Anti-BCMA heavy chain only antibody

Abstract

The present invention provides antibodies and pharmaceutical compositions for use in treating B-cell and plasma cell malignancies, as well as B-cell-related disorders, including autoimmune disorders, characterized by the expression or overexpression of human B-cell maturation antigens (BCMAs). [Solution] Disclosed are anti-BCMA heavy chain only antibodies (UniAbs) having a specific amino acid sequence, a method for producing such antibodies, compositions including pharmaceutical compositions containing such antibodies, and their uses for treating B cell damage characterized by BCMA expression.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is a result of U.S. Provisional Patent Application No. 62 / 522,295, filed on June 20, 2017. Claiming priority as of the filing date, the disclosure of this application is incorporated herein in its entirety by reference. To be absorbed.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is used by reference to this application. The entire text is used. The above ASCII copy was created on August 24, 2018. The file is named TNO-0003-WO_SL.txt and has a size of 47,761 bytes. ru. Field of Invention This invention relates to an anti-BCMA heavy chain only antibody (UniAb). This invention further relates to such A method for producing antibodies, a composition comprising a pharmaceutical composition containing such antibodies, and BCMA Regarding their use to treat B-cell disorders characterized by the expression of [specific gene / symptoms]. [Background technology]

[0003] B cell maturation antigen (BCMA) Tumor necrosis factor superfamily member 17 (TNFRSF17) (UniProt BCMA, also known as Q02223, is exclusively produced on plasma cells and plasmablasts. These are cell surface receptors. BCMA belongs to the tumor necrosis factor (TNF) superfamily. The two ligands, APRIL (proliferation-inducing ligand, TNFSF13, TALL-2, and Also known as TRDL-1, a high-affinity ligand for BCMA and B cell activity factor (BAFF) (BLyS, TALL-1, THANK, zTNF4, TNFSF2 (Also known as 0, and D8Ertd387e, a low-affinity ligand for BCMA) It is a receptor for [substance name]. APRIL and BAFF bind to BCMA and contribute to the survival of plasma cells. BCMA is a growth factor that promotes malignant plasma cells in human multiple myeloma (MM). It is highly expressed above. Antibodies that bind to BCMA include, for example, Gras et al. 1995, Int. Immunol. 7:1093-1106, WO200124811 And as described in WO200124812. Anti-BCMA antibodies that cross-react with TACI. This is described in WO2002 / 066516. Dual characteristics for BCMA and CD3 Heterosexual antibodies include, for example, US2013 / 0156769 A1 and US2015 / 0376 As described in 287 A1: Anti-BCMA antibody-MMAE or -MMAF conjugate It has been reported that this selectively induces the killing of multiple myeloma cells (Tai e t al., Blood 2014, 123(20):3128-38). Ali et al., Blood 2016, 128(13):1688-700 is a clinical trial (# NCT02215967) Chimeric antigen receptor (CAR) T cells targeting BCMA This has been reported to have resulted in remission of multiple myeloma in human patients.

[0004] Heavy chain only antibody In conventional IgG antibodies, the association of the heavy chain and light chain occurs between the constant region of the light chain and the constant CH1 domain of the heavy chain. This is partly due to the hydrophobic interaction between the yne and the heavy chain. The heavy-chain framework 2 (FR2) and framework 4 (FR4) areas also contribute to this purpose. There are additional residues.

[0005] However, sera of Camelidae (suborder Tylopoda including camels, dromedaries, and llamas) are known to contain a major type of antibody consisting only of paired H chains (heavy chain only antibodies or UniAbs) Camelidae (dromedaries, Bactrian camels, llamas, guanacos, alpacas, and vicuñas) UniAbs have a unique structure consisting of a single variable domain (VHH), hinge region, and two constant domains (CH2 and CH3), and they are highly homologous to the CH2 and CH3 domains of classical antibodies. These UniAbs lack the first domain of the constant region (CH1) present in the genome, but are spliced out during mRNA processing. The absence of the CH1 domain explains the absence of light chains in UniAbs, since this domain is the anchoring site for the constant domain of the light chain. Such UniAbs have naturally evolved to confer antigen-binding specificity and high affinity by the three CDRs from 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 polypeptide chains 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, 3 543-3554 (2003), Nuttall et al. Function an d Bioinformatics 55, 187-197 (2004), Dooley et al., 2009; Int. J. Biochem. Cell Biol. 41, 101-114 (2009)). et al., 2009; Int. J. Biochem. Cell Biol. 41, 101-114 (2009)). However, sera of Camelidae (suborder Tylopoda including camels, dromedaries, and llamas) are known to contain a major type of antibody consisting only of paired H chains (heavy chain only antibodies or UniAbs) Camelidae (dromedaries, Bactrian camels, llamas, guanacos, alpacas, and vicuñas) UniAbs have a unique structure consisting of a single variable domain (VHH), hinge region, and two constant domains (CH2 and CH3), and they are highly homologous to the CH2 and CH3 domains of classical antibodies. These UniAbs lack the first domain of the constant region (CH1) present in the genome, but are spliced out during mRNA processing. The absence of the CH1 domain explains the absence of light chains in UniAbs, et al.,Molecular Immunology 40,25-33(20 03)).

[0006] The ability of heavy-chain-only antibodies, lacking light chains, to bind to antigens was established in the 1960s (Jat). on et al.(1968)Biochemistry,7,4185-4195) Heavy chain immunoglobulins, physically separated from light chains, exhibit 80% antigenicity against tetrameric antibodies. It retained binding activity. Sitia et al. (1990) Cell, 60, 78 In 1-790, removing the CH1 domain from the rearranged mouse μ gene results in mammals In cell culture, it was demonstrated that antibodies are produced only from heavy chains lacking light chains. The antibody retained its VH binding specificity and effector function. Highly specific and affinity heavy chain antibodies are produced against various antigens through immunosensitization. It can be done (van der Linden, RH, et al. Bioch im.Biophys.Acta.1431,37-46(1999)), the VHH part is It can be easily cloned and expressed in yeast (Frenken, LGJ). ., et al. J. Biotechnol. 78, 11-21 (2000). The expression level, solubility, and stability are superior to those of the classical F(ab) or Fv fragments. (Ghahroudi, MA et al. FEBS Lett. 414) , 521-526 (1997). The λ (lambda) light (L) chain locus, and / or λ or κ (kappa) )) Mice in which the L chain locus is functionally silenced, and such mice The antibodies produced by [the agent] are U.S. Patent Nos. 7,541,513 and 8,367,88 It is described in issue 8. Recombinant production of heavy chain-only antibodies in mice and rats is, for example, WO2006008548, U.S. Patent Application Publication No. 20100122358, Nguy en et al.,2003,Immunology;109(1),93-101, Bruggemann et al.,Crit.Rev.Immunol.;2006 ,26(5):377-90, and Zou et al.,2007,J Exp Me This is reported in d;204(13):3271-3283. Zinc finger nuclea The generation of knockout rats by embryonic microinjection of -se is Geurts It is described in et al., 2009, Science, 325(5939):433. This includes antibodies containing only soluble heavy chains and heterologous heavy chain gene loci that produce such antibodies. Genic rodents are covered by U.S. Patent Nos. 8,883,150 and 9,365,655. It is described as a CAR-T structure containing a single-domain antibody as the binding (targeting) domain. For example, see Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al. al.,2014,Biochim Biophys Acta,1840:378- It is described in 386. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2001 / 024811 [Patent Document 2] International Publication No. 2001 / 024812 [Patent Document 3] International Publication No. 2002 / 066516 [Patent Document 4] U.S. Patent Application Publication No. 2013 / 0156769 [Patent Document 5] U.S. Patent Application Publication No. 2015 / 0376287 [Patent Document 6] U.S. Patent No. 7541513 [Patent Document 7] U.S. Patent No. 8,367,888 [Patent Document 8] International Publication No. 2006 / 008548 [Patent Document 9] U.S. Patent Application Publication No. 2010 / 0122358 [Patent Document 10] U.S. Patent No. 8883150 [Patent Document 11] U.S. Patent No. 9365655 [Non-patent literature]

[0008] [Non-Patent Document 1] Gras et al.,1995,Int.Immunol.7:1093-1106 [Non-Patent Document 2] Tai et al.,Blood 2014,123(20):3128-38 [Non-Patent Document 3] Ali et al.,Blood 2016,128(13):1688-700 [Non-Patent Document 4] Muyldermans,2001;J Biotechnol 74:277-302 [Non-Patent Document 5] Revets et al.,2005;Expert Opin Biol Ther 5:111-124 [Non-Patent Document 6] Nuttall et al.Eur.J.Biochem.270,3543-3554(2003)

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[0009] This invention relates to a heavy chain-only antibody that binds to human B cell maturation antigen (BCMA).

[0010] In one embodiment, the present invention relates to an anti-BCMA antibody that is a heavy chain-only antibody,

[0011] (a) Having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs. 1 to 7 CDR1 and / or

[0012] (b) Having two or fewer substitutions in any of the amino acid sequences of sequence numbers 8-11 CDR2, and / or

[0013] (c) Any of the amino acid sequences of sequence numbers 12-15 contains two or fewer substitutions. Regarding anti-BCMA antibodies that contain CDR3, heavy chain variable region, and are heavy chain-only antibodies. .

[0014] In one embodiment, the CDR1, CDR2, and CDR3 sequences are present in the human framework. do.

[0015] In another embodiment, the heavy chain-only antibody further inhibits the heavy chain constant region sequence in the absence of the CH1 sequence. It is included in.

[0016] In yet another embodiment, only the heavy chain antibody is,

[0017] (a) A CDR1 sequence selected from the group consisting of sequence numbers 1 to 7, and / or

[0018] (b) A CDR2 sequence selected from the group consisting of sequence numbers 8-11, and / or

[0019] (c) Includes a CDR3 sequence selected from the group consisting of sequence numbers 12-15.

[0020] In further embodiments, the heavy chain-only antibody is

[0021] (a) A CDR1 sequence selected from the group consisting of sequence numbers 1 to 7,

[0022] (b) A CDR2 sequence selected from the group consisting of sequence numbers 8-11,

[0023] (c) A CDR3 sequence selected from the group consisting of sequence numbers 12 to 15, and the above.

[0024] In further embodiments, the heavy chain-only antibody is,

[0025] (i) CDR1 sequence of SEQ ID NO: 1, CDR2 sequence of SEQ ID NO: 8, and C of SEQ ID NO: 12 DR3 sequence; or

[0026] (ii) The CDR1 sequence of sequence number 1, the CDR2 sequence of sequence number 8, and the CDR1 sequence of sequence number 14 CDR3 sequence; or

[0027] (iii) CDR1 sequence of sequence number 2, CDR2 sequence of sequence number 9, and sequence number 13 The CDR3 sequence of; or

[0028] (iv) The CDR1 sequence of sequence number 1, the CDR2 sequence of sequence number 8, and the sequence number 15 Includes the CDR3 sequence.

[0029] In a different embodiment, the heavy chain-only antibody is one of the sequences of SEQ ID NOs: 16-50 It includes a heavy chain variable region having at least 95% sequence identity.

[0030] In another embodiment, the heavy chain-only antibody is selected from the group consisting of SEQ ID NOs. 16-50. Includes a variable-strand region sequence.

[0031] In further embodiments, the heavy chain-only antibody is SEQ ID NOs. 16, 17, 18, 30, 34 and It includes a heavy chain variable region sequence selected from a group of 38.

[0032] The present invention further relates to a heavy chain-only antibody that binds to human B cell maturation antigen (BCMA),

[0033] (a) CDR1 sequence of the following equation

[0034] GFT X1 X2 X3 X4 X5

[0035] (In the formula,

[0036] X1 is V, I, or F.

[0037] X2 is either S or T,

[0038] X3 is either S or N,

[0039] X4 is either Y or S,

[0040] X5 is G or A),

[0041] (b) CDR2 sequence of the following equation

[0042] I X6 G X7 X8 X9 X10 T

[0043] (In the formula,

[0044] X6 is either R or S,

[0045] X7 is either S or D,

[0046] X8 is D, G, or S.

[0047] X9 is G or D,

[0048] X10 is S, T, or N), and

[0049] (c) CDR3 sequence of the following equation

[0050] AKQG X11 NDGPFD X12

[0051] (In the formula,

[0052] X11 is G or E,

[0053] (X12 is either Y or H) This relates to heavy chain-only antibodies, including heavy chain variable regions, including heavy chain variable regions.

[0054] The present invention further incorporates CDR1, CDR2, and CDR3 sequences into a human VH framework. The anti-BCMA antibody is limited to heavy chains containing the heavy chain variable region, and the CDR sequence is sequence number 1~ A heavy-chain-only anti-BCMA, containing two or fewer substitutions in a CDR sequence selected from a group of 15. Regarding antibodies.

[0055] In one embodiment, only the heavy chain antibody is present in the human VH framework as CDR1, CDR2 and The heavy chain variable region includes the CDR3 sequence, and the CDR sequence is a group consisting of sequence numbers 1 to 15. Selected from.

[0056] In another embodiment, a heavy chain-only antibody that binds to human B cell maturation antigen (BCMA) is:

[0057] (i) CDR1 sequence of SEQ ID NO: 1, CDR2 sequence of SEQ ID NO: 8, and C of SEQ ID NO: 12 DR3 sequence, or

[0058] (ii) The CDR1 sequence of sequence number 1, the CDR2 sequence of sequence number 8, and the CDR1 sequence of sequence number 14 CDR3 sequence, or

[0059] (iii) CDR1 sequence of sequence number 2, CDR2 sequence of sequence number 9, and sequence number 13 The CDR3 sequence of, or

[0060] (iv) The CDR1 sequence of sequence number 1, the CDR2 sequence of sequence number 8, and the sequence number 15 The CDR3 sequence,

[0061] Includes heavy chain variable regions within the human VH framework.

[0062] In all embodiments, the heavy chain-only antibody may have multispecificity, such as bispecificity. stomach.

[0063] In certain embodiments, the bispecific heavy chain-only anti-BCMA antibody is two different BCMA antibodies. For a protein, or for two different epitopes on the same BCMA protein It has binding affinity.

[0064] In other embodiments, only bispecific heavy chain anti-BCMA antibodies have effects such as T cell antigens. It has binding affinity to tar cells.

[0065] In further embodiments, the bispecific heavy chain-only anti-BCMA antibody is a binding parent to CD3. It has a Japanese character.

[0066] In various embodiments, multiple or bispecific anti-BCMA antibodies are expressed in CAR-T format. could be.

[0067] In another embodiment, the present invention relates to a pharmaceutically active composition comprising only heavy chain antibodies as described above herein. Related to objects.

[0068] In yet another aspect, the present invention relates to the treatment of B cell damage characterized by BCMA expression. With respect to the method, this method applies to subjects having such impairments as described above herein. This includes administering an antibody or a pharmaceutical composition.

[0069] In some embodiments, B-cell damage is, for example, multiple myeloma or systemic lupus erythema. It could be todes.

[0070] In a further embodiment, the present invention relates to a polynucleus encoding the anti-BCMA antibody described herein. Regarding leotide.

[0071] In further embodiments, the present invention relates to the poly(b) encoding the anti-BCMA antibody described herein. Regarding vectors containing nucleotides.

[0072] The present invention further comprises a polynucleotide encoding the anti-BCMA antibody described herein. Regarding cells containing vectors.

[0073] The present invention further provides a method for producing the antibody described herein, wherein the expression of a protein Under acceptable conditions, the polynucleotide encoding the anti-BCMA antibody described herein is included. Proliferating cells (e.g., host cells) and the cells and / or cell culture medium The present invention relates to a method comprising isolating antibodies from UniRat This involves immunizing animals with BCMA and identifying BCMA-binding heavy chain antibody sequences.

[0074] These and further embodiments, including examples, are described further in the remainder of this disclosure. The present invention provides, for example, the following items. (Item 1) This is a heavy chain-only antibody that binds to human B cell maturation antigen (BCMA), (a) Having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs. 1 to 7 CDR1 and / or (b) Having two or fewer substitutions in any of the amino acid sequences of sequence numbers 8-11 CDR2, and / or (c) Any of the amino acid sequences of sequence numbers 12-15 contains two or fewer substitutions. CDR3 A heavy chain-only antibody containing a heavy chain variable region. (Item 2) The aforementioned CDR1, CDR2, and CDR3 sequences are present in the human framework, item 1 Only the heavy chain antibodies described. (Item 3) A heavy chain-only antibody as described in item 1, further containing the heavy chain constant region sequence in the absence of the CH1 sequence. . (Item 4) (a) A CDR1 sequence selected from the group consisting of sequence numbers 1 to 7, and / or (b) A CDR2 sequence selected from the group consisting of sequence numbers 8-11, and / or (c) CDR3 sequences selected from the group consisting of sequence numbers 12-15 A heavy chain-only antibody, including one of the items 1-3 listed below. (Item 5) (a) A CDR1 sequence selected from the group consisting of sequence numbers 1 to 7, (b) A CDR2 sequence selected from the group consisting of sequence numbers 8-11, (c) A CDR3 sequence selected from the group consisting of sequence numbers 12-15 and Antibodies containing only the heavy chains listed in item 4. (Item 6) (i) CDR1 sequence of SEQ ID NO: 1, CDR2 sequence of SEQ ID NO: 8, and C of SEQ ID NO: 12 DR3 sequence; or (ii) The CDR1 sequence of sequence number 1, the CDR2 sequence of sequence number 8, and the CDR1 sequence of sequence number 14 CDR3 sequence; or (iii) CDR1 sequence of sequence number 2, CDR2 sequence of sequence number 9, and sequence number 13 The CDR3 sequence of; or (iv) The CDR1 sequence of sequence number 1, the CDR2 sequence of sequence number 8, and the sequence number 15 CDR3 sequence Antibodies containing only the heavy chains listed in item 5. (Item 7) It has at least 95% sequence identity with any of the sequences of sequence numbers 16-50. An antibody consisting only of the heavy chain described in any one of items 1 to 3, including the heavy chain variable region. (Item 8) Includes a heavy chain variable region sequence selected from the group consisting of sequence numbers 16-50, as described in item 7. Only the heavy chain of the antibody. (Item 9) Heavy chain variable regions selected from the group consisting of sequence numbers 16, 17, 18, 30, 34, and 38. Antibodies containing only the heavy chain sequence as described in item 8, including the regional sequence. (Item 10) (a) CDR1 sequence of the following equation GFT X1 X2 X3 X4 X5 (In the formula, X1 is V, I, or F. X2 is either S or T, X3 is either S or N, X4 is either Y or S, X5 is G or A), (b) CDR2 sequence of the following equation I X6 G X7 X8 X9 X10 T (In the formula, X6 is either R or S, X7 is either S or D, X8 is D, G, or S. X9 is G or D, X10 is S, T, or N), and (c) CDR3 sequence of the following equation AKQG X11 NDGPFD X12 (In the formula, X9 is G or E, (X10 is either Y or H) Human B cell maturation antigen (BCMA) includes, heavy chain variable region, heavy chain variable region, and Antibodies that bind only to the heavy chain. (Item 11) Heavy chain variable region containing CDR1, CDR2, and CDR3 sequences in the human VH framework A heavy chain-only antibody that binds to human B cell maturation antigen (BCMA), including the CDR The sequence has two or fewer substitutions in a CDR sequence selected from the group consisting of sequence numbers 1 to 15. The heavy chain-only antibody has the sequence described above. (Item 12) Heavy chain variable region containing CDR1, CDR2, and CDR3 sequences in the human VH framework The CDR sequence is selected from the group consisting of sequence numbers 1 to 15, as described in item 11. Only the heavy chain antibody is included. (Item 13) This is a heavy chain-only antibody that binds to human B cell maturation antigen (BCMA), (i) CDR1 sequence of SEQ ID NO: 1, CDR2 sequence of SEQ ID NO: 8, and C of SEQ ID NO: 12 DR3 sequence, or (ii) The CDR1 sequence of sequence number 1, the CDR2 sequence of sequence number 8, and the CDR1 sequence of sequence number 14 CDR3 sequence, or (iii) CDR1 sequence of sequence number 2, CDR2 sequence of sequence number 9, and sequence number 13 The CDR3 sequence of, or (iv) The CDR1 sequence of sequence number 1, the CDR2 sequence of sequence number 8, and the sequence number 15 CDR3 sequence A heavy chain-only antibody comprising a heavy chain variable region containing within a human VH framework. (Item 14) A multispecific antibody consisting only of heavy chains as described in any one of items 1 to 13. (Item 15) A bispecific heavy chain-only antibody as described in item 14. (Item 16) The heavy chain described in item 15 has binding affinity to two different BCMA proteins. Only antibodies. (Item 17) The term has binding affinity to two different epitopes on the same BCMA protein. Heavy chain-only antibodies as described in item 15. (Item 18) A heavy-chain-only antibody as described in item 14, which has binding affinity to effector cells. (Item 19) A heavy chain-only antibody as described in item 14, which has binding affinity to T cell antigens. (Item 20) A heavy chain-only antibody as described in item 19, which has binding affinity to CD3. (Item 21) A CAR-T format antibody consisting only of the heavy chain described in one of items 1-20. (Item 22) A pharmaceutical composition containing only heavy chain antibodies as described in any one of items 1 to 21. (Item 23) A method for treating B cell damage characterized by BCMA expression, wherein the affected For the target An antibody as described in any one of items 1 to 21 or a pharmaceutical composition as described in item 22. The method comprising administering the following. (Item 24) The method according to item 23, wherein the B-cell disorder is multiple myeloma. (Item 25) The method according to item 23, wherein the B-cell disorder is systemic lupus erythematosus. (Item 26) A polynucleotide encoding one of the antibodies listed in items 1-21. (Item 27) A vector containing the polynucleotides described in item 26. (Item 28) Cells containing the vector described in item 27. (Item 29) A method for producing an antibody described in any of items 1 to 21, The cells described in item 28 are grown under conditions that allow the expression of the aforementioned protein. , To isolate the antibody from the aforementioned cells and The method, including the method described above. (Item 30) A method for producing an antibody as described in any one of items 1 to 21, Immunizing UniRat animals with BCMA, Identifying the BCMA-binding heavy chain sequence and The method, including the method described above. [Brief explanation of the drawing]

[0075] [Figure 1-1] Only the 35 heavy chains of the present invention show the CDR1, CDR2, and CDR3 amino acid sequences of the anti-BCMA antibody. [Figure 1-2] Only the 35 heavy chains of the present invention show the CDR1, CDR2, and CDR3 amino acid sequences of the anti-BCMA antibody. [Figure 2-1] Only 35 heavy chains of the present invention represent the heavy chain variable region sequence of the anti-BCMA antibody. [Figure 2-2] Only 35 heavy chains of the present invention represent the heavy chain variable region sequence of the anti-BCMA antibody. [Figure 3]This column shows the binding of BCMA protein and BCMA-expressing cell lines. Column 1 shows the clone ID of the heavy-chain-only antibody (UniAb) tested. Column 2 shows the concentration of expressed UniAb in the supernatant. Column 3 shows the ELISA signal of human BCMA protein binding as a factor relative to the background. Column 4 shows the ELISA signal of human IgG1κ protein binding as a factor relative to the background. Column 5 shows the ELISA signal of human lambda protein binding as a factor relative to the background. Column 6 shows the ELISA signal of human serum albumin protein binding as a factor relative to the background. Column 7 shows the ELISA signal of baculovirus protein binding as a factor relative to the background. Column 8 shows the blocking percentage of April ligand protein to BCMA protein. Column 9 shows the average fluorescence intensity of cell binding to RPMI-8226 cells expressing BCMA on the cell surface. Column 10 shows the average fluorescence intensity of cell binding to NCI-H929 cells expressing BCMA on the cell surface. Column 11 shows the average fluorescence intensity of cell binding to HDLM2 cells that do not express BCMA on their cell surface. [Figure 4] The binding affinity of the monovalent and bivalent forms of the anti-BCMA heavy chain-only antibody 308902 was measured by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in dynamic mode. The binding affinity (Kd) for the monovalent form was 779 pM, and the Kd for the bivalent form was 53 pM. [Figure 5] This is a graph of the scFv CAR-T construct, the monospecific human VH CAR-T construct, and the bispecific human VH CAR-T construct. [Modes for carrying out the invention]

[0076] The implementation of this invention, unless otherwise indicated, falls within the scope of molecular biology (recombinant technology) in the art. It uses conventional techniques of microbiology, cell biology, biochemistry, and immunology (including techniques). Such technologies include "Molecular Cloning: A Laboratory M annual”, second edition(Sambrook et al.,19 89), “Oligonucleotide Synthesis” (MJGait ,ed.,1984), “Animal Cell Culture”(RIFre shney, ed., 1987), “Methods in Enzymology” ( Academic Press, Inc.), “Current Protocols in Molecular Biology” (FMAusubel et al. ,eds.,1987,and periodic updates), “PCR:Th e Polymerase Chain Reaction”,(Mullis et al., ed., 1994), “A Practical Guide to Mole cular Cloning” (Perbal Bernard V., 1988), “ Phage Display:A Laboratory Manual”(Barba s et al., 2001), Harlow, Lane and Harlow, Us. ing Antibodies:A Laboratory Manual:Porta ble Protocol No.I,Cold Spring Harbor Lab oratory (1998), and Harlow and Lane, Antibodi es:A Laboratory Manual,Cold Spring Harbo This is adequately explained in literature such as r Laboratory;(1988).

[0077] If a range of values ​​is provided, unless the context explicitly indicates otherwise, the lower limit is in units of 10 minutes. Up to 1, each intermediary value between the upper limit and lower limit of that range, and any other value within that display range. It is understood that the indicated value or intervening value is included in the present invention. A smaller range of these is included. The upper and lower limits of the range may be independently contained within a smaller range, and any value within the displayed range may be included. This is also included in this specification, subject to the specific exclusion limits of the intent. If it includes one or both of the upper and lower limits, the upper and lower limits that they encompass The scope of excluding either one or both of these is also included in the present invention.

[0078] Unless otherwise specified, antibody residues in this specification are numbered according to the Kabat numbering system. (For example, Kabat et al., Sequences of Immu) nological interest.5th Ed.Public Health Service,National Institutes of Health,Be Thesda, Md. (1991).

[0079] The following description will not include many specific details in order to provide a complete understanding of the present invention. However, the present invention may be implemented without one or more of these specific details. It will be obvious to those skilled in the art that this is possible. In other examples, to avoid obscuring the present invention Features and procedures that are readily apparent to those skilled in the art are not described.

[0080] All references cited herein, including patent applications and publications, are provided by reference to the same source. The whole thing is incorporated.

[0081] I. Definition "Includes" means that the listed elements are required for the composition / method / kit. However, other elements may be included to form a composition / method / kit, etc., within the scope of the claims. .

[0082] "Essentially consisting of..." means that the scope of the described composition or method is fundamental to the present invention. and to limit to specific materials or processes that do not substantially affect the novel features(s) It means that.

[0083] "~consists of" means the composition, method, or kit of any element specified in the claim. This means the absence of a process or ingredient.

[0084] When used herein, the term "monoclonal antibody" refers to substantially the same type of antibody. This refers to antibodies obtained from a group of bodies; in other words, individual antibodies contained in that group are present in trace amounts. They are identical except for any possible spontaneous mutations. Monoclonal antibodies are Furthermore, they are highly specific and target a single antigenic site. Conventional (polyclonal) antibody preparations containing different antibodies against a specific epitope are different from conventional (polyclonal) antibody preparations. In contrast, each monoclonal antibody targets a single determinant on the antigen.

[0085] The terms "heavy chain only antibody," "heavy chain antibody," and "UniAb" are used interchangeably. In its broadest sense, it refers to antibodies that lack the light chain of conventional antibodies. Homodimeric UniAb lacks a light chain. Therefore, lacking the VL domain, the antigen is a single domain, i.e., a heavy chain antibody. Recognized by the variable domain (VH) of the heavy chain. This term is not specifically limited. However, in the absence of the CH1 domain, the VH antigen-binding domain and the constant domains of CH2 and CH3 In, functional (antigen-binding) variants of such antibodies, soluble VH variants, one variable dom Ig containing a homodimer of yin (V-NAR) and five C-like constant domains (C-NAR) -NAR, and their functional fragments, as well as soluble single-domain antibodies (sUniDa b) is a homodimer antibody containing the above. In one embodiment, the heavy chain-only antibody is in framework 1 CDR1, Framework 2, CDR2, Framework 3, CDR3, and Frame It consists of a variable region antigen-binding domain consisting of workpiece 4. In one embodiment, only the heavy chain The antibody consists of an antigen-binding domain, at least a portion of the hinge region, and CH2 and C It consists of an H3 domain. In another embodiment, the heavy chain of the antibody has an antigen-binding domain. It consists of at least a portion of the hinge region and the CH2 domain. Further implementation Morphologically, heavy chain antibodies consist of the antigen-binding domain, at least a portion of the hinge region, and It consists of the CH3 domain. The CH2 and / or CH3 domains are disconnected. Heavy chain-only antibodies are also included herein. In further embodiments, the heavy chain is antigen-binding domain In, and at least one CH (CH1, CH2, CH3, or CH4) domain It is composed of these but does not include the hinge region. A heavy chain-only antibody is one in which the two heavy chains are otherwise co-located. It can be in the form of a dimer, either bonded or non-covalently bonded to each other by disulfide bonds. While chain-only antibodies may belong to the IgG subclass, they may also belong to the IgM, IgA, IgD, and IgE subclasses. Antibodies belonging to other subclasses, such as the class, are also included herein. In certain embodiments Heavy chain antibodies are IgG1, IgG2, IgG3, or IgG4 subtypes, especially Ig It is a G1 subtype. In one embodiment, the heavy chain only antibody described herein is a chimeric antigen receptor It is used as the binding (targeting) domain for (CAR).

[0086] As used herein, the term "BCMA" refers to a molecule preferentially expressed in differentiated plasma cells. It is a member of the tumor necrosis receptor superfamily, along with BCMA, CD269, and TNF. Regarding the human B cell maturation antigen, also known as RSF17 (UniProtQ02223) The extracellular domain of human BCMA is determined by UniProt to be amino acids 1-54 (or It consists of 5-51).

[0087] The terms "anti-BCMA heavy chain only antibody" and "BCMA heavy chain only antibody" refer to BCMA. This specification refers only to heavy chain antibodies defined above that bind immunologically. It is used inside.

[0088] The term "variable" as used in relation to antibodies refers to the arrangement of a particular portion of the antibody's variable domain. This refers to the fact that the bonds of each specific antibody against a particular antigen vary widely among antibodies. It is used in synthesis and specificity. However, variability is not limited to the entire variable domain of the antibody. It is not evenly distributed across. This is due to the hypercapsulation in the variable domains of both the light and heavy chains. It is concentrated in three segments called variable domains. The more highly conserved variable domains The remaining portion is called the framework region (FR). This is the original heavy and light chain variable domain. Each of these includes four FRs that primarily employ a β-sheet configuration, and these are located in three highly variable regions. These more interconnected, hypervariable regions connect the β-sheet structure, and in some cases form a part of it. It forms a loop. The hypervariable regions within each chain are brought close together by FR and held together. Therefore, together with the hypervariable region of the other chain, it contributes to the formation of the antigen-binding site of the antibody (Kabat). et al.,Sequences of Proteins of Immunol ogical Interest,5th Ed.Public Health Ser vice,National Institutes of Health,Bethe See sda, MD. (1991). The constant domain is directly involved in the binding of the antibody to the antigen. However, antibodies have various effector functions, such as their involvement in antibody-dependent cytotoxicity (ADCC). It exhibits the following characteristics.

[0089] When used herein, the term "hypervariable region" refers to the antibody responsible for antigen binding. This refers to anoacid residues. The hypervariable region is generally derived from the "complementarity-determining region" or "CDR". Mino acid residues (for example, residues 31-35 (H1) and 50-65 (H2) in the heavy chain variable domain) ), and 95-102(H3), Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institute lutes of health, Bethesda, MD. (1991) and / or Those residues derived from the "hypervariable loop," residues 26-32 (H1) in the heavy chain variable domain. 53-55 (H2), and 96-101 (H3), Chothia and Lesk Includes J.Mol.Biol.196:901-917(1987)). The "K region" or "FR" residues are those other than the hypervariable region residues as defined herein. This is a variant domain residue.

[0090] While exemplary CDR names are shown herein, those skilled in the art will understand the CDRs including Kabat definitions. It is understood that many definitions are commonly used ("Zhao et al. A g ermline knowledge based computational ap proach for determining antibody completion ntarity determining regions.”Mol Immunol (See .2010;47:694-700), this is based on sequence diversity, and most one It is commonly used. The Chothia definition is based on the position of the structure loop region (Chot hia et al. “Conformations of immunoglobulin in hypervariable regions.”Nature.1989;34 2:877-883). Important alternative CDR definitions include, but are not limited to, those of Honegge. r,“Yet another numbering scheme for immu noglobulin variable domains:an automatic modeling and analysis tool.”J Mol Biol. 2001;309:657-670, Ofran et al. identification of complementarity determination ining regions(CDRs) reveal peculiar char acteristics of CDRs and B cell epitopes. ”J Immunol.2008;181:6230-6235, Almagro“Id entification of differences in the speci ficity-determining residues of antibodie s that recognize antigens of different s ize:implications for the rational design of antibody repertoires.”J Mol Recognit .2004;17:132-143, and Padlanet al. “Identifi cation of specificity-determining residu es in antibodies.”FasebJ.1995;9:133-139 This includes the disclosures thereof, each of which is specifically incorporated herein by reference. To be absorbed.

[0091] The term "two or fewer substitutions" in an amino acid sequence refers to two or one substitution in a reference amino acid sequence. Or, as used herein, to mean zero substitutions.

[0092] Regarding the reference polypeptide sequence, the "amino acid sequence identity percentage (%)" is any Conservative substitutions are not taken into consideration as part of sequence identity to obtain the highest possible sequence identity percentage. After sequence alignment and the introduction of gaps as necessary, the reference polypeptide is prepared. It is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the sequence. Alignment, which aims to determine the percentage of amino acid sequence identity, is performed using this technique. Various methods that fall within the scope of skill in the field, for example, publicly available computers Computer software, such as BLAST, BLAST-2, ALIGN, or Mega This can be achieved using lign (DNASTAR) software. Therefore, it is necessary to obtain the maximum alignment over the full length of the sequences being compared. This includes any algorithm that is considered appropriate for performing array alignment. The data can be determined. However, for the purposes of this specification, sequence comparison The computer program ALIGN-2 is used to generate amino acid sequence identity percentage values.

[0093] "Isolated" antibodies are those identified, separated, and / or recovered from components of their natural environment. These are materials that interfere with the diagnostic or therapeutic use of antibodies due to their natural environmental contamination. These include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. This may include: In a preferred embodiment, the antibody is purified to the following degree: (1) Lowry When determined by law, the antibody content exceeds 95% by weight, most preferably exceeding 99% by weight. (2) Spinning cup sequencer To obtain at least 15 residues of the N-terminal or internal amino acid sequence using (or) (3) Reduction to a sufficient degree, or (3) using Coomassie blue, or preferably silver staining. Alternatively, under non-reducing conditions, use SDS-PAGE until homogeneous. The isolated antibody contains Because at least one component of the natural environment is absent, the antibodies of Insights in recombinant cells It includes. However, usually, isolated antibodies are prepared by at least one purification step. It will be manufactured.

[0094] The antibodies of the present invention include multispecific antibodies. Multispecific antibodies have two or more binding specificities. The term "multiple specificity" specifically refers to "bispecificity" and "triple specificity," High-order independent specific binding affinity, such as high-order polyepitope specificity, and tetravalent antibodies and antibody fragments. This includes. Specifically, "multispecific" antibodies are antibodies that contain combinations of different binding entities. This includes antibodies containing multiple identical binding entities. "Multispecific antibodies," "Multispecific single-chain antibodies only." The terms “antibody” and “multispecific UniAb” are used in the broadest sense herein. This includes all antibodies that have more than one binding specificity.

[0095] When used herein, the term "valence" refers to a specific number of binding sites in an antibody molecule. cormorant.

[0096] A "polyvalent" antibody has two or more binding sites. Therefore, "bivalent," "trivalent," and The term "tetravalent" refers to two binding sites, three binding sites, and four binding sites, respectively. This refers to the presence of. Therefore, the bispecific antibody according to the present invention is at least bivalent and It can be trivalent, tetravalent, or otherwise polyvalent.

[0097] A wide variety of methods and protein structures are known, and bispecific monoclonal antibodies (BsMAB) is used in the preparation of triplicate antibodies and other similar antibodies.

[0098] The terms "bispecific triple-chain antibody-like molecule" or "TCA" are used herein to mean three different types of molecules. Antibody-like molecules that contain, or consist of, polypeptide subunits. It is used to point to, two of which are antigen-binding regions and at least one CH domain A monoclonal antibody containing one heavy chain and one light chain, or such antibody chain This heavy / light chain pair contains, essentially consists of, functional antigen-binding fragments. It has binding specificity to the first antigen. The third polypeptide subunit is CH1 In the absence of the domain, F containing the CH2 and / or CH3 and / or CH4 domains Antibodies containing only the heavy chain with the c portion, as well as antibodies with a different epitope for the second antigen or the first antigen. Essentially, or consisting of, an antigen-binding domain that binds to an epitope. The binding domain originates from the variable region of the antibody heavy or light chain, or has sequence identity with it. It possesses. Some of these variable regions are the VH and / or VL gene segments, D and Variable region may be encoded by the JH gene segment or the JL gene segment. This refers to the reconstituted VHDJH, VLDJH, VHJL, or VLJL gene segment. It can be encoded by. The TCA protein utilizes only heavy chain antibodies as defined above. do.

[0099] The term "chimeric antigen receptor" or "CAR" refers to the transmembrane domain and intracellular sigma Desired binding specificity to the monoclonal transmission domain (e.g., monoclonal antibodies or other receptors) In this specification, it is used most broadly to refer to artificial receptors that fuse the antigen-binding domain of Gandr. Typically, the receptor is monochromatic to create a chimeric antigen receptor (CAR). It is used to fuse the specificity of a single antibody onto T cells. (J Natl Canc er Inst,2015;108(7):dvj439, and Jackson et al. al.,Nature Reviews Clinical Oncology,201 6;13:370-383.) Representative monospecific and human VH extracellular binding domains Figure 5 shows a comparison of the bispecific CAR-T construct with the scFv CAR-T construct.

[0100] "Human ideotype" refers to a type of immunoglobulin ideotype in the variable region of the heavy and / or light chain. This refers to polypeptide sequence epitopes present on antibodies. It is also known as "human ideotype." When used herein, the term refers to the naturally occurring sequence of human antibodies, as well as naturally occurring human antibodies. It contains both a synthetic sequence that is substantially identical to the polypeptide found in the antibody. "Substantially" means This means that the degree of amino acid sequence identity is at least about 85% to 95%. More specifically, the degree of amino acid sequence identity is over 90%, more preferably over 95%.

[0101] A "chimeric antibody" or "chimeric immunoglobulin" is a substance containing at least two different Ig antibodies. Immunoglobulin molecules containing amino acid sequences from the gene locus, for example, the human Ig gene locus The transgene contains the encoded portion and the portion encoded by the rat Ig locus. This refers to a chimeric antibody. A chimeric antibody is a tiger antibody that has a non-human Fc region or an artificial Fc region. This includes immunogenic antibodies and human ideotypes. Such immunoglobulins are These can be isolated from animals of the present invention that have been engineered to produce such chimeric antibodies.

[0102] The "effector function" of an antibody is the Fc region of the antibody (the Fc region of the natural sequence or amino acids). This refers to the biological activity attributed to the sequence variant Fc region. An example of antibody effector function is... This includes C1q binding, complement-dependent cytotoxicity, Fc receptor binding, and antibody-dependent cell-mediated cytotoxicity. (ADCC), phagocytosis, and downregulation of cell surface receptors (e.g., B cell receptor; BCR) It includes the character for "honored".

[0103] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to the non-specific cytotoxic cells expressing Fc receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognizing an antibody bound to a target cell and subsequently causing lysis of the target cell, indicating a cell-mediated reaction. NK cells, which are primary cells for mediating ADCC, express only Fc γRIII, while monocytes express FcγRI, FcγRII, and FcγRII II. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu.Rev.Immunol 9:457-92(1991). To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay as described in U.S. Patent No. 5,500,362 or No. 5,821,337 can be performed. Effector cells useful for such an assay include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656(1998).

[0104] "Human effector cells" are leukocytes expressing one or more FcRs and performing effector functions. Preferably, this cell expresses at least FcγRIII and performs the ADCC effector function. Examples of human leukocytes via ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, It contains a sphere, and PBMC and NK cells are preferred. Effector cells can be isolated from their natural sources, such as blood or PBMC, as described herein.

[0105] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., a polypeptide (e.g., an antibody)) complexed with a homologous antigen. To evaluate complement activation, for example, a CDC assay as described in Gazzano-Santoro et al., J.I mmunol.Methods 202:163(1996) can be performed.

[0106] "Binding affinity" refers to the total strength of the non-covalent binding 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 the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigens more quickly and tend to remain bound.

[0107] As used herein, "Kd" or "Kd value" refers to, in a kinetic format, using an Oct et QK384 instrument (Fortebio Inc., Menlo Park, CA) This refers to the dissociation constant determined by BioLayer interferometry. For example, anti-Mau The mouse-Fc fusion antigen is loaded onto the Fc sensor, and then immersed in an antibody-containing well to determine the concentration. The residual association rate (KON) is measured. The antibody dissociation rate (KOFF) is measured in the final step. The sensor is immersed in a well containing only the buffer solution. Kd is the ratio of koff / kon. (For further details, see Concepcion, J, et al., Comb.) Chem High Throughput Screen,12(8),791-80 (See 0.2009).

[0108] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. In this case, the antigen has several or many different epitopes, and many different antibodies This term specifically includes linear epitopes and conformational epitopes.

[0109] "Epitope mapping" refers to the binding sites or epitopes of antibodies on those target antigens. This is the process of identifying the antibody epitope. The antibody epitope is either a linear epitope or a conformational epitope. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are discontinuous in protein sequences, but their three-dimensional structure... It is formed from amino acids that bind during the folding process into its structure.

[0110] "Polyepitope specificity" refers to the ability to identify two or more different targets, whether the same or different. This refers to the ability to specifically bind to a particular epitope.

[0111] When two antibodies recognize the same or sterically overlapping epitopes, the antibody acts as a reference antibody. and binds to an "essentially identical epitope". Two epitopes are identical or sterically overlapping To determine whether it binds to an epitope that does, the most widely used rapid method is a competitive assay, which can be configured in any format using a labeled antigen or a labeled antibody . Usually, the antigen is immobilized on a 96-well plate, and the ability of the unlabeled antibody to interfere with the binding of the labeled antibody is measured using a radioactive or enzyme label .

[0112] The terms "treatment", "treating", etc. are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect can be preventive in terms of completely or partially preventing the disease or its symptoms, and / or therapeutic in terms of partially or completely curing the disease and / or side effects resulting therefrom. "Treatment", as used herein, encompasses any treatment of a disease in a mammal and includes (a) preventing the onset of the disease in a subject that is susceptible to but has not yet been diagnosed as having the disease, (b) inhibiting the disease, i.e., preventing its expression, or (c) alleviating the disease, i.e., causing regression of the disease. Therapeutic agents can be administered before, during or after the onset of a disease or injury . Treatment of an ongoing disease is of particular interest when the treatment stabilizes or alleviates the undesirable clinical symptoms of the patient. Such treatment is desirably carried out before complete loss of function in the affected tissue . The present therapy can be administered during the symptomatic stage of the disease and, in some cases, after the symptomatic stage of the disease .

[0113] ​"Therapeutic dose" refers to the amount of activator needed to provide a therapeutic benefit to a subject. For example, a "therapeutic dose" is the amount that improves or otherwise causes improvement in pathological symptoms. To improve the progression of a disease, or to alleviate a disease-related physiological condition or resistance. This is the amount that induces it.

[0114] The terms “subject,” “individual,” and “patient” are used interchangeably in this specification. Refers to mammals being evaluated and / or treated for therapeutic purposes. In one embodiment, Mammals are humans. The terms "subject," "individual," and "patient" refer to those who have cancer. This includes, but is not limited to, individuals with autoimmune diseases, individuals with pathogen infections, etc. It will not be used. The subjects may be humans, but other mammals, especially humans, can be used as laboratory models for diseases. This may include useful mammals such as mice and rats.

[0115] The term "CD3" refers to the human CD3 protein multiple subunit complex. The 3-protein multi-subunit complex consists of six distinct polypeptide chains. These are CD3γ chain (SwissProt P09693) and CD3δ chain (SwissProt t P04234), two CD3ε chains (SwissProt P07766) and one It contains the CD3ζ chain homodimer (SwissProt 20963), which is a T cell. It associates with receptor α and β chains. Unless otherwise specified, the term "CD3" is arbitrary. CD3 variants, isoforms, and those naturally expressed by cells (including T cells), Alternatively, transfect the genes or cDNA that encode those polypeptides. This includes homologous species that can be expressed on cells.

[0116] "BCMA x CD3 antibody" is a multispecific heavy chain-only antibody, for example, a bispecific heavy chain-only antibody. This contains two different antigen-binding regions, one of which is the antigen BCMA It binds specifically to [the target molecule], and one of these molecules specifically binds to CD3.

[0117] The term "pharmaceutical preparation" refers to a preparation that enables the biological activity of the active ingredient to be effective. It is in such a form, and is unacceptably toxic to the subjects to whom the formulation will be administered. This refers to a preparation that does not contain any further ingredients. Such preparations are sterile. An "acceptable excipient" (vehicle, additive) is one that provides an effective dose of the active ingredient used. This refers to substances that can be reasonably administered to target mammals for the purpose of [achieving a specific condition].

[0118] "Sterile" preparations are either sterilized or contain no living microorganisms and their cells. Does not contain or does not contain the ingredient. "Frozen" formulations are those prepared at temperatures below 0°C. .

[0119] A "stable" formulation is one in which the proteins within it maintain their physical stability and / or This refers to substances that inherently retain chemical stability and / or biological activity. Preferably, the formulation has physical and chemical stability when stored, as well as biological stability. It essentially retains its activity. The storage period is generally based on the intended shelf life of the formulation. Selected. Various analytical techniques for measuring protein stability are available in this field. It is capable of, for example, Peptide and Protein Drug Delive ry,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 over a selected period of time. Stability is evaluated by assessing aggregate formation (e.g., using size exclusion chromatography, turbidity). By measurement and / or visual inspection; cation exchange chromatography - Imaging capillary isoelectric focusing (icIEF) or capillary zone electrophoresis This method involves evaluating charge heterogeneity using dynamics; amino-terminated or carboxy-terminated Column analysis; mass spectrometry; SDS-PAGE analysis comparing reduced antibodies with complete antibodies; Petitometabolism (e.g., by trypsin or LYS-C) analysis; biological activity of antibodies Qualitative and / or determinative methods including evaluating antigen-binding function; It can be evaluated quantitatively. Instability includes aggregation, deamidation (e.g., Asn deamidation), Oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), cleavage / hydrolysis / interruption. Fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s) One or more of the following are observed: N-terminal extension, C-terminal processing, glycosylation differences. It may be accompanied by.

[0120] II. Detailed explanation Anti-BCMA antibody This invention provides a family of closely related heavy chain-only antibodies that bind to human BCMA. The antibodies of this family are defined herein and use a set of CDR sequences shown in Figure 1. This includes, and also the heavy chain variable region (VH) sequences provided in sequence numbers 16-50 shown in Figure 2. is exemplified by. The family of antibodies provides many advantages that contribute to their usefulness as clinical therapeutics (s). Antibodies include members with a range of binding affinities that enable the selection of a specific sequence with the desired binding affinity. Appropriate antibodies can be selected from those provided herein for development and therapeutic or other use, including but not limited to use as bispecific or trispecific antibodies, or as part of a CAR-T construct such as that shown in FIG. 5. The determination of affinity for a candidate protein can be carried out using methods known in the art such as Biacore measurements. Members of the antibody family can have an affinity for BCMA with a Kd of from about 10

[0121] to about 10 and include any value within those ranges. The selection of affinity is based on biological evaluation for modulation, such as BCMA biological activity including inhibition, in vitro assays, preclinical models, and clinical trials. including but not limited to use as bispecific or trispecific antibodies, or as part of a CAR-T construct such as that shown in FIG. 5.

[0122] The determination of affinity for a candidate protein can be carried out using methods known in the art such as Biacore measurements. Members of the antibody family can have an affinity for BCMA with a Kd of from about 10 to about 10 , -11 , , -6 , -9 , , -6 , -10 , , -11 , 8 , -10 , - , , -9 , -9 , , -10 , -9 , -8 , -8 , -8 6 to about 10 -11 and can include any value within those ranges. The selection of affinity is based on biological evaluation for modulation, such as BCMA biological activity including inhibition, in vitro assays, preclinical models, and clinical trials. to about 10 -6 to about 10 -10 to about 10 -6 to about 10 -9 to about 10 -6 to about 10 - 8 to about 10 -8 to about 10 -11 to about 10 -8 to about 10 -10 to about 10 -8 to about 10 -9 to about 10 -9 to about 10 -11 to about 10 -9 to about 10 -10 and also The selection of affinity is based on biological evaluation for modulation, such as BCMA biological activity including inhibition, in vitro assays, preclinical models, and clinical trials. such as inhibition, in vitro assays, preclinical models, and clinical trials, including BCMA biological activity This can be confirmed by evaluating its properties and potential toxicity.

[0123] The members of the antibody family described herein are cynomolgus monkey BCMA protein and Although not cross-reactive, if desired, cynomolgus monkey BCMA protein or any other It can be manipulated to provide cross-reactivity with BCMAs of animal species.

[0124] The family of BCMA-specific antibodies described herein is CDR within the human VH framework. 1. Contains a VH domain containing CDR2 and CDR3 sequences. The CDR sequence is, for example, CDR1, CDR2 and CDR1 of the provided exemplary variable region sequences described in SEQ ID NOs. 16-50 For each of DR3, amino acid residues 26-35, 53-59, and 98-117 They can be located within the preceding and succeeding regions. Generally, the order of the array remains the same, but different frames Those skilled in the art will understand that if the C sequence is selected, the CDR sequence may be in a different position. It will probably happen.

[0125] The CDR1, CDR2, and CDR3 sequences of the anti-BCMA antibody of the present invention are as follows: This can be encompassed, where X represents a variable amino acid, which is a specific A as shown below. It could be a amino acid.

[0126] CDR1

[0127] GFT X1 X2 X3 X4 X5

[0128] (In the formula,

[0129] X1 is V, I, or F.

[0130] X2 is either S or T,

[0131] X3 is either S or N,

[0132] X4 is either Y or S,

[0133] X5 is either G or A.

[0134] In one embodiment, X1 is V or I, X2 is S, X3 is S, and X4 is Y is G, and X5 is G. In another embodiment, CDR1 is selected from the sequence of sequence numbers 1 to 6. Selected. In yet another embodiment, CDR1 includes the sequence of sequence number 1 or sequence number 2. In further embodiments, CDR1 includes the sequence GFTVSSYG (sequence number 1).

[0135] CDR2

[0136] I X6 G X7 X8 X9 X10 T

[0137] (In the formula,

[0138] X6 is either R or S,

[0139] X7 is either S or D,

[0140] X8 is D, G, or S.

[0141] X9 is G or D,

[0142] X10 is S, T, or N).

[0143] In one embodiment, X6 is D and X7 is G. In another embodiment, X8 is S. In yet another embodiment, X9 is G and X10 is T. In this embodiment, X9 is G and X10 is S. In a further embodiment, X9 is G, X10 is T. In another embodiment, CDR2 is selected from the sequence of sequence numbers 8-11. In yet another embodiment, CDR2 includes an array of sequence number 8 or sequence number 9. In a specific embodiment, CDR2 includes the sequence IRGSDGST (sequence number 8).

[0144] CDR3

[0145] AKQG X11 NDGPFD X12

[0146] (In the formula,

[0147] X11 is G or E,

[0148] X12 is either Y or H.

[0149] In one embodiment, X11 is G and X12 is Y. In another embodiment, X11 is In another embodiment, X11 is E and X12 is H. In further embodiments, X11 is E and X12 is Y. Then, CDR3 is selected from the sequence of sequence numbers 12-15. In another embodiment, CDR 3 includes the sequence of sequence numbers 12, 14, or 15. In yet another embodiment, CDR3 is Includes the sequence AKQGENDGPFDH (sequence number 14).

[0150] Representative CDR1, CDR2, and CDR3 sequences are shown in Figure 1.

[0151] In one embodiment, the anti-BCMA heavy chain antibody of the present invention contains the CDR1 sequence of SEQ ID NO: 1, It includes the CDR2 sequence at column number 8 and the CDR3 sequence at sequence number 12 or sequence number 14.

[0152] In another embodiment, the anti-BCMA heavy chain only antibody of the present invention corresponds to the CDR1 sequence of SEQ ID NO: 2. The CDR2 sequence of sequence number 8 or sequence number 9, and sequence numbers 12, 13, 14, or 15 It includes the CDR3 sequence.

[0153] In a further embodiment, the anti-BCMA heavy chain only antibody of the present invention is CDR1 compound of SEQ ID NO: 1 The sequence includes the column, the CDR2 sequence of sequence number 8, and the CDR3 sequence of sequence number 14.

[0154] In further embodiments, the anti-BCMA antibody of the present invention is equivalent to the weight of SEQ ID NOs. 16-50 (Figure 2). Contains any of the amino acid sequences in the chain variable region.

[0155] In further embodiments, the anti-BCMA heavy chain only antibody of the present invention is the heavy chain of SEQ ID NO: 34. Includes variable region sequence (Antibody 308902).

[0156] In some embodiments, the CDR sequences of the anti-BMA antibody of the present invention are sequence numbers 1-15 ( In any one of the figures in Figure 1), the CDR1, CDR2 and / or CDR3 sequences or This refers to one or two amino acids compared to the CDR1, CDR2, and CDR3 sequences as a set. This includes substitutions. In some embodiments, the above amino acid substitution(s) are the same as shown in the formula above. In comparison, one or two of the amino acid positions 4-6 of CDR1 and / or CD One or two amino acid positions 2, 4-7 of R2, and / or CDR3 The no-acid position is one or two of positions 5 and 12. In some embodiments, this specification The single-chain anti-BCMA antibody in the book is less likely to be found in any of the heavy chain variable region sequences shown in Figure 2. At least 85% identity, at least 90% identity, at least 95% identity, at least Both include heavy chain variable region sequences with 98% identity or at least 99% identity. It will become that.

[0157] In some embodiments, bispecific antibodies or multispecific antibodies are provided, and they This includes, but is not limited to, three-chain bispecific antibodies, and any configuration discussed herein. It may have any of the following. Bispecific antibodies are antibodies that are specific to proteins other than BCMA. Both include a heavy chain variable region.

[0158] When the protein of the present invention is a bispecific antibody, one of the binding sites is specific to human BCMA. They are heterogeneous, and the other arm is target cells, tumor-associated antigens, target antigens, such as integrins. However, it may be specific to pathogen antigens, checkpoint proteins, etc. Target cells are specific Specifically, this includes cancer cells such as hematological malignancies, for example, B-cell tumors, as described below.

[0159] Single-stranded polypeptide, double-stranded polypeptide, triple-stranded polypeptide, quadruple-stranded polypeptide The present invention includes, but is not limited to, a large number of, bispecific antibodies of various forms. It is within the range. The bispecific antibodies used herein specifically refer to those on plasma cells (PCs) and T cells that bind to CD3 and BCMA, which is selectively expressed on multiple myeloma (MM). It contains bispecific antibodies (anti-BCMA × anti-CD3 antibodies). Such antibodies transport BCMA. It induces potent T cell-mediated killing of transporting cells, and as described below, tumors, especially hematological tumors. It can be used to treat ulcers, such as B-cell tumors.

[0160] Bispecific antibodies against CD3 and BCMA are, for example, WO2007117600. WO2009132058, WO2012066058, WO2012143498, W O2013072406, WO2013072415, and WO2014122144, It is also described in US20170051068.

[0161] Pharmaceutical composition Another aspect of the present invention relates to one or more antibodies of the present invention mixed with a suitable pharmaceutically acceptable carrier. The objective is to provide a pharmaceutically acceptable composition containing the body. The carrier may, but is not limited to, hold an adjuvant, solid carrier, water, buffer, or therapeutic component. To that end, examples of other carriers, or combinations thereof, used in the art are provided.

[0162] The antibody pharmaceutical composition used in accordance with the present invention contains a protein having a desired purity. Any pharmaceutically acceptable carrier, excipient, or in the form of a lyophilized formulation or aqueous solution. Prepared for storage by mixing with a stabilizer (e.g., Remington's P pharmaceutical sciences 16th edition,Osol (See A. Ed. (1980)). Permitted carriers, excipients, or stabilizers are specified in the recipe. The ent is non-toxic at the dosage and concentration used, and the buffer (phosphate, citrate, and Antioxidants and preservatives (such as oxyacids and other organic acids), ascorbic acid, and methionine. Ammonium tadecyldimethylbenzyl chloride, hexamethonium chloride, benzalkonium chloride Um, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkyl Parabens (such as methyl or propylparaben), catechol, resorcinol, cyanoparabens (e.g., chlorohexanol, 3-pentanol, and m-cresol), low molecular weight (approximately 10%) Polypeptides (less than 1 unit), proteins (serum albumin, gelatin, or immunoglobulins) (e.g., ), hydrophilic polymers (e.g., polyvinylpyrrolidone), amino acids (glycine, glutaramide) (e.g., mine, asparagine, histidine, arginine, or lysine), monosaccharides, disaccharides , and other carbohydrates (including glucose, mannose, or dextrin), kire Drugs (such as EDTA), sugars (sucrose, mannitol, trehalose, or sorbitol), (e.g., bitols), salt-forming counterions (e.g., sodium), metal complexes (e.g., Zn-proteins) (Citrate complex), and / or nonionic surfactants (TWEEN®, PLURONI Contains CS (trademark) or polyethylene glycol (PEG), etc.

[0163] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and suitable Manufactured under Good Manufacturing Practice (GMP) conditions. The pharmaceutical composition is available in unit dosage forms (i.e., single doses). It may be provided as a dosage for administration. The formulation is determined by the selected route of administration. The antibody of the present invention may be administered by intravenous injection or infusion, or subcutaneously. For injection administration, the antibody of the present invention is preferably administered to reduce discomfort at the injection site. It can be formulated as an aqueous solution in a physiologically compatible buffer. The solution is as described above. It may contain a carrier, excipient, or stabilizer. Alternatively, the antibody may be used before use. To provide a suitable vehicle, for example, a freeze-dried form for re-preparation using a sterile, pyrogen-free, water-free vehicle. It is possible.

[0164] Anti-BCMA antibody preparations 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 include, for example, US This is described in 20160355591 and US20160166689.

[0165] How to use The pharmaceutical compositions described herein are characterized by the expression or overexpression of BCMA in B cells and morphogenetic molecules. Used to treat septic cell malignancies and B-cell related disorders, including autoimmune disorders. It is possible.

[0166] Such B cell-related disorders include malignancies of B cells and plasma cells, as well as autoimmune disorders. Disorders include plasmacytoma, Hodgkin lymphoma, follicular lymphoma, and unsevered small cell lymphoma. Burkitt lymphoma, endemic Burkitt lymphoma, sporadic Burkitt lymphoma, marginal zone lymphoma, extranodal mucosal lymphoma Membrane-associated lymphoid lymphoma, nodular monocytic B-cell lymphoma, splenic lymphoma, mantle cell lymphoma Lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, immunoblastic lymphoma, primary Mediastinal B-cell lymphoma, pulmonary B-cell vascular lymphoma, small lymphocytic lymphoma, undetermined malignancy B-cell proliferation of sexually transmitted diseases, lymphomatous granulomatosis, post-transplant lymphoproliferative disorders, immunomodulatory disorders, joint problems Rheumatism, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid antibody syndrome, Shaga Sjögren's disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjögren's syndrome, Pemphigus plaque, scleroderma, multiple sclerosis, antiphospholipid antibody syndrome, ANCA-associated vasculitis, good Dopasture disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis, heavy chain This includes diseases, primary or immune cell-associated amyloidosis, or monoclonal gammasis. However, it is not limited.

[0167] Plasma cell disorders characterized by BCMA expression include multiple myeloma (MM). This refers to B cells characterized by the monoclonal proliferation and accumulation of abnormal plasma cells in the bone marrow compartment. It is a malignant tumor. Current treatments for MM often result in remission, but almost all patients The patient eventually experiences a relapse and dies. Immune intervention of myeloma cells in the context of allogeneic hematopoietic stem cell transplantation. There is substantial evidence of sexual removal, however, this approach is highly toxic and incurable. Few patients are cured. Some monoclonal antibodies are in preclinical and early stages. Clinical trials have shown promise in treating MM, but any specific substance for MM is not available. The consistent clinical efficacy of clonal antibody therapy has not been definitively proven. Therefore, MM New therapies, including immunotherapy, are in great need (for example, Carpenter et al., Clin Cancer Res 2013,19(8):2048-2 (See 060).

[0168] Overexpression or activation of BCMA by the proliferation-inducing ligand APRIL occurs in vivo. BCMA is known to accelerate the progression of multiple myeloma (MM). BCMA is used in mice. It has also been shown that it promotes the proliferation of xenografted MM cells containing p53 mutations in vivo. The activity of the APRIL / BCMA pathway is involved in MM disease development, and in tumor cells and the bones that support them. Because it plays a central role in drug resistance through bidirectional interactions with the medullary microenvironment, B CMA has been identified as a target for the treatment of MM. For further details, see the example below. Yu-Tsu Tai et al.,Blood 2016;127(25):32 See 25-3236.

[0169] Another B-cell disorder involving plasma cells, i.e., BCMA expression, is also known as lupus. It is known as systemic lupus erythematosus (SLE). SLE is a systemic autoimmune disease. This affects every part of the body, causing the immune system to attack the body's own cells and tissues, leading to chronic illness. It causes sexual inflammation and tissue damage. This is because antibody-immune complexes coagulate and further... This is a type III hypersensitivity reaction that triggers an immune response (Inaki & Lee, Na t Rev Rheumatol 2010;6:326-337).

[0170] The anti-BCMA heavy chain only antibody (UniAb) of the present invention is effective in treating MM, SLE, and BCMA Treatment of other B cell disorders or plasma cell disorders characterized by the present, such as those listed above. It can be used to develop therapeutic drugs for this purpose. In particular, the present invention's anti-BCMA heavy Chain-only antibodies (UniAb), used alone or in combination with other MM treatments, are effective against MM. It is a potential treatment.

[0171] In one embodiment, the antibody herein has a heavy chain only, i.e., an anti-BCMA antibody-CAR structure. Only the heavy chain may be the morphology of the anti-BCMA antibody-CAR transduced T cell structure.

[0172] The effective amount of the composition of the present invention for the treatment of disease depends on the means of administration, the target site, and the physiological state of the patient. The condition, whether the patient is human or animal, other drugs administered, and whether the treatment is preventive. It varies due to many different factors, including whether it is present or therapeutic. Typically, patients have However, companion animals other than humans, such as dogs, cats, and horses, are also included. It can also treat laboratory animals such as herons, mice, and rats. The therapeutic dosage is safe. It can be titrated to optimize its properties and effectiveness.

[0173] The dosage level can be easily determined by those skilled in the art, and if necessary, For example, it can be modified if necessary to alter the subject's response to treatment. The active ingredient can be combined with a carrier material to produce a single dosage form. The dosage varies depending on the host being treated and the specific mode of administration. Generally, the unit dosage is approximately 1 mg. Contains between approximately 500 mg of active ingredients.

[0174] In some embodiments, the therapeutic dose of the drug is approximately 0.0001 to 10 times the host's body weight. It may be 0 mg / kg, and more commonly in the range of 0.01 to 5 mg / kg. For example, The dosage is 1 mg / kg body weight, 10 mg / kg body weight, or within the range of 1 to 10 mg / kg. It may be within the range. An example treatment plan is once every two weeks, once a month, or every 3-6 months. The treatment is administered once a month. The therapeutic substance of the present invention is usually administered on multiple occasions. Single dose The intervals between these measurements can be weekly, monthly, or yearly. The intervals also depend on the blood levels of the patient's treatment entity. If specified by measuring the bell, it may be irregular. Alternatively, according to the present invention The treatment can be administered as a sustained-release formulation, in which case frequent administration is not necessary. No administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient. do.

[0175] Typically, the composition is prepared as an injectable liquid solution or suspension, and Furthermore, a solid form suitable for dissolving or suspending in a liquid vehicle before injection can also be prepared. The pharmaceutical compositions described herein can be used as is, or as solid (e.g., lyophilized) compositions. After reconstitution, it is suitable for intravenous or subcutaneous administration. The preparation is also enhanced as described above. Due to the adjuvant effect, liposomes or polylactides, polyglycolides, Alternatively, it can be emulsified or encapsulated in microparticles such as copolymers. Langer, Science 249:1527, 1990 and Hanes, Advanced Dr ug Delivery Reviews 28:97-119, 1997. The drug of the present invention. The agent can be formulated in a way that allows for sustained or pulsed release of the active ingredient. It can be administered in the form of an intravenous injection or implant preparation. Pharmaceutical composition It is generally sterile, substantially isotonic, and meets all the standards of the U.S. Food and Drug Administration. Formulated in full compliance with Good Manufacturing Practice (GMP) regulations.

[0176] The toxicity of antibodies and antibody structures described herein is not applicable in cell cultures or experimental animals. According to standard pharmaceutical procedures, for example, LD 50 (A lethal dose for 50% of the population) is LD 100 This can be determined by determining (a dose that is lethal to 100% of the population). The dose-to-toxicity ratio is the therapeutic index. This is observed in their cell culture assays and animal studies. The data obtained from the experiment is used to prescribe a dosage range that is non-toxic for use in humans. It can be used. The antibody dosage described herein is preferably such that it has little toxicity. It is within the range of circulating concentrations that include an effective dose. The dosage depends on the dosage form and usage. Depending on the route of administration used, this range may vary. Accurate prescription, route of administration, and administration The dosage may be selected by individual physicians, taking into consideration the patient's condition.

[0177] The administration composition generally consists of an antibody dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. It may contain a body or other abrasives. Various aqueous carriers, such as buffered saline solution, may be used. These solutions are sterile and generally do not contain undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The product is a pharmaceutically acceptable auxiliary substance necessary to approximate physiological conditions. (pH adjusters, buffers and toxicity adjusters, for example, sodium acetate, sodium chloride, chloride These may contain potassium, calcium chloride, and sodium lactate, etc. The concentration of the active ingredient in the drug can vary widely, depending on the specific mode of administration selected and the patient's needs. The choice will likely be based primarily on factors such as body fluid volume, viscosity, and body weight (for example, Remingt). on's Pharmaceutical Science(15th ed.,198 0) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds. .,1996)).

[0178] A kit including the activator and formulation thereof, and instructions for use, also falls within the scope of the present invention. The kit may also include at least one additional reagent, such as a chemotherapy drug. The kit typically includes a label indicating the intended use of the kit's contents. The term includes any word that is on the kit, supplied with the kit, or otherwise included in the kit. This includes all accompanying documents or records.

[0179] The present invention, as fully described herein, will not depart from the spirit or scope of the invention. It will become apparent to those skilled in the art that various changes and modifications can be made. [Examples]

[0180] Example 1 Genetically modified rats that express only heavy chain antibodies. The human-rat IgH gene locus is constructed and assembled in several parts. This involved modifying and ligating the rat C region gene downstream of human JH, and human VH6-D Upstream addition of fragmentary regions was included. Subsequently, two B genes with separate populations of human VH genes were formed. AC[BAC6 and BAC3] are human VH6, all D, all JH, and modified Encodes assembled and modified regions, including rat Cγ2a / 1 / 2b(ΔCH1). It was administered simultaneously with a BAC called Georg.

[0181] Transgenic gene with an artificial heavy chain immunoglobulin locus in an unreconfigured stereochemistry Nick rats were created. IgG2a(ΔC H 1), IgG1(ΔC H 1) IgG2b (ΔC H 1) The gene is C H One fragment was missing. Constant region genes IgE, IgA, and 3 The enhancer was included in Georg BAC. Transgenic rat RT- PCR and serum analysis (ELISA) are used to identify transgenic immunoglobulin loci. We revealed the expression of antibodies only from heavy chains of various isotypes in serum, as well as their reproductive rearrangement. Lancegenic rats were previously described in U.S. Patent Publication 2009 / 0098134 A1. Rats with the described mutant endogenous heavy chain and light chain gene loci were crossed. The analysis of the substance involved inactivation of rat immunoglobulin heavy and light chain expression, as well as human V, D, Furthermore, it showed high levels of expression of heavy chain antibodies containing a variable region encoded by the J gene. Immunization of transgenic rats generates a high-titer serum response of antigen-specific heavy chain antibodies. They released it. Transgenic rats expressing heavy chain antibodies with human VDJ regions It was called UniRat.

[0182] Example 2 immunity Immunity mediated by the recombinant extracellular domain of BCMA. Twelve UniRat animals (6 HC27 and 6 HC28) were recombinant human BCMA Immunization was performed using protein. Titermax / Alhydrogel adjuvant was used. Animals were immunized according to a standard protocol. The recombinant extracellular domain of BCMA was R& Purchased from D Systems, diluted with sterile saline, and combined with an adjuvant. The immunogen was combined with Titermax and Alhydrogel adjuvant. The primary immunization (priming) with an immunogen during Titermax was administered to both legs. Subsequent booster immunization was performed in the presence of Alhydrogel, and booster immunization was performed 3 days before harvesting. The procedure was performed using immunogens in BS. Serum was collected from rats at the time of final blood collection to determine serum titer. I took it.

[0183] Serum titer results The binding activity to a single 1:500 serum titer dilution was found in huBCM produced in eukaryotic cells. A+Fc protein and cynoBCMA+Fc protein, and their respective strains of E. coli. The two human BCMA proteins derived from wheat germ are tested by ELISA. Furthermore, serum samples were tested for two extratarget proteins, HSA and human IgG1. Furthermore, serum from all animals is used to create NCI-H929 cells (BCMA+, lambda-). We will perform an assay to check for binding to [the target organism].

[0184] Typically, this is linked to serum reactivity levels to NCI-H929 cells (BCMA+, lambda-). Since a significant spread of the results is observed, the relevance of these results is related to a subset of animals. Confirmed by ELISA-bound data created using cynoBCMA+Fc protein. A positive signal for binding to a substance indicates the ECD or F of a molecule also found in human immunogens. This may reflect binding to either of the c-parts. In both assay types, these may be observed before immunization. Analysis of serum collected from animals does not show reactivity to immunogens or untargeted proteins. It was.

[0185] Example 3 Gene construction, expression, and binding assays Only the heavy chain antibodies highly expressed in lymph node cells are encoded in the cDNA gene. Selected for construction and cloned into expression vectors. Subsequently, their heavy chain sequences were used in U The niAb heavy chain only (CH1 deletion, no light chain) was expressed in HEK cells as an antibody.

[0186] Figure 3 shows the results of the assay testing the binding of the anti-BCMA heavy chain-only antibody according to the present invention.

[0187] The supernatants of the six antibodies were tested for binding to human BCMA in a standard ELISA assay. The test was conducted. Binding to recombinant BCMA protein was determined to be by Abcam(ab50089) or Human BCMA ECD obtained was used for measurement by ELISA. The protein was used at a concentration of 2 μg / mL to capture 50 ng / mL of UniAb. The binding of niAb is due to the goat anti-human IgG HRP complex antibody (ThermoFisher 3 1413) was used for detection. All antibodies were detected using 0.05% Tween-20 and 1% powdered milk. Diluted with 1x TBS containing [unclear].

[0188] Off-target binding to baculovirus protein extract (BVP) for diluents By modifying the sample to use 1X PBS and 1% dried milk powder, the above ELISA was performed. The BVP extract was obtained from INSERM (Nantes, France). . Binding to baculovirus particles (in the assay conducted by the inventors, background noise) (More than 5 times) is correlated with a decrease in the half-life of antibodies in humans and monkeys, and is associated with a lower level of antibody activity in human tissue. This suggests affinity interaction (Hotzel et al., mAbs 4:6, pp. 753-760, 2012).

[0189] To enable off-target binding of human IgG1, UniAb is used to capture human IgG1 kappa. The ELISA used, followed by the detection of kappa chains using a goat anti-human kappaHRP complex antibody, was performed. The evaluation was based on the source (Southern Biotech 2060-05).

[0190] The supernatant of six test anti-BCMA antibodies was also found in RPMI-8226 cells (BCMA+, Lamb). The binding to (Da+) was tested by flow cytometry, and all 35 anti-BCMA The antibody supernatant was also tested for binding to H929 cells (BCMA+, lambda-). The last column in Figure 3 shows the binding of T cells to Hodgkin lymphoma (HDLM2) cells. However, this is because BCMA is not expressed on the cell surface.

[0191] The sample was placed in an EMD Millipore Guava easyCyte 8HT instrument. The data was measured using flow cytometry and analyzed using guavaSoft. Antibodies were detected using goat anti-human IgG F(ab')2 conjugated with PE (Souther (n Biotech 2042-09). Dilute all antibodies with PBS containing 1% BSA. Positive staining was determined by comparing it with staining using a human IgG1 isotype control. It was determined that the NCI-H929 and RPMI-8226 cell lines express human BCMA. This is a human multiple myeloma strain, but it is from the American Type Culture Collection (A It was obtained from TCC and cultured according to ATCC's recommendations.

[0192] The six UniAbs were further tested in a recombinant protein ELISA assay using APRI The ability to block L (ligand) / BCMA (receptor) binding was evaluated. To evaluate the blocking of receptor / ligand interactions between CMA and APRIL, Replace human BCMA (Sino Biological 10620-H03H) directly The samples were spread onto a plate and then incubated with a series of dilutions of each UniAb. , HA-tagged recombinant APRIL protein (RnD Systems 5860-AP -010) is incubated with BCMA / antibody complex, and APRI against BCMA L binding is performed using chicken anti-HA antibody (Abcam ab1190) conjugated with HRP. It was detected by using RnD systems' anti-BCMA antibody (AF193) for BCMA / A It was used as a positive control for PRIL blocking.

[0193] In Figure 3, column 8 shows the blocking of April ligand protein against BCMA protein. The graph shows the percentage of cells that express BCMA on the cell surface. Column 9 shows RPMI-8 cells. This shows the average fluorescence intensity of cell binding to 226 cells. Column 10 shows BCMA on the cell surface. This shows the average fluorescence intensity of cell binding to NCI-H929 cells expressing the gene. Column 11 shows This shows the average fluorescence intensity of cell binding to HDLM2 cells that do not express BCMA on the cell surface. vinegar.

[0194] Further off-target binding assays were performed on untreated baculovirus particles (BVP). However, none of the UniAb samples tested showed positive binding.

[0195] Figure 4 is essentially Concepcion, J, et al., Comb Chem H igh Throughput Screen,12(8),791-800,2009 As described, Octet QK384 instrument (Fortebio Inc., Measurements were taken using biolayer interferometry with Menlo Park (CA) in dynamic mode. Only the monovalent and bivalent forms of anti-BCMA heavy chains show binding affinity to antibody 308902. The binding affinity (Kd) of the nucleotide in its nucleotide form was 779 pM, while the Kd of the divalent form was 53 pM.

[0196] Preferred embodiments of the present invention have been shown and described herein, but such embodiments It will be obvious to those skilled in the art that these are provided only as examples. Numerous variations, modifications, and substitutions may be conceived without departing from the present invention. Various alternatives to the embodiments of the present invention described in the details may be adopted in the implementation of the present invention. It should be understood that this may be used. The following claims define the scope of the present invention and patent application It is intended to include methods and structures within the scope of the request, as well as their equivalents.

Claims

1. This is a heavy chain-only antibody that binds to human B cell maturation antigen (BCMA), (a) Having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs. 1 to 7 CDR1 and / or (b) Having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs. 8 to 11 CDR2 and / or (c) Having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs. 12-15 CDR3 A heavy chain-only antibody containing a heavy chain variable region.

2. The CDR1, CDR2, and CDR3 sequences are present in the human framework, according to the claim. The heavy chain-only antibody described in 1.

3. The heavy chain according to claim 1, further comprising a heavy chain constant region sequence in the absence of the CH1 sequence, is only resistant. body.

4. (a) A CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 to 7, and / or (b) A CDR2 sequence selected from the group consisting of SEQ ID NOs: 8 to 11, and / or (c) CDR3 sequence selected from the group consisting of sequence numbers 12 to 15 A heavy chain-only antibody according to any one of claims 1 to 3, comprising:

5. (a) A CDR1 sequence selected from the group consisting of sequence numbers 1 to 7, (b) A CDR2 sequence selected from the group consisting of sequence numbers 8 to 11, (c) A CDR3 sequence selected from the group consisting of sequence numbers 12 to 15 and A heavy chain-only antibody according to claim 4, comprising the above.

6. (i) CDR1 sequence of SEQ ID NO: 1, CDR2 sequence of SEQ ID NO: 8, and C of SEQ ID NO: 12 DR3 sequence; or (ii) The CDR1 sequence of sequence number 1, the CDR2 sequence of sequence number 8, and the CDR1 sequence of sequence number 14 CDR3 sequence; or (iii) CDR1 sequence of SEQ ID NO: 2, CDR2 sequence of SEQ ID NO: 9, and SEQ ID NO: 13 The CDR3 sequence; or (iv) CDR1 sequence of SEQ ID NO: 1, CDR2 sequence of SEQ ID NO: 8, and SEQ ID NO: 15 CDR3 array A heavy chain-only antibody according to claim 5, comprising the above.

7. It has at least 95% sequence identity with any of the sequences of sequence numbers 16 to 50. A heavy chain-only antibody according to any one of claims 1 to 3, comprising a heavy chain variable region.

8. The claim 7 includes a heavy chain variable region sequence selected from the group consisting of sequence numbers 16 to 50. Only the heavy chain antibody is included.

9. Heavy chain variable region selected from the group consisting of sequence numbers 16, 17, 18, 30, 34, and 38. A heavy chain-only antibody according to claim 8, comprising a regional sequence.

10. (a) CDR1 array of the following formula G F T X1 X2 X3 X4 X5 (In the formula, X1 is V, I, or F, X2 is S or T, X3 is S or N, X4 is either Y or S, X5 is G or A), (b) CDR2 sequence of the following equation I X6 G X7 X8 X9 X10 T (In the formula, X6 is either R or S. X7 is S or D, X8 is D, G, or S, X9 is G or D, X10 is S, T, or N), and (c) CDR3 array of the following formula A K Q G X11 N D G P F D X12 (In the formula, X9 is G or E, X10 is either Y or H. Human B cell maturation antigen (BCMA) includes a heavy chain variable region, a heavy chain variable region, and Antibodies that bind only to the heavy chain.

11. Heavy chain variable region containing CDR1, CDR2, and CDR3 sequences in the human VH framework A heavy chain-only antibody that binds to human B cell maturation antigen (BCMA), including the CDR The sequence has two or fewer substitutions in a CDR sequence selected from the group consisting of sequence numbers 1 to 15. The heavy chain-only antibody has the sequence described above.

12. Heavy chain variable region containing CDR1, CDR2, and CDR3 sequences in the human VH framework The CDR sequence is selected from the group consisting of sequence numbers 1 to 15, according to claim 11. Only the heavy chain antibodies listed.

13. This is a heavy chain-only antibody that binds to human B cell maturation antigen (BCMA), (i) CDR1 sequence of SEQ ID NO: 1, CDR2 sequence of SEQ ID NO: 8, and C of SEQ ID NO: 12 DR3 sequence, or (ii) The CDR1 sequence of sequence number 1, the CDR2 sequence of sequence number 8, and the CDR1 sequence of sequence number 14 CDR3 sequence, or (iii) CDR1 sequence of SEQ ID NO: 2, CDR2 sequence of SEQ ID NO: 9, and SEQ ID NO: 13 The CDR3 sequence, or (iv) CDR1 sequence of SEQ ID NO: 1, CDR2 sequence of SEQ ID NO: 8, and SEQ ID NO: 15 CDR3 array A heavy chain-only antibody comprising a heavy chain variable region containing within a human VH framework.

14. A heavy chain-only antibody according to any one of claims 1 to 13, which is multispecific.

15. A bispecific heavy chain-only antibody according to claim 14.

16. The heavy protein according to claim 15, which has binding affinity to two different BCMA proteins. Antibodies consisting only of the chain.

17. It has binding affinity to two different epitopes on the same BCMA protein. A heavy chain-only antibody as described in item 15.

18. A heavy chain-only antibody according to claim 14, having binding affinity to effector cells.

19. A heavy chain-only antibody according to claim 14, having binding affinity to a T cell antigen.

20. A heavy chain-only antibody according to claim 19, having binding affinity to CD3.

21. A heavy chain-only antibody according to any one of claims 1 to 20, in CAR-T format.

22. A pharmaceutical composition comprising only the heavy chain antibody described in any one of claims 1 to 21.

23. A method for treating B cell damage characterized by BCMA expression, wherein the patient has the said damage For the target The antibody according to any one of claims 1 to 21 or the pharmaceutical composition according to claim 22 The method comprising administering the following.

24. The method according to claim 23, wherein the B cell disorder is multiple myeloma.

25. The method according to claim 23, wherein the B cell disorder is systemic lupus erythematosus.

26. A polynucleotide encoding an antibody according to any one of claims 1 to 21.

27. A vector comprising the polynucleotide described in claim 26.

28. A cell comprising the vector according to claim 27.

29. A method for producing an antibody according to any one of claims 1 to 21, Proliferate the cells according to claim 28 under conditions that allow the expression of the aforementioned protein. and, To isolate the antibody from the aforementioned cells and The method, including the method described above.

30. A method for producing an antibody according to any one of claims 1 to 21, Immunizing UniRat animals with BCMA, Identifying the BCMA-binding heavy chain sequence and The method, including the method described above.

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