, the CH2 domain includes a variant human IgG4 CH2 domain containing the F234A mutation, the L235A mutation, or both the F234A and L235A mutations. In some embodiments, the CH3 domain includes the sequence of the wild-type human IgG4 CH3 domain (SEQ ID NO: 38). In some embodiments, the CH3 domain includes a variant human IgG4 CH3 domain (SEQ ID NO: 39) containing the T366W mutation. In some embodiments, the CH3 domain includes a variant human IgG4 CH3 domain (SEQ ID NO: 40) containing the T366S mutation, the L368A mutation, and the Y407V mutation. In some embodiments, the hinge region includes the sequence of the wild-type human IgG4 hinge region (SEQ ID NO: 32). In some embodiments, the hinge region includes a variant human IgG4 hinge region sequence containing the S228P mutation (SEQ ID NO: 33).

[0026] In some embodiments, the multispecific antibody further comprises a variant human IgG4 CH2 domain containing the F234A and L235A mutations. In some embodiments, the multispecific antibody further comprises a variant human IgG4 CH3 domain containing the T366W mutation. In some embodiments, the multispecific antibody further comprises a variant human IgG4 CH3 domain containing the T366S, L368A, and Y407V mutations.

[0027] In some embodiments, the second binding unit further comprises a light chain constant region. In some embodiments, the light chain constant region comprises a human V-kappa constant region sequence. In some embodiments, the light chain constant region comprises a human V-lambda constant region sequence. In some embodiments, the multispecific antibody is bispecific.

[0028] Aspects of the present invention include (a) a first binding unit that binds to the La protein, comprising (i) a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 4 or 7, a CDR2 sequence of SEQ ID NO: 5, and a CDR3 sequence of SEQ ID NO: 6 or 8 in the human VH framework; and (ii) a light chain variable region comprising a light chain variable region comprising a CDR1 sequence of SEQ ID NO: 9, a CDR2 sequence of SEQ ID NO: 10, and a CDR3 sequence of SEQ ID NO: 11 in the human VL framework; and (b) a second binding unit that binds to BCMA, comprising (i) a second binding unit comprising an antigen-binding domain of an anti-BCMA heavy chain only antibody, comprising a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 2, and a CDR3 sequence of SEQ ID NO: 3 in the human VH framework, wherein the antigen-binding domain of the anti-BCMA heavy chain only antibody is monovalent or bivalent in structure, and comprising a multispecific antibody.

[0029] In some embodiments, the heavy chain variable region and light chain variable region of the first binding unit are located on a common polypeptide subunit of the multispecific antibody and linked by a linker sequence. In some embodiments, the heavy chain variable region and light chain variable region of the first binding unit are located on different polypeptide subunits of the multispecific antibody.

[0030] In some embodiments, the heavy chain variable region of the first binding unit includes (i) the CDR1 sequence of SEQ ID NO: 4, the CDR2 sequence of SEQ ID NO: 5, and the CDR3 sequence of SEQ ID NO: 6; or (ii) the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 5, and the CDR3 sequence including the sequence of SEQ ID NO: 8. In some embodiments, the human VL framework is a human V-kappa framework. In some embodiments, the human VL framework is a human V-lambda framework. In some embodiments, the heavy chain variable region of the first binding unit includes a sequence having at least 95% identity with SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the heavy chain variable region of the first binding unit includes the sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the light chain variable region of the first binding unit includes a sequence having at least 95% identity with SEQ ID NO: 16 or SEQ ID NO: 17. In some embodiments, the light chain variable region of the first binding unit includes the sequence of SEQ ID NO: 16 or SEQ ID NO: 17. In some embodiments, the antigen-binding domain of the anti-BCMA heavy chain-only antibody includes a variable region sequence having at least 95% identity with the sequence of SEQ ID NO: 12. In some embodiments, the antigen-binding domain of the anti-BCMA heavy chain-only antibody includes a variable region sequence containing the sequence of SEQ ID NO: 12.

[0031] In some embodiments, the antigen-binding domain of the anti-BCMA heavy chain-only antibody is a bivalent structure and includes a linker sequence. In some embodiments, the linker sequence includes a G4S linker sequence.

[0032] In some embodiments, the first binding unit further includes a heavy chain constant region. In some embodiments, the heavy chain constant region includes a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the CH2 domain includes the sequence of the wild-type human IgG4 CH2 domain (SEQ ID NO: 36). In some embodiments, the CH2 domain includes a variant human IgG4 CH2 domain containing the F234A mutation, the L235A mutation, or both the F234A and L235A mutations. In some embodiments, the CH3 domain includes the sequence of the wild-type human IgG4 CH3 domain (SEQ ID NO: 38). In some embodiments, the CH3 domain includes a variant human IgG4 CH3 domain (SEQ ID NO: 39) containing the T366W mutation. In some embodiments, the CH3 domain includes a variant human IgG4 CH3 domain (SEQ ID NO: 40) containing the T366S mutation, the L368A mutation, and the Y407V mutation. In some embodiments, the hinge region includes the sequence of the wild-type human IgG4 hinge region (SEQ ID NO: 32). In some embodiments, the hinge region includes a variant human IgG4 hinge region sequence containing the S228P mutation (SEQ ID NO: 33).

[0033] In some embodiments, the multispecific antibody further comprises a variant human IgG4 CH2 domain containing the F234A and L235A mutations. In some embodiments, the multispecific antibody further comprises a variant human IgG4 CH3 domain containing the T366W mutation. In some embodiments, the multispecific antibody further comprises a variant human IgG4 CH3 domain containing the T366S, L368A, and Y407V mutations.

[0034] In some embodiments, the first binding unit further includes a light chain constant region. In some embodiments, the light chain constant region includes a human V-kappa constant region sequence. In some embodiments, the light chain constant region includes a human V-lambda constant region sequence.

[0035] In some embodiments, the second binding unit further includes a heavy chain constant region sequence in the absence of the CH1 sequence. In some embodiments, the heavy chain constant region sequence includes a CH2 domain and a CH3 domain, but does not include a CH1 domain. In some embodiments, the CH2 domain includes the sequence of the wild-type human IgG4 CH2 domain (SEQ ID NO: 36). In some embodiments, the CH2 domain includes a variant human IgG4 CH2 domain containing the F234A mutation, the L235A mutation, or both the F234A mutation and the L235A mutation. In some embodiments, the CH3 domain includes the sequence of the wild-type human IgG4 CH3 domain (SEQ ID NO: 38). In some embodiments, the CH3 domain includes a variant human IgG4 CH3 domain (SEQ ID NO: 39) containing the T366W mutation. In some embodiments, the CH3 domain includes a variant human IgG4 CH3 domain (SEQ ID NO: 40) containing the T366S mutation, the L368A mutation, and the Y407V mutation. In some embodiments, the hinge region includes a sequence of the wild-type human IgG4 hinge region (SEQ ID NO: 32). In some embodiments, the hinge region includes a variant human IgG4 hinge region sequence containing the S228P mutation (SEQ ID NO: 33).

[0036] In some embodiments, the multispecific antibody further comprises a variant human IgG4 CH2 domain containing the F234A and L235A mutations. In some embodiments, the multispecific antibody further comprises a variant human IgG4 CH3 domain containing the T366W mutation. In some embodiments, the multispecific antibody further comprises a variant human IgG4 CH3 domain containing the T366S, L368A, and Y407V mutations. In some embodiments, the multispecific antibody is bispecific.

[0037] Aspects of the present invention include a bispecific triple-chain antibody-like molecule that binds to BCMA and La proteins, comprising (a) a first heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 18; (b) a second heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 24; and (c) a first light-chain polypeptide subunit comprising the sequence of SEQ ID NO: 20.

[0038] Aspects of the present invention include a bispecific triple-chain antibody-like molecule that binds to BCMA and La proteins, comprising (a) a first heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 18; (b) a second heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 26; and (c) a first light-chain polypeptide subunit comprising the sequence of SEQ ID NO: 20.

[0039] Aspects of the present invention include a bispecific triple-chain antibody-like molecule that binds to BCMA and La proteins, comprising (a) a first heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 21; (b) a second heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 24; and (c) a first light-chain polypeptide subunit comprising the sequence of SEQ ID NO: 23.

[0040] Aspects of the present invention include a bispecific triple-chain antibody-like molecule that binds to BCMA and La proteins, comprising (a) a first heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 21; (b) a second heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 26; and (c) a first light-chain polypeptide subunit comprising the sequence of SEQ ID NO: 23.

[0041] Aspects of the present invention include a bispecific triple-chain antibody-like molecule that binds to BCMA and La proteins, comprising (a) a first heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 19; (b) a second heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 25; and (c) a first light-chain polypeptide subunit comprising the sequence of SEQ ID NO: 20.

[0042] Aspects of the present invention include a bispecific triple-chain antibody-like molecule that binds to BCMA and La proteins, comprising (a) a first heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 19; (b) a second heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 27; and (c) a first light-chain polypeptide subunit comprising the sequence of SEQ ID NO: 20.

[0043] Aspects of the present invention include a bispecific triple-chain antibody-like molecule that binds to BCMA and La proteins, comprising (a) a first heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 22; (b) a second heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 25; and (c) a first light-chain polypeptide subunit comprising the sequence of SEQ ID NO: 23.

[0044] Aspects of the present invention include a bispecific triple-chain antibody-like molecule that binds to BCMA and La proteins, comprising (a) a first heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 22; (b) a second heavy-chain polypeptide subunit comprising the sequence of SEQ ID NO: 27; and (c) a first light-chain polypeptide subunit comprising the sequence of SEQ ID NO: 23.

[0045] Aspects of the present invention include pharmaceutical compositions comprising antibodies as described herein.

[0046] Aspects of the present invention include a method for treating B-cell damage characterized by BCMA expression, comprising administering an antibody or pharmaceutical composition described herein to a subject having the said damage.

[0047] Aspects of the present invention include the use of the antibodies described herein in the preparation of a pharmaceutical agent for the treatment of B-cell disorders characterized by BCMA expression.

[0048] Aspects of the present invention include antibodies described herein for use in the treatment of B-cell damage characterized by BCMA expression.

[0049] In some embodiments, the disorder is multiple myeloma (MM). In some embodiments, the disorder is an autoimmune disorder. In some embodiments, the autoimmune disorder is systemic lupus erythematosus (SLE). In some embodiments, the autoimmune disorder is rheumatoid arthritis (RA). In some embodiments, the autoimmune disorder is multiple sclerosis (MS).

[0050] Aspects of the present invention include polynucleotides encoding the antibodies described herein, vectors containing the polynucleotides described herein, and cells containing the vectors described herein.

[0051] Aspects of the present invention include a method for producing an antibody as described herein, comprising growing cells as described herein under conditions that allow antibody expression, and isolating the antibody from the cells.

[0052] Aspects of the present invention include a method for producing an antibody as described herein, comprising immunizing a UniRat animal with BCMA and identifying a BCMA-binding heavy chain sequence.

[0053] One aspect of the present invention is a method of treatment comprising administering an effective dose of the antibody or pharmaceutical composition described herein to an individual in need.

[0054] These aspects and further aspects are described in the remainder of this disclosure, including the examples. [Brief explanation of the drawing]

[0055] [Figure 1] This is a table providing the amino acid sequence of the indicated protein. [Figure 2] This is a table providing the amino acid sequence of the indicated protein. [Figure 3] This is a table providing the amino acid sequence of the indicated protein. [Figure 4-1] This is a table providing the amino acid sequence of the indicated protein. [Figure 4-2] This is a table providing the amino acid sequence of the indicated protein. [Figure 5] This is a table providing the amino acid sequence of the indicated protein. [Figure 6-1] This is a table providing the amino acid sequence of the indicated protein. [Figure 6-2] This is a table providing the amino acid sequence of the indicated protein. [Figure 7] Panel A is a schematic diagram of a triple-chain antibody-like molecule containing three polypeptide subunits. The first binding unit is formed from a first light-chain polypeptide subunit and a first heavy-chain polypeptide subunit. The second heavy-chain polypeptide contains a variable region domain consisting only of a bivalent heavy chain, which provides the second binding unit. Panel B is a schematic diagram of a triple-chain antibody-like molecule containing three polypeptide subunits. The first binding unit is formed from a first light-chain polypeptide subunit and a first heavy-chain polypeptide subunit. The second heavy-chain polypeptide contains a variable region domain consisting only of a monovalent heavy chain, which provides the second binding unit. [Figure 8] Panel A is a schematic diagram of a CAR-T structure containing the anti-BCMA extracellular binding domain described herein. Panel B is a graph showing antigen-specific binding and activation of the CAR construct containing the anti-BCMA extracellular binding domain described herein, as tested in the indicated cell lines. [Figure 9] Panel A is a graph showing the average fluorescence intensity (MFI) of the test antibody that binds to MM1.S cells. Panel B is a graph showing the average fluorescence intensity (MFI) of the test antibody that binds to H929 cells. [Figure 10-1] Panel A is a graph showing the A450-A570 values ​​for anti-X**BCMA_F7E that binds to BCMA in the presence of the BCMA agonist APRIL. Panel B is a graph showing the A450-A570 values ​​for anti-X**BCMA_F7E_F7E that binds to BCMA in the presence of the BCMA agonist APRIL. Panel C is a graph showing the A450-A570 values ​​for anti-X**GP120_F8A that binds to BCMA in the presence of the BCMA agonist APRIL. [Figure 10-2]Panel A is a graph showing the A450-A570 values ​​for anti-X**BCMA_F7E that binds to BCMA in the presence of the BCMA agonist APRIL. Panel B is a graph showing the A450-A570 values ​​for anti-X**BCMA_F7E_F7E that binds to BCMA in the presence of the BCMA agonist APRIL. Panel C is a graph showing the A450-A570 values ​​for anti-X**GP120_F8A that binds to BCMA in the presence of the BCMA agonist APRIL. [Modes for carrying out the invention]

[0056] Unless otherwise specified, the implementation of this invention utilizes conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. Such techniques are documented in literature such as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (FMAusubel et al., eds., 1987 and regularly updated); "PCR: The Polymerase Chain Reaction" (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001); and Harlow, Lane and Harlow, "Using Antibodies: A Laboratory Manual: Portable Protocol No. I," Cold Spring Harbor. This is adequately explained in Laboratory (1998) and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988).

[0057] Where a range of values ​​is specified, the intermediate values ​​between the upper and lower limits of this range (up to one-tenth of the lower limit unit, unless otherwise clearly indicated in the context) and any other specified or intermediate values ​​within this specified range are included in the present invention. The upper and lower limits of these smaller ranges may independently be included in this smaller range and are also included in the present invention, subject to any limitations specifically excluded in this specified range. If a specified range includes one or both limits, the range excluding one or both of these included limits is also included in the present invention.

[0058] Unless otherwise indicated, antibody residues in this specification 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)).

[0059] The following description includes many specific details to provide a more detailed understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be carried out without one or more of these specific details. In other examples, features and procedures that are well known to those skilled in the art are not described in order to avoid obscuring the invention.

[0060] All references cited throughout this disclosure, including patent applications and publications, are incorporated herein by reference in their entirety.

[0061] I. Definition "Includes" means that the listed elements are required in the composition / method / kit, but other elements may be included to form the composition / method / kit etc. within the claims.

[0062] "Substantially consisting of" means limiting the scope of the described composition or method to specific materials or processes that do not substantially affect the fundamental and novel features of the subject invention.

[0063] "Consists of" means excluding elements, processes, or components not specified in the claims from the composition, method, or kit.

[0064] In this specification, antibody residues are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used to refer to residues in the variable domain (approximately residues 1 to 113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used to refer to residues in the constant region of the immunoglobulin heavy chain (e.g., the EU index reported in Kabat et al., cited above). "EU index in Kabat" refers to the numbering of residues in the human IgG1 EU antibody. Unless otherwise stated in this specification, references to residue numbers in the variable domain of an antibody mean residue numbering according to the Kabat numbering system. Unless otherwise stated in this specification, references to residue numbers in the constant domain of an antibody mean residue numbering according to the EU numbering system.

[0065] Antibodies, also called immunoglobulins, traditionally contain at least one heavy chain and one light chain, and the sequences of the amino-terminal domains of the heavy and light chains are variable; therefore, they are generally called variable region domains or variable heavy chain (VH) or variable light chain (VL) domains. Traditionally, the two domains associate to form a specific binding region, but as discussed herein, specific binding can also be obtained using a variable sequence of the heavy chain alone, and various non-natural structures of antibodies are known and used in the art.

[0066] "Functional" or "biologically active" 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 capable of exhibiting one or more of their intrinsic activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding molecule, e.g., a TCA, may have the ability to specifically bind to an antigen, and this binding may then induce or modify cellular or molecular events such as signal transduction or enzymatic activity. A functional antibody or other binding molecule, e.g., a TCA, may also block ligand activation of a receptor or act as an agonist or antagonist. The ability of an antibody or other binding molecule, e.g., a TCA, to exhibit one or more of its intrinsic activities depends on several factors, including proper folding and assembly of the polypeptide chain.

[0067] The term "antibody" as used herein is used in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, triple-chain antibodies, triple-chain antibody-like molecules (TCAs), single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments insofar as they exhibit the desired biological activity (Miller et al (2003) Jour. Of Immunology 170:4854-4861). Antibodies may be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, or derived from other species.

[0068] The term antibody may refer to a polypeptide comprising an immunologically active portion of any full-length heavy chain, full-length light chain, intact immunoglobulin molecule, or polypeptide subunit thereof, i.e., an antigen-binding site that immunely binds to an antigen or a portion thereof of a target antigen. Such targets include, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass of immunoglobulin molecules comprising a modified subclass having a modified Fc portion that reduces or enhances effector cell activity. The light chain of the antibody may be a kappa light chain (V-kappa) or a lambda light chain (V-lambda). The immunoglobulin may originate from any species. In one embodiment, the immunoglobulin is of human origin.

[0069] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for any naturally occurring variations that may be present. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. Monoclonal antibodies according to the present invention can be prepared, for example, by the hybridoma method first described in Kohler et al. (1975) Nature 256:495, and also by recombinant protein preparation methods (see, for example, U.S. Patent No. 4,816,567).

[0070] When used in relation to antibodies, the term "variable" refers to the fact that the sequence of a particular portion of the antibody's variable domain differs significantly between antibodies, and that this particular portion is used in the binding and specificity of each antibody to its particular antigen. However, variability is not evenly distributed throughout the antibody's variable domain. It is concentrated in three segments called hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). The natural heavy and light chain variable domains each contain four FRs connected by three hypervariable regions, which mostly take on a β-sheet structure, forming loop connections and sometimes forming parts of the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRs and, together with the hypervariable region from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domain does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cytotoxicity.

[0071] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody responsible for antigen binding. The hypervariable region generally includes amino acid residues derived from the “complementarity-determining region” or “CDR” (e.g., residues 31–35(H1), 50–65(H2), and 95–102(H3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or amino acid residues derived from the “hypervariable loop” (e.g., residues 26–32(H1), 53–55(H2), and 96–101(H3) of the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901–917 (1987)).

[0072] The term "CDR" and its plural form "CDRs" refer to complementarity-determining regions, of which three constitute the binding properties of the light chain variable region (CDRL1, CDRL2, and CDRL3) and three constitute the binding properties of the heavy chain variable region (CDRH1, CDRH2, and CDRH3). CDRs contribute to the functional activity of antibody molecules and are separated by amino acid sequences containing scaffold or framework regions. The precise definition of CDR boundaries and lengths follows various classification and numbering systems.

[0073] While exemplary CDR names are given herein, those skilled in the art will understand that several definitions of CDRs are commonly used, including Kabat's 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). Chothia's definition is based on the location of the structural loop region (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature. 1989;342:877-883).Interesting alternative definitions of CDRs, though not limited to them, include: 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 Examples include those disclosed in J.1995;9:133-139. Each of these documents is explicitly incorporated herein by reference.

[0074] In a particular embodiment, “CDR” means the complementarity-determining region of an antibody, as defined in Lefranc, MP et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999).

[0075] A "framework region" or "FR" residue is a variable domain residue other than a hypervariable region / CDR residue as defined herein.

[0076] The terms “heavy chain only antibody” and “heavy chain antibody” are used interchangeably herein and broadly refer to antibodies that lack the light chain of conventional antibodies. Specifically, this term includes, but is not limited to, homodimeric antibodies containing a VH antigen-binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain; functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NARs containing a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NAR), their functional fragments; and soluble single-domain antibodies (sUniDabs). TM) are included. In one embodiment, the heavy chain-only antibody consists of a variable region antigen-binding domain comprising Framework 1, CDR1, Framework 2, CDR2, Framework 3, CDR3, and Framework 4. In another embodiment, the heavy chain-only antibody consists of an antigen-binding domain, at least a portion of the hinge region, and CH2 and CH3 domains in the absence of the CH1 domain. In another embodiment, the heavy chain-only antibody consists of an antigen-binding domain, at least a portion of the hinge region, and the CH2 domain. In yet another embodiment, the heavy chain-only antibody consists of an antigen-binding domain, at least a portion of the hinge region, and the CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. In a further embodiment, the heavy chain-only antibody consists of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, and does not include a hinge region. In a further embodiment, the heavy chain-only antibody consists of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain and at least a portion of the hinge region. Heavy-chain-only antibodies may be in the form of dimers, where two heavy chains are disulfide-bonded to each other, or otherwise covalently or noncovalently bonded to each other. While heavy-chain-only antibodies may belong to the IgG subclass, antibodies belonging to other subclasses, such as the IgM, IgA, IgD, and IgE subclasses, are also included herein. In certain embodiments, the heavy-chain antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 or IgG4 subtype. In one embodiment, the heavy-chain antibody is of the IgG4 subtype, with one or more CH domains modified to alter the effector function of the antibody. In another embodiment, the heavy-chain antibody is of the IgG1 subtype, with one or more CH domains modified to alter the effector function of the antibody. Modifications of CH domains that alter effector function are further described herein. Non-limiting examples of heavy-chain antibodies are, for example, described in International Publication No. 2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0077] In some embodiments, the antibodies herein (e.g., heavy chain only antibodies) are used as the binding (targeting) domain of a chimeric antigen receptor (CAR). This definition specifically includes UniAb TM human heavy chain only antibodies produced by a human immunoglobulin transgenic rat (UniRat TM ) called. UniAb TM The variable region (VH) of is called UniDabs TM and is a versatile building block that can be linked to the Fc region or serum albumin for the development of novel therapeutic agents with increased multispecificity, potency and extended half-life. The homodimeric UniAb TM lacks a light chain and thus a VL domain, so the antigen is recognized by a single domain, namely the variable domain of the heavy chain of the heavy chain antibody (antigen-binding domain) (VH or VHH). In some embodiments, the antibodies herein are multispecific (e.g., bispecific) antibodies comprising a first binding unit having binding affinity for a first target antigen (e.g., an antigen on a target cell, e.g., BCMA) and a second binding unit having binding affinity for a second target antigen (e.g., an antigen on a universal chimeric antigen receptor (CAR) complex). Thus, in some embodiments, the antibodies described herein can functionalize a universal CAR complex by providing binding affinity for a specific antigen target (e.g., BCMA).

[0078] As used herein, “intact antibody chain” refers to an antibody chain comprising a full-length variable region and a full-length constant region (Fc). An intact “conventional” antibody, in secreted IgG, includes an intact light chain and an intact heavy chain, as well as the light chain constant domain (CL) and the CH1, hinge, CH2, and CH3 of the heavy chain constant domain. Other isotypes, such as IgM or IgA, may have different CH domains. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. An intact antibody may have one or more “effector functions” that refer to biological activity 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. Steady-state region variants include those that alter the effector profile, binding to Fc receptors, and other parameters.

[0079] Depending on the amino acid sequence of the heavy chain's FC (constant domain), antibodies and various antigen-binding proteins can be assigned to different "classes." The heavy chain Fc region has five major classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The FC constant domains corresponding to various classes of antibodies can be called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of various classes of immunoglobulins are well known. The Ig form includes hinged or hingeless forms (Roux et al (1998) J.Immunol.161:4083-4090; Lund et al (2000) Eur.J.Biochem.267:7246-7256; U.S. Patent Application Publication No. 2005 / 0048572; U.S. Patent Application Publication No. 2004 / 0229310). The light chains of antibodies derived from any vertebrate species can be assigned to one of two types called κ (kappa) and λ (lambda) based on the amino acid sequence of their constant domains. Antibodies according to embodiments of the present invention may contain kappa light chain sequences or lambda light chain sequences.

[0080] A "functional Fc region" possesses the "effector function" of a naturally occurring Fc region. Non-limiting examples of effector function include C1q binding; CDC; Fc receptor binding; ADCC; ADCP; and downregulation of cell surface receptors (e.g., B cell receptors). Such effector functions generally require an Fc region that interacts with receptors, such as FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors and low-affinity FcRn receptors, and can be evaluated using various assays known in the art. A "dead" or "silenced" Fc region is one that has been mutated to retain activity with respect to the extension of its serum half-life, for example, but does not activate high-affinity Fc receptors or has reduced affinity for Fc receptors.

[0081] A "natural sequence Fc region" contains an amino acid sequence identical to that of a naturally occurring Fc region. Examples of natural sequence human Fc regions include the natural sequence human IgG1 Fc region (non-A and A allotypes), 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 naturally occurring variants.

[0082] The "variant Fc region" includes an amino acid sequence that differs from the amino acid sequence of the natural sequence Fc region by modification of at least one amino acid, preferably by substitution of one or more amino acids. Preferably, the variant Fc region has at least one amino acid substitution, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions in the natural sequence Fc region or the parent polypeptide Fc region, compared to the natural sequence Fc region or the parent polypeptide Fc region. The variant Fc region as described herein preferably has at least about 80% homology, most preferably at least about 90% homology, and more preferably at least about 95% homology with the natural sequence Fc region and / or the parent polypeptide Fc region.

[0083] The variant Fc sequence may contain three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncanet al., (1988) Nature 332:563). Two amino acid substitutions at the complement C1q binding site at EU index positions 330 and 331 reduce complement binding (see Tao et al., J.Exp.Med.178:661 (1993) and Canfield and Morrison, J.Exp.Med.173:1483 (1991)). Substitutions at positions 233–236 of human IgG1 or IgG2 residues, and substitutions at positions 327, 330, and 331 of IgG4 residues, significantly reduce ADCC and CDC (e.g., Armour KL. et al., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL. Et al., 2001. J Biol Chem, 276(9):6591-604). The human IgG4 Fc amino acid sequence (UniProtKB number P01861) is shown herein as Sequence ID No. 28. Silenced IgG1 is described, for example, Boesch, AW, et al., “Highly parallel characterization of IgG Fc binding interactions,” Mabs, 2014. 6(4):p.915–27, and these disclosures are incorporated herein by reference in their entirety.

[0084] Other Fc variants are possible, including but not limited to those in which a region capable of forming a disulfide bond is deleted, or in which a specific amino acid residue is removed from the N-terminus of the native Fc, or in which a methionine residue is added. Therefore, in some embodiments, one or more Fc moieties of the conjugated compound may contain one or more mutations in the hinge region to eliminate the disulfide bond. In yet another embodiment, the hinge region of Fc may be completely removed. In yet another embodiment, the conjugated compound may contain an Fc variant.

[0085] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector function by substituting (mutating), deleting, or adding amino acid residues to result in complement binding or Fc receptor binding. For example, deletions may occur at complement binding sites, such as C1q binding sites. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Publications 97 / 34631 and 96 / 32478. In addition, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

[0086] In some embodiments, the antibody contains a hinge region sequence of wild-type human IgG4 (SEQ ID NO: 32). In some embodiments, the antibody contains a variant human IgG4 hinge region sequence containing the S228P mutation (SEQ ID NO: 33).

[0087] In some embodiments, the antibody comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 36). In some embodiments, the antibody comprises a variant human IgG4 CH2 domain sequence (SEQ ID NO: 37) containing the F234A mutation, the L235A mutation, or both the F234A mutation and the L235A natural mutation.

[0088] In some embodiments, the antibody comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 38). In some embodiments, the antibody comprises a variant human IgG4 CH3 domain sequence containing the T366W mutation (SEQ ID NO: 39), which may optionally be referred to herein as an IgG4 CH3 knob sequence. In some embodiments, the antibody comprises a variant human IgG4 CH3 domain sequence containing the T366S mutation, the L368A mutation, and the Y407V mutation (SEQ ID NO: 40), which may optionally be referred to herein as an IgG4 CH3 hole sequence. The IgG4 CH3 mutations described herein can be used in any preferred manner to place a “knob” in the first heavy chain constant region of the first monomer in the antibody dimer and a “hole” in the second heavy chain constant region of the second monomer in the antibody dimer, thereby promoting the proper pairing (heterodimization) of desired pairs of heavy chain polypeptide subunits in the antibody.

[0089] The above-mentioned mutations in the hinge region, CH2 domain, and CH3 domain can be incorporated into the antibody of the present invention in any combination. In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region containing the S228P mutation, F234A mutation, L235A mutation, and T366W mutation (knob). In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region containing the S228P mutation, F234A mutation, L235A mutation, T366S mutation, L368A mutation, and Y407V mutation (hole).

[0090] The term "Fc region-containing antibody" refers to an antibody that contains 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 recombination of the nucleic acid encoding the antibody. Therefore, an antibody having an Fc region according to the present invention may include an antibody having K447 or an antibody without K447.

[0091] Aspects of the present invention include, but are not limited to, bispecific and tripspecific conjugated compounds having multispecific structures. A wide variety of methods and protein compositions are known and are used in bispecific monoclonal antibodies (BsMABs), tripspecific antibodies, and the like.

[0092] Aspects of the present invention include antibodies comprising a heavy chain-only variable region having a monovalent or bivalent structure. As used herein, the term “monovalent structure” as used with respect to a heavy chain-only variable region domain means that there is only one heavy chain-only variable region domain and that it has a single binding site (see Figure 7, Panel B). On the other hand, the term “bivalent structure” as used with respect to a heavy chain-only variable region domain means that there are two heavy chain-only variable region domains (each having a single binding site) that are linked by a linker sequence (see Figure 7, Panel A). Non-limiting examples of linker sequences are discussed further herein and are not limited to, but include GS linker sequences of various lengths. When the heavy chain-only variable region has a bivalent structure, each of the two heavy chain-only variable region domains may have binding affinity to the same antigen or to different antigens (e.g., to different epitopes of the same protein; to two different proteins, etc.). However, unless otherwise specified, a heavy chain-only variable region described as a "bivalent structure" is understood to contain two identical heavy chain-only variable region domains linked by a linker sequence, and each of the two identical heavy chain-only variable region domains has binding affinity to the same target epitope.

[0093] Various methods have been developed to produce polyvalent artificial antibodies by recombinant fusion of the variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains of a polypeptide are linked by a polypeptide linker. One non-limiting example of such a polypeptide linker is a GS linker having an amino acid sequence of four glycine residues followed by one serine residue, the sequence of which is repeated n times (where n is an integer in the range of 1 to about 10, e.g., 2, 3, 4, 5, 6, 7, 8, or 9). Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 34) (n=1) and GGGGSGGGGS (SEQ ID NO: 35) (n=2). In some embodiments, the variable domain of the first heavy chain only and the variable domain of the second heavy chain only are linked by the same heavy chain polypeptide subunit of the antibody. They are linked together to form a bivalent structure of the heavy chain-only variable region domain. In some embodiments, the heavy chain variable region and the light chain variable region are linked together by the same heavy chain polypeptide subunit of the antibody (i.e., located in a common polypeptide subunit of the antibody) to form an scFv structure of the heavy chain and light chain variable region domains. In some embodiments, the heavy chain variable region and the light chain variable region are located in different polypeptide subunits of the antibody to form a binding unit having the conventional antibody structure of the heavy chain variable region and the light chain variable region. Other suitable linkers can also be used, for example, described in Chen et al., Adv Drug Deliv Rev. 2013 October 15;65(10):1357-69, the disclosure of which is incorporated herein by reference in whole.

[0094] The terms “triple-chain antibody-like molecule” or “TCA” are used herein to refer to an antibody-like molecule comprising, substantially comprising, or comprising three polypeptide subunits, two of which comprise a functional antigen-binding fragment of a monoclonal antibody, comprising one heavy chain and one light chain or an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity to a first antigen. The third polypeptide subunit comprises an Fc portion comprising CH2, and / or CH3, and / or CH4 domains in the absence of a CH1 domain, and one or more antigen-binding domains (e.g., two antigen-binding domains) that bind to an epitope of a second antigen or a different epitope of a first antigen, wherein such binding domains consist of, substantially comprising, or comprising only a heavy chain antibody, derived from or having sequence identity with a variable region of the antibody heavy chain or light chain. A portion of such a variable region is V H and / or V L Gene segments, D and J H Gene segment or J L It can be encoded by a gene segment. The variable region is reconstructed V H DJ H , V L DJ H , V H J L or V L J L It can be encoded by a gene segment. The TCA protein utilizes only the heavy chain of the antibody as defined above.

[0095] TCA-conjugated compounds, when used herein, refer to single-chain antibodies that include the heavy chain constant region CH2 and / or CH3 and / or CH4 but do not include the CH1 domain, using either a "heavy chain-only antibody," "heavy chain antibody," or "heavy chain polypeptide." In one embodiment, the heavy chain antibody consists of an antigen-binding domain, at least a portion of the hinge region, and the CH2 and CH3 domains. In another embodiment, the heavy chain antibody consists of an antigen-binding domain, at least a portion of the hinge region, and the CH2 domain. In yet another embodiment, the heavy chain antibody consists of an antigen-binding domain, at least a portion of the hinge region, and the CH3 domain. Heavy chain antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. In further embodiments, the heavy chain antibody consists of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, and does not include the hinge region. Heavy-chain-only antibodies may be in the form of a dimer in which two heavy chains are disulfide-bonded or otherwise covalently or noncovalently bonded to each other, and may optionally include an asymmetric interface between two 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 such as the 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, particularly an IgG1 or IgG4 subtype. Non-limiting examples of TCA-conjugated compounds are described, for example, in International Publication No. 2017 / 223111 and International Publication No. 2018 / 052503, and these disclosures are incorporated herein by reference in their entirety.

[0096] Heavy chain antibodies constitute about a quarter of the IgG antibodies produced by camelids, such as camels and llamas (Hamers-Casterman C., et al. Nature. 363:446-448 (1993)). These antibodies are formed from two heavy chains but lack a light chain. As a result, the variable antigen-binding region is called the VHH domain, representing the smallest naturally occurring intact antigen-binding site, with a length of only about 120 amino acids (Desmyter, A., et al. J. Biol. Chem. 276:26285-26290 (2001)). Highly specific and affinity heavy chain antibodies can be generated against various antigens by immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH portion can be easily cloned and expressed in yeast (Frenken, LGJ, et al. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility, and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, MA et al. FEBS Lett. 414, 521-526 (1997)). It has also been shown that sharks have a single VH-like domain called VNAR in their antibodies. (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)).

[0097] As used herein, 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 that is preferentially expressed in differentiated plasma cells. The extracellular domain of human BCMA consists of amino acids 1-54 (or 5-51), according to UniProt. The term “BCMA” includes BCMA proteins of any human or non-human animal species, specifically human BCMA and BCMA of non-human animals.

[0098] The terms “anti-BCMA heavy chain only antibody” and “BCMA heavy chain only antibody” are used herein to refer to the heavy chain only antibodies defined above that bind immunospecifically to BCMA. The term “human BCMA” as used herein includes any variant, isoform, and species homolog of human BCMA (UniProt Q02223), regardless of its source or preparation method. Therefore, “human BCMA” includes human BCMA naturally expressed by cells and BCMA expressed in cells transfected with the human BCMA gene.

[0099] The terms “anti-BCMA heavy chain only antibody,” “BCMA heavy chain only antibody,” “anti-BCMA heavy chain antibody,” and “BCMA heavy chain antibody” are used interchangeably herein and refer to heavy chain only antibodies that bind immunospecifically to BCMA, including human BCMA as defined above. This definition is not limited to, but may also apply to transgenic rats or transgenic mice expressing human immunoglobulins, for example, human anti-BCMA UniAb as defined above. TM UniRat produces TM This includes human heavy chain antibodies produced by transgenic animals.

[0100] The "amino acid sequence identity percentage (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum possible sequence identity percentage, without considering conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve the maximum possible alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the amino acid sequence identity % value is generated using the sequence comparison computer program ALIGN-2.

[0101] "Isolated" antibodies are those identified, separated, and / or recovered from components of their natural environment. Contaminations from the natural environment are substances that may interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody is purified (1) to more than 95% by weight, most preferably more than 99% by weight, by Lowry determination; (2) to a sufficient extent to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer; or (3) to homogenize by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Since isolated antibodies do not contain at least one component of their natural environment, they may contain antibodies present in situ within recombinant cells. However, isolated antibodies are typically prepared by at least one purification step.

[0102] Examples of antibodies of the present invention include multispecific antibodies. Multispecific antibodies have two or more binding specificities. The term "multispecificity" specifically includes "bispecificity" and "trispecificity," as well as higher-order independent specific binding affinities such as higher-order polyepitope specificity, and tetravalent antibodies and antibody fragments. TM The terms "multispecific binding compound" and "multispecific binding compound" are used herein in their broadest sense and encompass any antibody having two or more binding specificities. Specifically, the multispecific anti-BCMA antibodies of the present invention include antibodies that immunospecifically bind to a single epitope of a BCMA protein, such as human BCMA, and to an epitope of a different protein, such as the CD3 protein. Specifically, the multispecific anti-BCMA antibodies of the present invention include antibodies that immunospecifically bind to two or more non-overlapping epitopes of a BCMA protein, such as human BCMA. Specifically, the multispecific anti-BCMA antibodies of the present invention also include antibodies that immunospecifically bind to an epitope of a BCMA protein, such as human BCMA, and to an epitope of a different protein, such as the human La protein. Specifically, the multispecific anti-BCMA antibodies of the present invention also include antibodies that immunospecifically bind to two or more non-overlapping or partially overlapping epitopes of a BCMA protein, such as human BCMA, and to an epitope of a different protein, such as the human La protein.

[0103] Examples of antibodies of the present invention include monospecific antibodies having a single binding specificity. Specifically, monospecific antibodies include antibodies containing a single binding specificity and antibodies containing two or more binding units having the same binding specificity. "Monospecific antibody," "Monospecific heavy chain antibody," "Monospecific heavy chain only antibody," and "Monospecific UniAb" TMThe term "monospecific heavy chain anti-BCMA antibody" is used herein in its broadest sense and encompasses any antibody having a single binding specificity. Specifically, the monospecific heavy chain anti-BCMA antibodies of the present invention include antibodies (monovalent and monospecific) that immunospecifically bind to a single epitope of a BCMA protein, such as human BCMA. Specifically, the monospecific heavy chain anti-BCMA antibodies of the present invention also include antibodies (e.g., multivalent antibodies) that have two or more binding units that immunospecifically bind to an epitope of a BCMA protein, such as human BCMA. For example, a monospecific antibody according to an embodiment of the present invention may include a heavy chain-only variable region (i.e., a tandem anti-BCMA heavy chain-only variable region) containing two heavy chain-only antigen-binding domains, where each of the two antigen-binding domains binds to the same epitope of the BCMA protein (i.e., bivalent and monospecific).

[0104] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. This term specifically includes linear epitopes and structural epitopes.

[0105] Epitope mapping is the process of identifying the sites or epitopes on an antibody's target antigen where the antibody binds. Antibody epitopes can be linear epitopes or structural epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Structural epitopes are formed from discontinuous amino acids in the protein sequence that come together when the protein folds into a three-dimensional structure.

[0106] "Polyepitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. As described above, the present invention includes, in particular, anti-BCMA heavy chain antibodies having polyepitope specificity, i.e., anti-BCMA heavy chain antibodies that bind to one or more non-overlapping epitopes on BCMA proteins such as human BCMA; and anti-BCMA heavy chain antibodies that bind to one or more epitopes on BCMA proteins and epitopes on different proteins such as human La protein. 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. A pair of antibodies or antigen-binding regions on a multispecific antibody that recognize a non-overlapping epitope and target the same antigen can bind to that antigen simultaneously without competing for binding.

[0107] When two antibodies recognize the same or sterically overlapping epitopes, they bind to the "essentially identical epitope" as the reference antibody. The most rapid and widely used method for determining whether two epitopes bind to the same or sterically overlapping epitopes is a competitive assay, which can be composed of any number of different forms using labeled antigens or labeled antibodies. Typically, the antigen is immobilized on a 96-well plate, and the ability of an unlabeled antibody to block the binding of the labeled antibody is measured using radiolabeling or enzymatic labeling.

[0108] As used herein, the term "valence" refers to a specific number of binding sites in an antibody molecule.

[0109] A "monovalent" antibody has one binding site. Therefore, a monovalent antibody is also monospecific.

[0110] A "polyvalent" antibody has 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 antibody according to the present invention is at least bivalent and may be trivalent, tetravalent, or other polyvalent. A bivalent antibody according to embodiments of the present invention may have two binding sites to the same epitope (i.e., bivalent, single paratopic) or to two different epitopes (i.e., bivalent, double paratopic).

[0111] A wide variety of methods and protein structures are known and used for preparing bispecific monoclonal antibodies (BsMABs), tripspecific antibodies, and the like.

[0112] The terms “chimeric antigen receptor” or “CAR” are used herein in their broadest sense to refer to an artificial receptor that conjugates a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) to a transmembrane domain and an intracellular signaling domain. Typically, this receptor is used to conjugate the specificity of a monoclonal antibody to a T cell to create a chimeric antigen receptor (CAR). (J Natl Cancer Inst, 2015;108(7):dvj439; and Jackson et al., Nature Reviews Clinical Oncology, 2016;13:370-383). CAR-T cells are T cells genetically engineered to produce 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, each containing at least an extracellular domain, a transmembrane domain, and at least one cytoplasmic domain.

[0113] The term "human antibody" is used herein to include antibodies having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human antibodies herein may include amino acid residues not encoded by a human germline immunoglobulin sequence, for example, mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo. Specifically, the term "human antibody" includes heavy-chain-only antibodies having a human heavy-chain variable region sequence, produced by transgenic animals, such as transgenic rats or mice, particularly the UniRat antibodies defined above. TM UniAb produced by TM It includes.

[0114] "Chimera antibody" or "chimeric immunoglobulin" means an immunoglobulin molecule containing amino acid sequences derived from at least two different Ig gene loci, for example, a transgenic antibody containing a portion encoded by the human Ig gene locus and a portion encoded by the rat Ig gene locus. Chimeric antibodies include transgenic antibodies and human idiotypes having non-human Fc regions or artificial Fc regions. Such immunoglobulins can be isolated from animals of the present invention that have been engineered to produce such chimeric antibodies.

[0115] As used herein, the term “effector cell” refers to an immune cell involved in the effector phase of an immune response, as opposed to the recognition and activation phases of the immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, can induce antibody-dependent cell-mediated cytotoxicity (ADCC). For example, monocytes and macrophages expressing FcR are involved in the specific death of target cells and the presentation of antigens to other components of the immune system or their binding to antigen-presenting cells. In some embodiments, effector cells can phagocytose target antigens or target cells.

[0116] "Human effector cells" are leukocytes that express receptors such as T cell receptors or FcR 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, with NK cells being preferred. Effector cells can be isolated from their natural sources, such as blood or PBMCs as described herein.

[0117] The term “immune cells” is used herein in its broadest sense and is not limited to cells of bone marrow or lymphoid origin, such as lymphocytes (e.g., T cells including B cells and cytolytic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0118] Antibody "effector function" refers to the biological activity resulting from the antibody's Fc region (either the natural sequence Fc region or the amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors (e.g., B cell receptors, BCRs).

[0119] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells, subsequently causing lysis of the target cells. NK cells, the primary cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), p. 464, Table 3. To evaluate the ADCC activity of a target molecule, an in vitro ADCC assay (e.g., those described in U.S. Patent No. 5,500,362 or 5,821,337) can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or furthermore, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, such as those disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0120] Complement-dependent cell injury, or CDC, refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) that has formed a complex with a congener antigen. To assess complement activation, a CDC assay can be performed, such as the one described in Gazzano-Santoro et al., J.Immunol.Methods 202:163 (1996).

[0121] "Binding affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by its dissociation constant (Kd). Affinity can be measured using common methods known in the art. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind more quickly to antigens and remain bound.

[0122] As used herein, "Kd" or "Kd value" refers to the dissociation constant determined by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in dynamic mode. For example, a mouse Fc fusion antigen is loaded onto an anti-mouse Fc sensor, which is then immersed in an antibody-containing well to measure the concentration-dependent binding rate (kon). The antibody dissociation rate (koff) is measured in the final step, in which the sensor is immersed in a well containing only buffer. Kd is the koff / kon ratio. (For further details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0123] As used herein, the terms “specifically interacting,” “specifically binding,” or “specifically binding” mean that the binding domain exhibits a measurable affinity for a particular target protein or antigen and generally does not show significant reactivity with non-target proteins or antigens. “Measurable affinity” is approximately 10 -6 It contains a bond with M(KD) or a bond having a stronger affinity. Preferably, the bond affinity is about 10 -12 ~10 -8 M, 10 -12 ~10 -9 M, 10 -12~10 -10 M, 10 -11 ~10 -8 M, preferably about 10 -11 ~10 -9 If M is present, the binding is considered specific. Whether the binding domain specifically reacts with or binds to a target protein or antigen can be easily tested, in particular, by comparing the reaction of the binding domain to the target protein or antigen with the reaction of the binding domain to a non-target protein or antigen. Preferably, the binding domain of the present invention substantially does not bind to or is unable to bind to a non-target protein or antigen.

[0124] The terms "substantially non-binding" or "unable to bind" mean that the binding domain of the present invention does not bind to a non-target protein or antigen, that is, if binding to a target protein or antigen is set to 100%, it does not exhibit a reactivity of more than 30%, preferably more than 20%, more preferably more than 10%, and particularly preferably more than 9%, 8%, 7%, 6%, or 5% to a non-protein or antigen.

[0125] The terms “treatment,” “to treat,” etc., are used herein to mean generally obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. As used herein, “treatment” encompasses the treatment of a disease in mammals and includes (a) preventing the development of the disease in a subject that may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., preventing its onset; or (c) alleviating the disease, i.e., causing its regression. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease, where the treatment stabilizes or alleviates undesirable clinical symptoms in the patient, is of particular interest. Such treatment is preferably carried out before the complete loss of function in the affected tissue. Treatment of a subject may be administered during, and possibly after, the symptomatic stage of the disease.

[0126] The "therapeutic dose" refers to the amount of active agent necessary to provide a therapeutic benefit to a subject. For example, the "therapeutic dose" is the amount that induces, improves, or causes improvement in disease-related pathological symptoms, disease progression, or physiological condition, or improves resistance to the disorder.

[0127] In relation to the present invention, the terms "B-cell neoplasm" or "mature B-cell neoplasm" are not limited to, but include, all lymphoid leukemias and lymphomas, chronic lymphocytic leukemia, acute lymphoblastic leukemia, prolymphoblastic leukemia, precursor B-lymphoblastic leukemia, hairy cell leukemia, small lymphocytic lymphoma, B-cell prolymphoblastic lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma. This includes (DLBCL), multiple myeloma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell tumors such as plasma cell myeloma, plasmacytoma, monoclonal immunoglobulinemia, heavy chain disease, MALT lymphoma, nodal marginal zone B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, lymphomatoid granulomatosis, non-Hodgkin lymphoma, Hodgkin lymphoma, hairy cell leukemia, primary exudative lymphoma, and AIDS-associated non-Hodgkin lymphoma.

[0128] The term "characterized by BCMA expression" broadly refers to any disease or disorder in which BCMA expression is associated with or involved in one or more pathological processes characteristic of the disease or disorder. Such disorders include, but are not limited to, B-cell neoplasms.

[0129] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to mammals being evaluated for treatment and / or being treated. In one embodiment, the mammal is a human. The terms “subject,” “individual,” and “patient” include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. While the subject may be a human, it may also include other mammals, particularly mammals useful as laboratory models for human diseases, such as mice and rats.

[0130] The term "pharmaceutical preparation" refers to a preparation that is in a form that enables the effective biological activity of the active ingredient and does not contain any additional ingredients that would be unacceptably toxic to the subject to which the preparation is administered. Such preparations are sterile. A "pharmaceutically acceptable" excipient (vehicle, additive) is one that can be reasonably administered to the target mammal to provide an effective dose of the active ingredient used.

[0131] A “sterile” preparation is sterile, or contains no or substantially no living microorganisms and their spores. A “frozen” preparation is kept below 0°C.

[0132] A “stable” formulation is one in which the proteins within it substantially retain their physical and / or chemical stability and / or biological activity during storage. Preferably, the formulation retains its physical and chemical stability and its biological activity during storage. The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are outlined, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. Stability can be qualitatively and / or quantitatively assessed by a variety of different methods, including: evaluation of aggregate formation (e.g., by measuring turbidity using size exclusion chromatography and / or by visual inspection); assessment of charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; analysis of amino-terminus or carboxy-terminus sequences; mass spectrometry; SDS-PAGE analysis comparing reduced intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; and evaluation of the biological activity or antigen-binding function of the antibody. Instability may include one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine, N-terminal elongation, C-terminal processing, and differences in glycosylation.

[0133] II. Detailed explanation Anti-BCMA antibody The present invention provides antibodies comprising heavy chain-only antibodies (UniAbs) that bind to human BCMA, but is not limited to the following. The anti-BCMA UniAbs of the present invention comprise a set of CDR sequences as defined herein and shown in Figure 1, and are exemplified by the heavy chain variable region (VH) sequence provided as SEQ ID NO: 12 shown in Figure 2. These antibodies offer many benefits that contribute to their usefulness as clinical therapeutic agents. The antibodies comprise members with a range of binding affinities, and it is possible to select specific sequences with desired binding affinities.

[0134] The anti-BCMA antibodies provided herein do not cross-react with cynomolgus monkey BCMA protein, but can be modified to provide cross-reactivity with cynomolgus monkey BCMA protein or BCMA of any other animal species, if necessary.

[0135] In some embodiments, the anti-BCMA UniAb antibody herein comprises a VH domain containing CDR1, CDR2, and CDR3 sequences within a human VH framework. For example, CDR1, CDR2, and CDR3 may be located in regions around amino acid residues 26–35, 53–59, and 98–117 of the provided exemplary variable region sequence described in SEQ ID NO: 12. While the CDR sequences may be in different positions depending on the selected framework sequence, the order of the sequences will generally remain the same, as will be understood by those skilled in the art.

[0136] In some embodiments, the anti-BCMA antibody includes a variable region comprising a CDR1 sequence containing two or fewer amino acid substitutions in the sequence of SEQ ID NO: 1, and / or a CDR2 sequence containing two or fewer substitutions in the sequence of SEQ ID NO: 2, and / or a CDR3 sequence containing two or fewer substitutions in the sequence of SEQ ID NO: 3.

[0137] In some embodiments, the anti-BCMA antibody includes a variable region comprising a CDR1 sequence containing two or fewer substitutions in the sequence of SEQ ID NO: 1, a CDR2 sequence containing two or fewer substitutions in the sequence of SEQ ID NO: 2, and a CDR3 sequence containing two or fewer substitutions in the sequence of SEQ ID NO: 3.

[0138] In some embodiments, the anti-BCMA antibody includes a variable region comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 2, and the CDR3 sequence of SEQ ID NO: 3.

[0139] In some embodiments, the anti-BCMA antibody includes a variable region consisting only of heavy chains containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the anti-BCMA antibody includes a variable region consisting only of heavy chains in a monovalent or bivalent structure.

[0140] In further embodiments, the anti-BCMA antibody of the present invention comprises a monovalent or bivalent heavy chain variable region amino acid sequence of SEQ ID NO: 12 (Figure 2).

[0141] In some embodiments, the anti-BCMA antibody preferably comprises a CDR sequence containing two or fewer amino acid substitutions to the CDR1, CDR2, and / or CDR3 sequences in any one of SEQ ID NOs 1-3 (Figure 1), and binds to BCMA. In some embodiments, the anti-BCMA antibody preferably comprises a heavy chain variable region sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity with the heavy chain variable region sequence (SEQ ID NO: 12) shown in Figure 2, and binds to BCMA.

[0142] In some embodiments, the anti-BCMA antibody preferably includes a heavy chain variable domain (VH) in which the CDR3 sequence has 80% or more amino acid-level sequence identity to SEQ ID NO: 3, for example, at least 85%, at least 90%, at least 95%, or at least 99%, and binds to BCMA.

[0143] In some embodiments, the anti-BCMA antibody preferably contains a heavy chain variable domain (VH) that has 85 percent (85%) or more sequence identity at the amino acid level to the complete set (combination) of CDR1, 2, and 3 of SEQ ID NOs: 1-3, and binds to BCMA.

[0144] The present invention provides multispecific antibodies that bind to human BCMA and human La protein. The multispecific antibodies of the present invention may comprise a first binding unit that binds to BCMA and a second binding unit that binds to human La protein. In some embodiments, the binding unit that binds to human La protein comprises a set of CDR sequences as defined herein and shown in Figure 1, and is exemplified by the heavy chain variable region (VH) sequences of SEQ ID NOs. 14-15 and the light chain variable region (VL) sequences of SEQ ID NOs. 16, 17, provided and shown in Figure 3. In some embodiments, the antibody that binds to human La protein binds to an epitope comprising the amino acid sequence KPLPEVTDEY (SEQ ID NOs. 42). In some embodiments, the antibody that binds to human La protein binds to an epitope comprising the amino acid sequence VEKEALKKIIEDQQESLNKW (SEQ ID NOs. 43). Antibodies that bind to human La protein are described, for example, in International Publication No. 2016 / 030414, U.S. Patent Application Publication No. 2020 / 0181228, U.S. Patent Application Publication No. 2020 / 0131262, and U.S. Patent Application Publication No. 2017 / 0240612, the disclosures of which are incorporated herein by reference in their entirety.

[0145] The antibodies described herein offer many advantages that contribute to their usefulness as clinical therapeutic agents. The antibodies comprise members with a range of binding affinities, and it is possible to select specific sequences with desired binding affinities.

[0146] Suitable antibodies may be selected from those provided herein for development, therapeutic or other uses, for example, bispecific or trispecific antibodies or for use as part of a CAR-T structure, but are not limited to those provided herein.

[0147] Affinity determination for candidate proteins can be performed using methods known in the art, such as Biacore assays. The target antibody has an affinity of approximately 10 to human La protein. -6 ~about 10 -11 For example, not limited to, but approximately 10-6 ~about 10 -10 , about 10 -6 ~about 10 -9 , about 10 -6 ~about 10 -8 , about 10 -8 ~about 10 -11 , about 10 -8 ~about 10 -10 , about 10 -8 ~about 10 -9 , about 10 -9 ~about 10 -11 , about 10 -9 ~about 10 -10 Alternatively, it may have an affinity of any Kd value within these ranges. The selection of affinity can be confirmed by biological evaluations to modulate, for example, increase, the desired activity (e.g., the desired binding affinity between the universal CAR structure and the binding unit of the target multispecific antibody), including in vitro assays, preclinical models and clinical trials, and assessment of potential toxicity.

[0148] In some embodiments, the anti-human La protein binding unit of the target antibody comprises a VH domain containing CDR1, CDR2, and CDR3 sequences in a human VH framework, and a VL domain containing CDR1, CDR2, and CDR3 sequences in a human VL framework, e.g., a human V kappa framework or a human V lambda framework. The CDR sequences may, for example, be located in regions around amino acid residues 26-35, 53-59, and 98-117 of the provided exemplary variable region sequences described in SEQ ID NOs. 14-17, respectively. While the CDR sequences may be in different positions when different framework sequences are selected, it will be understood by those skilled in the art that the sequence order generally remains the same.

[0149] In some embodiments, the anti-human La protein binding unit of the target antibody includes a heavy chain variable region comprising a CDR1 sequence containing two or fewer substitutions in either the sequence of SEQ ID NO: 4 or 7, and / or a CDR2 sequence containing two or fewer substitutions in the sequence of SEQ ID NO: 5, and / or a CDR3 sequence containing two or fewer substitutions in either the sequence of SEQ ID NO: 6 or 8, and a light chain variable region comprising a CDR1 sequence containing two or fewer substitutions in the sequence of SEQ ID NO: 9, and / or a CDR2 sequence containing two or fewer substitutions in the sequence of SEQ ID NO: 10, and / or a CDR3 sequence containing two or fewer substitutions in the sequence of SEQ ID NO: 11.

[0150] In some embodiments, the anti-human La protein binding unit of the target antibody includes a heavy chain variable region comprising a CDR1 sequence containing two or fewer substitutions in either the sequence of SEQ ID NO: 4 or 7, a CDR2 sequence containing two or fewer substitutions in the sequence of SEQ ID NO: 5, and a CDR3 sequence containing two or fewer substitutions in either the sequence of SEQ ID NO: 6 or 8, and a light chain variable region comprising a CDR1 sequence containing two or fewer substitutions in the sequence of SEQ ID NO: 9, a CDR2 sequence containing two or fewer substitutions in the sequence of SEQ ID NO: 10, and a CDR3 sequence containing two or fewer substitutions in the sequence of SEQ ID NO: 11.

[0151] In one embodiment, the anti-human La protein binding unit of the antibody of the present invention comprises a heavy chain containing the CDR1 sequence of SEQ ID NO: 4, the CDR2 sequence of SEQ ID NO: 5, and the CDR3 sequence of SEQ ID NO: 6, and a light chain containing the CDR1 sequence of SEQ ID NO: 9, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 11. In another embodiment, the anti-human La protein binding unit of the antibody of the present invention comprises a heavy chain containing the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 5, and the CDR3 sequence of SEQ ID NO: 8, and a light chain containing the CDR1 sequence of SEQ ID NO: 9, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 11.

[0152] In further embodiments, the anti-human La protein binding unit of the antibody of the present invention includes the heavy chain variable region amino acid sequence of SEQ ID NO: 14 and the light chain variable region amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-human La protein binding unit of the antibody of the present invention includes the heavy chain variable region amino acid sequence of SEQ ID NO: 15 and the light chain variable region amino acid sequence of SEQ ID NO: 17.

[0153] In some embodiments, the heavy chain CDR sequence in the anti-human La protein binding unit of the antibody of the present invention includes two or fewer amino acid substitutions to any one of the CDR1, CDR2, and / or CDR3 sequences of SEQ ID NOs.4 to 8 (Figure 1). In some embodiments, the anti-human La protein binding unit of the antibody of the present invention includes a heavy chain variable region sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity with the heavy chain variable region sequences (SEQ ID NOs.14 and 15) shown in Figure 3.

[0154] In some embodiments, the light chain CDR sequence in the anti-human La protein binding unit of the antibody of the present invention contains two or fewer amino acid substitutions to any one of the CDR1, CDR2, and / or CDR3 sequences of SEQ ID NOs.9 to 11 (Figure 1). In some embodiments, the anti-human La protein binding unit of the antibody of the present invention contains a light chain variable region sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity with the light chain variable region sequences (SEQ ID NOs.16 and 17) shown in Figure 3.

[0155] In some embodiments, multispecific (e.g., bispecific) antibodies are provided, which may have any of the structures described herein, including, but not limited to, a triple-chain bispecific antibody or a triple-chain bispecific antibody-like molecule (bispecific TCA). The bispecific antibody includes a heavy chain variable region of the antibody that is specific to at least a protein other than BCMA.

[0156] When the protein of the present invention is a bispecific antibody, one binding unit may be specific to human BCMA, while the other binding unit may be specific to target cells (e.g., effector cells, e.g., T cells), tumor-associated antigens, target antigens such as integrins, pathogen antigens, checkpoint proteins, proteins on the universal CAR structure, etc. Target cells specifically include cancer cells such as hematological malignancies, e.g., B-cell tumors, as described below. In some embodiments, the bispecific antibody comprises a first binding unit that binds to BCMA and a second binding unit that binds to human La protein.

[0157] Various forms of multispecific antibodies are within the scope of the present invention and include, for example, single-chain polypeptides, double-chain polypeptides, triple-chain polypeptides, quadruple-chain polypeptides, and complexes thereof. Multispecific (e.g., bispecific) antibodies as used herein specifically include T-cell bispecific antibodies that bind to BCMA and CD3 selectively expressed on plasma cells (PCs) and multiple myeloma (MM) cells (anti-BCMA × anti-CD3 antibodies). Multispecific (e.g., bispecific) antibodies as used herein also specifically include universal CAR bispecific antibodies that bind to BCMA and antigens on universal CAR structures selectively expressed on plasma cells (PCs) and multiple myeloma (MM) cells (anti-BCMA × anti-La protein antibodies). Such antibodies can induce potent T-cell or CAR T-cell-mediated death of BCMA-expressing cells and can be used to treat tumors, particularly hematological malignancies such as B-cell tumors as described below, and autoimmune disorders characterized by the presence of autoreactive plasma cells, as further described herein.

[0158] Bispecific antibodies against CD3 and BCMA are described, for example, in International Publication Brochures 2007 / 117600, 2009 / 132058, 2012 / 066058, 2012 / 143498, 2013 / 072406, 2013 / 072415, and 2014 / 122144, as well as in U.S. Patent Application Publication US2017 / 0051068. Universal chimeric antigen receptors containing human La protein are described, for example, in International Publication Brochure 2016 / 030414, the disclosures of which are incorporated herein by reference in their entirety.

[0159] Antibody preparation The multispecific antibodies of the present invention can be prepared by methods known in the art. In preferred embodiments, the antibodies described herein are produced by transgenic animals in which endogenous immunoglobulin genes are knocked out or inactivated, such as transgenic mice and rats, preferably rats. In preferred embodiments, the antibodies described herein are produced in UniRat®. The endogenous immunoglobulin genes of UniRat® are silenced, and a diverse, naturally optimized repertoire of fully human HCAbs is expressed using the human immunoglobulin heavy chain trans locus. While rat endogenous immunoglobulin loci can be knocked out or silenced using various techniques, in UniRat®, the endogenous rat heavy chain J locus, light chain Cκ locus, and light chain Cλ locus are inactivated using zinc finger (endo) nuclease (ZNF) technology. ZNF constructs for microinjection into oocytes can produce IgH and IgL knockout (KO) strains. For further details, see, for example, Geurts et al., 2009, Science 325:433. The characteristics of Ig heavy chain knockout rats are reported in Menoret et al., 2010, Eur.J.Immunol. 40:2932-2941. An advantage of ZNF technology is that non-homologous ends, linked to silence a gene or locus by deletions of up to a few kb, can also provide target sites for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). Human heavy chain antibodies produced with UniRat® are UniAb TM These antibodies are capable of binding to epitopes that cannot be attacked by conventional antibodies. Due to their high specificity, affinity, and small size, they are ideal for both single-specificity and multi-specificity applications.

[0160] UniAb TMIn addition, the camelid VHH framework and heavy chain-only antibodies lacking mutations, as well as their functional VH regions, are specifically included herein. Such heavy chain-only antibodies can be produced in transgenic rats or mice containing the complete human heavy chain-only locus, as described, for example, in International Publication No. 2006 / 008548, but other transgenic mammals such as rabbits, guinea pigs, and rats may also be used, with rats and mice being preferred. Heavy chain-only antibodies (including their VHH or VH functional fragments) can also be produced by recombinant DNA technology, such as the expression of encoding nucleic acids in suitable eukaryotic or prokaryotic hosts, including mammalian cells (e.g., CHO cells), Escherichia coli (E. coli), or yeast.

[0161] The heavy-chain-only antibody domains combine the advantages of antibodies and small molecule drugs, can be monovalent or polyvalent, have low toxicity, and are cost-effective to manufacture. Due to their small size, these domains are easily administered, including orally or topically, and are characterized by high stability, including gastrointestinal stability. Their half-lives can be tailored to the desired use or indication. Furthermore, the VH and VHH domains of HCAbs can be manufactured in a cost-effective manner.

[0162] In a particular embodiment, UniAb TMThe heavy chain antibody of the present invention, comprising the above, has a native amino acid residue at the first position of the FR4 region (position 101 of the amino acid according to the Kabat numbering system) that is substituted with another amino acid residue, and this amino acid residue can disrupt a surface-exposed hydrophobic patch that contains or is bound to the native amino acid residue at that position. Such a hydrophobic patch is usually embedded at the interface with the antibody light chain constant region, but in HCAb it is surface-exposed, at least partially, due to undesirable aggregation and light chain binding of the HCAb. The substituted amino acid residue is preferably charged, more preferably positively charged, and is, for example, 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 has high antigen-binding affinity and solubility under physiological conditions in the absence of aggregation.

[0163] As part of the present invention, in an ELISA protein and cell binding assay, UniRat binds to human BCMA. TM Human anti-BCMA heavy chain antibodies (UniAb) that have a unique sequence derived from animals. TM The identified heavy chain variable region (VH) sequences (see, for example, Figure 2) were positive for binding to human BCMA protein and / or BCMA+ cells, and negative for binding to cells that do not express BCMA.

[0164] Heavy chain antibodies that bind to non-duplicated epitopes on BCMA proteins, such as UniAb TMNon-competitive antibodies can be identified by competitive binding assays, such as enzyme-linked immunosorbent assays (ELISA) or flow cytometry competitive binding assays. For example, competition between a known antibody that binds to the target antigen and the antibody of interest can be used. Using this approach, a set of antibodies can be divided into those that compete with the reference antibody and those that do not. Non-competitive antibodies are identified as binding to a distinct epitope that does not overlap with the epitope to which the reference antibody binds. Often, one antibody is immobilized, the antigen is bound, and a second labeled (e.g., biotinylated) antibody is tested in an ELISA assay for its ability to bind to the captured antigen. This can also be done using surface plasmon resonance (SPR) platforms, including ProteOn XPR36 (BioRad, Inc.), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and MX96 SPR imager (Ibis Technologies BV), as well as biolayer interference platforms such as Octet Red384 and Octet HTX (ForteBio, Pall Inc.). For further details, please refer to the examples in this specification.

[0165] Typically, an antibody "competes" with a reference antibody if it causes a reduction of about 15–100% in the binding of the reference antibody to the target antigen, as determined by standard techniques, such as the competitive binding assay described above. In various embodiments, the relative inhibition rates are 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%, and at least about 95% or more.

[0166] Pharmaceutical composition Another aspect of the present invention is to provide a pharmaceutical composition comprising one or more antibodies of the present invention mixed with a suitable pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers used herein include, but are not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to hold therapeutic components, or combinations thereof.

[0167] The antibody pharmaceutical compositions used in accordance with this disclosure are prepared by mixing a protein of desired purity, such as in the form of a lyophilized preparation or aqueous solution, with an optional pharmaceutically acceptable carrier, excipient, or stabilizer for storage (see Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin Proteins such as cellulose, gelatin, or immunoglobulins; hydrophilic polymers (such as polyvinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0168] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage forms (i.e., dosage forms for single-dose administration). The formulation depends on the chosen route of administration. Antibodies as defined herein may be administered intravenously by injection or infusion or subcutaneously. For injectable administration, antibodies as defined herein may be formulated in aqueous solution, preferably with a physiologically compatible buffer, to reduce injection site discomfort. The solution may contain carriers, excipients, or stabilizers, as described above. Alternatively, antibodies may be in a lyophilized form to be prepared with a suitable vehicle (e.g., sterile water free of pyrogens) before use.

[0169] Anti-BCMA antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used with the protein of the present invention. Subcutaneous antibody formulations are described, for example, in U.S. Patent Publication No. 2016 / 0355591 and U.S. Patent Publication No. 2016 / 0166689.

[0170] How to use The antibodies and pharmaceutical compositions described herein can be used to treat B-cell and plasma cell malignancies and B-cell related disorders, including autoimmune disorders characterized by the expression or overexpression of BCMAs.

[0171] Such B-cell-related disorders include B-cell and plasma cell malignancies and autoimmune disorders, such as, but are not limited to, plasmacytoma, Hodgkin lymphoma, follicular lymphoma, small cell non-incisional lymphoma, endemic Burkitt lymphoma, sporadic Burkitt lymphoma, marginal zone lymphoma, extranodal mucosal-associated lymphoid tissue lymphoma, nodal monocytic B-cell lymphoma, splenic lymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B-cell lymphoma, pulmonary B-cell vascular lymphoma, small lymphocytic lymphoma, and malignancy of unknown grade. These include B cell proliferation, lymphomatous granulomatosis, 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, Sjögren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture disease, Kawasaki disease, autoimmune hemolytic anemia and rapidly progressive glomerulonephritis, heavy chain disease, primary or immune cell-associated amyloidosis, or monoclonal immunoglobulinemia.

[0172] Multiple myeloma (MM) is a plasma cell disorder characterized by BCMA expression. MM is a B-cell malignancy characterized by the monoclonal proliferation and accumulation of abnormal plasma cells in the bone marrow compartment. Current treatments for MM often result in remission, but almost all patients eventually relapse and die. While there is substantial evidence of immune-mediated elimination of myeloma cells with allogeneic hematopoietic stem cell transplantation, this approach is highly toxic and patients rarely achieve a cure. Although several monoclonal antibodies have shown promise for the treatment of MM in preclinical and early clinical trials, the consistent clinical efficacy of any monoclonal antibody therapy for MM has not ultimately been demonstrated. Therefore, there is a great need for new therapies for MM, including immunotherapy (see, e.g., Carpenter et al., Clin Cancer Res 2013, 19(8):2048-2060).

[0173] Overexpression or activation of BCMA by its proliferation-inducing ligand, APRIL, is known to promote the progression of human multiple myeloma (MM) in vivo. BCMA has also been shown to promote in vivo proliferation of xenografted MM cells with p53 mutations in mice. Because the activity of the APRIL / BCMA pathway plays a central 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 target for MM treatment. For further details, see, for example, Yu-Tsu Tai et al., Blood 2016;127(25):3225-3236.

[0174] Another B-cell disorder involving plasma cells that express BCMA is systemic lupus erythematosus (SLE), also known as lupus. SLE is a systemic autoimmune disorder that can affect any part of the body, represented by an immune system that attacks the body's own cells and tissues, resulting in chronic inflammation and tissue damage. It is a type III hypersensitivity reaction in which antibody-immune complexes precipitate and trigger a further immune response (Inaki & Lee, Nat Rev Rheumatol 2010;6:326-337).

[0175] The anti-BCMA heavy chain-only antibody (UniAb) of the present invention can be used to develop therapeutic agents for the treatment of MM, SLE, and other B-cell disorders or plasma cell disorders characterized by BCM expression, such as those listed above. In particular, the anti-BCMA heavy chain-only antibody (UniAb) of the present invention is a candidate for treating MM, either alone or in combination with other MM treatments.

[0176] In one embodiment, the antibodies described herein may be in the form of a heavy-chain-only anti-BCMA antibody-CAR structure, i.e., a heavy-chain-only anti-BCMA antibody-CAR transduced T cell structure. CARs having antigen specificity for BCMA and methods of using them are described, for example, in International Publication No. 2019 / 006072, the disclosure of which is incorporated herein by reference in its entirety.

[0177] In some embodiments, the antibodies herein are multispecific (e.g., bispecific) and comprise a first binding unit having binding affinity to BCMA and a second binding unit having binding affinity to the La protein present on the universal chimeric antigen receptor (CAR) complex on effector cells (e.g., T cells). Thus, the multispecific antibodies of the present invention can be used to functionalize the universal CAR complex by binding to it by the first binding unit and providing binding affinity to BCMA by the second binding unit. The method may further include treating a subject in need of treatment for a disease or disorder characterized by BCMA expression by administering an effective amount of a cell therapy comprising a plurality of cells containing the universal CAR complex functionalized to bind to BCMA using the multispecific antibodies of the present invention, thereby treating the disease or disorder of the subject.

[0178] The effective dose of the composition of the present invention for the treatment of disease varies depending on many different factors, including the means of administration, the target site, the patient's physiological state, whether the patient is human or animal, and whether the administration and treatment of other drugs are prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, such as companion animals like dogs, cats, and horses, and laboratory mammals like rabbits, mice, and rats, can also be treated. The therapeutic dose can be titrated to optimize safety and efficacy.

[0179] Dosage levels can be easily determined by clinicians with standard skills and can be modified as needed, for example, to alter the subject's response to treatment. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the host being treated and the specific mode of administration. A unit dosage form generally contains approximately 1 mg to 500 mg of the active ingredient.

[0180] In some embodiments, the therapeutic dose of the drug may be in the range of approximately 0.0001 to 100 mg / kg of host body weight, more typically 0.01 to 5 mg / kg. For example, the dose may be in the range of 1 mg / kg body weight, 10 mg / kg body weight, or 1 to 10 mg / kg. Exemplary treatment regimens involve administration every two weeks, once a month, or once every three to six months. The therapeutic substance of the present invention is usually administered multiple times. The interval between single doses may be weekly, monthly, or yearly. The interval may also be irregular, as indicated by measuring the blood levels of the therapeutic substance in the patient. Alternatively, the therapeutic substance of the present invention can be administered as a sustained-release formulation, in which case the frequency of administration needs to be lower. The dose and frequency vary depending on the half-life of the polypeptide in the patient.

[0181] Typically, the compositions are prepared as injectable solutions or suspensions, and solid forms suitable for solutions or suspensions in a liquid vehicle prior to injection may also be prepared. The pharmaceutical compositions described herein are suitable for direct or intravenous or subcutaneous administration after reconstitution of solid (e.g., lyophilized) compositions. Preparations may also be emulsified or encapsulated in liposomes or microparticles such as polylactides, polyglycolides, or copolymers to enhance the adjuvant effect, as described above. (Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997). The agents of the present invention may be administered in the form of depot injections or implantable formulations that can be formulated in a manner that allows for sustained or pulsed release of the active ingredient. The pharmaceutical compositions are generally sterile, substantially isotonic, and formulated in full compliance with all Good Manufacturing Practice (GMP) rules of the U.S. Food and Drug Administration.

[0182] The toxicity of the antibodies and antibody structures described herein can be determined by standard pharmaceutically procedures in cell culture or experimental animals, for example, by determining the LD50 (lethal dose in 50% of the population) or LD100 (lethal dose in 100% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used when formulating dose ranges that are not toxic for human use. The dosage of the antibodies described herein is preferably within the range of circulating concentrations that include effective doses with little to no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage may be selected by individual physicians, taking into account the patient's condition.

[0183] The composition for administration generally contains an antibody or other ablative agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous carriers, such as buffered saline, can be used. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, and toxicity modifiers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentration of the active agent in these formulations can vary widely and is selected based on the chosen specific mode of administration and the patient's needs, mainly on fluid volume, viscosity, body weight, etc. (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0184] The present invention also includes the active agent and its formulation, as well as a kit comprising instructions for use. The kit may further contain at least one additional agent, such as a chemotherapeutic agent. The kit typically includes a label indicating the intended use of the kit's contents. The term "label" includes any written or recorded material on or supplied with the kit, or accompanying the kit.

[0185] The present invention will be fully described here, but it will be apparent to those skilled in the art that various modifications and alterations can be made without departing from the spirit or scope of the present invention. [Examples]

[0186] The following embodiments are provided to illustrate specific embodiments and should not be construed as limiting the scope of this disclosure.

[0187] Example 1: CAR-T-mediated T cell activation by human tumor cell lines CAR-T cell activity was measured by transfecting Jurkat T lymphocytes with anti-BCMA CAR and 6x NFAT TK nanoluciferase reporters. Transfected Jurkat cells were co-cultured for 24 hours with BCMA-positive NCI-H929, U266, and Daudi or BCMA-negative K562 cells. Luciferase activity was measured using the Promega Nano-Glo Luciferase Assay System (catalog number N1110), and the data were normalized to the co-culture containing CAR-transfected Jurkat and BCMA-negative K562 cell lines. Statistical significance was determined using an unpaired two-sided t-test.

[0188] The results are shown in Figure 8, Panel B.

[0189] Figure 8, Panel A is a schematic diagram of the CAR-T structure containing the anti-BCMA extracellular binding domain with the VH sequence of Sequence ID No. 12 (clone ID number 316302). Figure 8, Panel B shows the T cell activity of Jurkat cells transfected with the NFAT luciferase reporter and anti-BCMA316302CAR by NCI-H929 (*p=0.04), U266 (***p=0.0008), and Daudi (*p=0.03) cells. Since no luciferase reporter signal was detected in co-culture of BCMA CAR Jurkat cells with BCMA-negative K562 cell lines, these results indicate that T cell activation was specific to BCMA target binding. Furthermore, no luciferase reporter signal was detected in incubation of Jurkat cells transfected with the reporter instead of the CAR with BCMA-positive NCI-H929, U266, and Daudi cells.

[0190] Example 2: Antibody that binds to BCMA-expressing cells Antibody binding to BCMA-expressing MM1.S and H929 cells was evaluated by flow cytometry. The test antibodies included a bispecific construct that binds to the La protein (referred to herein as "AntiX") and BCMA (referred to herein as "BCMA_F7E"). As described elsewhere in this specification, the bispecific construct according to embodiments of the present invention may include a bivalent format BCMA-binding domain (also referred to herein as "BCMA_F7E_F7E") as shown on the right side of panel A in Figure 7, or a monovalent format BCMA-binding domain (also referred to herein as "BCMA_F7E") as shown on the right side of panel B in Figure 7.

[0191] Binding experiments were performed using the following constructs: anti-X**BCMA_F7E, a bispecific antibody with a monovalent arm targeting BCMA (including SEQ ID NO: 12) and an La protein-binding arm; anti-X**BCMA_F7E_F7E, a bispecific antibody with a bivalent arm targeting BCMA (with a bivalent structure including SEQ ID NO: 12) and an La protein-binding arm; and anti-X**AGP120_F8A, a bispecific negative control antibody containing an La protein-binding arm and an arm that binds to GP120. Briefly, 300,000 to 500,000 cells in 150 μl of FACS buffer (1×PBS, 2% FBS, 1 mM EDTA) were co-incubated with the test antibodies listed in Table 1 and a commercially available anti-BCMA antibody (Biolegend, 19F2) at a rate of 0.5 μl / test. The cells were then washed twice and incubated with an anti-IgG(PE) secondary antibody. After two further washes, the cells were processed using a Cytoflex instrument (Beckman Coulter) and analyzed using the FlowJo software package. Table 1, panel A in Figure 9, and panel B in Figure 9 show the mean fluorescence intensity (MFI) of anti-IgG4 secondary antibodies in MM1.S and H929 cells.

[0192] [Table 1]

[0193] Example 3: Competitive ELISA using APRIL and test antibodies The bispecificity of BCMA binding affinity was tested in a competitive ELISA with APRIL (also known as BAFF), a natural agonist of BCMA. In this assay, BCMA bound to the plate surface was co-incubated with various concentrations of test antibody and APRIL, respectively. Clear flat-bottom non-sterile 96-well plates (Corning, catalog no. 3455) containing 10 ng of recombinant BCMA (R&D Systems, catalog no. 193-BC) per well (in 100 μL of PBS) were incubated overnight. The plates were washed and incubated with 1× Blocker BSA (Thermo Scientific, catalog no. 37525).

[0194] The test antibody and recombinant APRIL (R&D Systems, catalog number 560-AP) were incubated with BCMA-coated plates at the concentrations listed in Table 2. The plates were washed and incubated with mouse anti-human IgG-HRP antibody (Southern Biotech, catalog number 9200-05). The plates were washed and spread with Pierce TMB substrate kit (Thermo Fisher, catalog number 34021) according to the manufacturer's instructions. Absorbance was measured at 450 nm and 570 nm within 15 minutes. Table 2 shows the A450-A570 values ​​for each sample. Panels A-C in Figure 10 are graphs showing the A450-A570 values ​​for the bispecific antibody constructs AntiX**BCMA_F7E (Panel A), AntiX**BCMA_F7E_F7E (Panel B), and AntiX**GP120_F8A (Panel C), respectively. More antibody binding is indicated by higher A450-A570 values. Even at higher APRIL concentrations compared to the bispecific antibody construct concentration, the anti-X**BBCMA_F7E_F7E construct containing a bivalent BCMA-binding VH sequence shows significant binding. The anti-X**BCMA_F7E_F7E construct showed stronger binding than the anti-X**BCMA_F7E bispecific antibody construct containing a monovalent BCMA-binding VH sequence.

[0195] Table 2

Claims

1. An antibody that binds to BCMA, CDR1 sequence containing the sequence of sequence number 1; CDR2 sequence containing the sequence of Sequence ID No. 2; and CDR3 sequence containing sequence of sequence number 3 An antibody comprising a first binding unit that includes a variable region containing a variable region.

2. The antibody according to claim 1, wherein the CDR1, CDR2, and CDR3 sequences are present within the human VH framework.

3. The antibody according to claim 1, which is monospecific.

4. The antibody according to claim 1, wherein the variable region is a variable region consisting only of heavy chains.

5. The antibody according to any one of claims 1 to 4, wherein the variable region comprises a sequence having at least 95% sequence identity with respect to SEQ ID NO:

12.

6. The antibody according to any one of claims 1 to 5, wherein the variable region includes SEQ ID NO:

12.

7. The antibody according to any one of claims 1 to 6, wherein the variable region has a monovalent or bivalent structure.

8. A CAR-T cell comprising a CAR containing an anti-BCMA extracellular binding domain, The anti-BCMA extracellular binding domain CDR1 sequence containing the sequence of sequence number 1; CDR2 sequence containing the sequence of Sequence ID No. 2; and CDR3 sequence containing sequence of sequence number 3 CAR-T cells containing a variable region including [specific region].

9. The CAR-T cell according to claim 8, wherein the CDR1, CDR2, and CDR3 sequences are present within the human VH framework.

10. The CAR-T cell according to claim 8, wherein the anti-BCMA extracellular binding domain is monospecific.

11. The CAR-T cell according to claim 8, wherein the variable region is a variable region consisting only of heavy chains.

12. The CAR-T cell according to any one of claims 8 to 11, wherein the variable region includes a sequence having at least 95% sequence identity with respect to sequence number 12.

13. The CAR-T cell according to any one of claims 8 to 12, wherein the variable region includes sequence number 12.

14. The CAR-T cell according to any one of claims 8 to 13, wherein the variable region has a monovalent or bivalent structure.

15. A CDR1 sequence containing the sequence of Sequence ID 1; CDR2 sequence containing the sequence of Sequence ID No. 2; and CDR3 sequence containing sequence of sequence number 3 A polynucleotide encoding an anti-BCMA binding unit that includes a variable region.

16. An antibody according to any one of claims 1 to 7, or a polynucleotide encoding the CAR of a CAR-T cell according to any one of claims 10 to 14.

17. An antibody comprising only a heavy chain that binds to BCMA, comprising a first heavy chain polypeptide subunit having the sequence of SEQ ID NO: 24, 25, 26, or 27.

18. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 7 or 17.

19. A method for treating a disease or disorder characterized by the expression of BCMA, comprising administering to a subject having the disorder an antibody according to any one of claims 1 to 7 or 17, or a pharmaceutical composition according to claim 18.

20. Use of the antibody according to any one of claims 1 to 7 or 17 in the manufacture of a pharmaceutical product for the treatment of a disease or disorder characterized by BCMA expression.

21. The antibody according to any one of claims 1 to 7 or 17, for use in the treatment of a disease or disorder characterized by BCMA expression.

22. A pharmaceutical composition comprising CAR-T cells according to any one of claims 8 to 14.

23. A method for treating a disease or disorder characterized by BCMA expression, comprising administering to a subject having the disorder CAR-T cells described in any one of claims 8 to 14, or the pharmaceutical composition described in claim 22.

24. Use of CAR-T cells according to any one of claims 8 to 14 in the manufacture of a pharmaceutical product for the treatment of a disease or disorder characterized by BCMA expression.

25. CAR-T cells according to any one of claims 8 to 14, for use in the treatment of a disease or disorder characterized by BCMA expression.

26. A kit for treating a disease or disorder characterized by BCMA expression, comprising an antibody according to any one of claims 1 to 7 or 17, or CAR-T cells according to any one of claims 8 to 14.

27. ​​A kit for treating a disease or disorder characterized by BCMA expression, comprising the pharmaceutical composition according to claim 18 or 22.

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