Anti-BCMA antibodies, bispecific antigen binding molecules that bind to BCMA and CD3, and uses thereof

Abstract

To provide monoclonal antibodies that immunospecifically bind to B cell maturation antigen (BCMA), multispecific antibodies that immunospecifically bind to BCMA and cluster determining factor 3 (CD3), and methods of production and use of the antibodies.SOLUTION: Provided herein are antibodies that immunospecifically bind to BCMA. Also described are related polynucleotides capable of encoding the provided BCMA-specific antibodies or antigen-binding fragments, cells expressing the provided antibodies or antigen- binding fragments, as well as associated vectors and detectably labeled antibodies or antigen-binding fragments. The provided antibodies may be used to diagnose, treat, or monitor BCMA-expressing cancer progression, regression, or stability; to determine whether or not a patient should be treated for cancer; or to determine whether or not a subject is afflicted with BCMA-expressing cancer and thus may be amenable to treatment with a BCMA-specific anti-cancer therapeutic, such as the multispecific antibodies against BCMA and CD 3 described herein.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application is a continuation of U.S. Provisional Patent Application No. 62 / 206,246, filed August 17, 2015. No. 6,399,433, filed on Oct. 23, 2003, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a sequence listing that has been submitted electronically in ASCII format. The ASCII copy is available at: It was created on August 15, and the file name is PRD3383USNP_SL.txt. Its size is 87,341 bytes.

[0003] Technical Field The disclosure provided herein relates to a molecule that immunospecifically binds to B cell maturation antigen (BCMA). Monoclonal antibodies that immunospecifically bind to BCMA and cluster determinant factor 3 (CD3) The present invention relates to multispecific antibodies that are capable of binding to human leukocytes and methods for producing and using such antibodies. [Background technology]

[0004] B cell maturation antigens include BCMA, CD269, and TNFRSF17 (UniProt Q0 2223), also known as the tumor necrosis receptor serogroup, is preferentially expressed on differentiated plasma cells. It is a member of the oper family [Laabi et al. (1992) EMBO J 11(11):3897~3904;Madry et al.(1998)Int Immunol 10(11):1693-1702]. BCMA is non-glycosylated. BCMA is a type I transmembrane protein involved in the maturation, proliferation, and survival of B cells. It is a receptor for two ligands of the TNF superfamily, which are expressed by BC APRIL (proliferation-inducing ligand, CD256, TN FSF13), and the B cell activating factor BAFF, a low affinity ligand for BCMA (THANK, BlyS, B-lymphocyte stimulatory factor, TALL-1, and zTNF4). APRIL and BAFF show structural similarity and overlapping but distinct receptor binding specificities. The negative regulator TACI also binds to both BAFF and APRIL. Ligation of BAFF to BCMA and / or TACI activates the transcription factor NF-κB. , pro-survival Bcl-2 family members (e.g., Bcl-2, Bcl-xL, Bc lw, Mcl-1, A1) and increased the expression of pro-apoptotic factors (e.g., Bid It inhibits apoptosis by decreasing the expression of several proteins, including Bad, Bik, and Bim. This combined effect promotes B cell differentiation, proliferation, survival, and antibody production. (Rickert RC et al., Immunol Rev (2011) 244 (1):115-133). Consistent with this finding, BCMA also It also supports the growth and survival of malignant human B cells, such as multiple myeloma (MM) cells. reported that MM cell lines and freshly isolated MM cells express BCMA and TACI on their cell surface. protein and their expression of BAFF-R protein on the cell surface varies Novak et al. (2004) Blood 10 3(2):689~694).

[0005] Multiple myeloma (MM) is the second most common hematologic malignancy and accounts for 2% of all cancer deaths. MM is a heterogeneous disease, most commonly caused by chromosomal translocations, particularly t(11;14), t( 4;14), t(8;14), del(13), and del(17) (Drac h et al.,(1998)Blood 92(3):802~809;Gertz et al.,(2005)Blood 106(8):2837~2840;Fac on et al., (2001) Blood 97(6):1566~1571). M Patients with M disease suffer from bone marrow infiltration, bone destruction, renal failure, immunodeficiency, and the psychosocial burden of a cancer diagnosis. As of 2006, the 5-year relative survival rate for MM was Approximately 34% of patients with MM have a history of refractory disease, highlighting the fact that MM is a difficult disease with no current treatment options. I decided to do so.

[0006] The use of anti-BCMA antibodies in the treatment of lymphoma and multiple myeloma is disclosed in WO 2005 / 023366. 02066516 and 2010104949. The antibody can be, for example, Gras MP. et al. Int Immunol. 7 (1999) 95) 1093-1106, International Publication No. 200124811, and International Publication No. 2001248 It is described in issue 12. Summary of the Invention [Problem to be solved by the invention]

[0007] Nevertheless, BCMA, BAFF-R, and TACI, the TNF receptor superfamily, -family of B cell receptors and their ligands BAFF and APRIL Despite the fact that it is a therapeutic target in the fight against cancer, There is a need to have more options available for the treatment of medical conditions. [Means for solving the problem]

[0008] As used herein, antibodies and antigen-binding fragments thereof that immunospecifically bind to BCMA are Also provided are compositions comprising the BCMA-specific antibodies and antigen-binding fragments thereof. and related polynucleotides capable of expressing the provided antibodies and antigen-binding fragments. The present invention relates to a method for the production of a detectably labeled antibody or antigen-binding fragment thereof, comprising administering to the patient a detectable antibody or antigen-binding fragment thereof, and administering to the patient a detectable antibody or antigen-binding fragment thereof. In addition, methods for using the provided antibodies and antigen-binding fragments are also described. For example, BCMA-specific antibodies and antigen-binding fragments can be produced using the To diagnose or monitor the progression, regression, or stability of CMA-expressing cancers; determining whether a subject should be treated or whether the subject is suffering from a BCMA-expressing cancer and thus, the BCMA-specific anti-cancer therapeutic agents, e.g., The subject invention may be suitable for treatment with the multispecific antibodies against BCMA and CD3 described herein.

[0009] Provided herein are multispecific antibodies that immunospecifically bind to BCMA and CD3 and Further provided are multispecific antigen-binding fragments of the BCMAxCD 3. Related polynucleotides capable of encoding multispecific antibodies, expressing the provided antibodies Also described are cells comprising the polypeptide, as well as associated vectors and detectably labeled multispecific antibodies. Additionally, methods of using the provided multispecific antibodies are described. The BCMA×CD3 multispecific antibody is a potential treatment for the progression, regression, or stability of BCMA-expressing cancers. to diagnose or monitor the patient's condition, to determine whether the patient should be treated for cancer, or to This can be used to determine whether elephants are suffering from BCMA-expressing cancer, and therefore BCMA-specific anti-cancer therapeutics, such as the BCMA×CD3 multispecific antibodies described herein. It may be suitable for treatment by the body.

[0010] BCMA specific antibody Described herein are recombinant antibodies and antigen-binding fragments specific for BCMA. In some embodiments, the BCMA-specific antibodies and antigen-binding fragments are human. In some embodiments, the BCMA-specific antibodies and antigen-binding fragments In some embodiments, the BCM binds to human BCMA and cynomolgus BCMA. A-specific antibodies and antigen-binding fragments are derived from 1 BCMA extracellular domain (ECD). This BCMA-specific antibody or antigen-binding fragment binds to an epitope that contains one or more residues. The fusion fragment has an IC of at least 5.9 nM as measured by ELISA. 50 in May inhibit APRIL binding.

[0011] Table 1 provides a summary of examples of some BCMA-specific antibodies described herein.

[0012] [Table 1]

[0013] In some embodiments, the CDR1, CDR2 and / or CDR3 of any one of the antibodies listed in Table 1 are and a heavy chain comprising CDR3. In some embodiments, the CDR1 of any one of the antibodies listed in Table 1 is provided. A heavy chain comprising CDR1, CDR2, and CDR3, and any one of the CDRs of the antibodies listed in Table 1. and a light chain comprising DR1, CDR2, and CDR3. The original binding fragment is provided.

[0014] The IgG class has four isotypes in humans: IgG1, IgG2, and IgG3. These isotypes are divided into IgG1, IgG2, and IgG3. These isotypes differ in the amino acid sequence of the Fc region. They share over 95% homology with each other, but differ primarily in the amino acid composition and structure of the hinge region. The Fc region is responsible for effector functions, such as antibody-dependent cellular cytotoxicity (ADCC). and mediates complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody Fc receptors on the surface of immune effector cells, such as natural killer and macrophages. These immune effector cells also mediate phagocytosis or lysis of target cells. In CDC, antibodies induce the target cell to trigger the complement cascade on the cell surface. The antibodies described herein are of the IgG isotype. The IgG antibody includes an antibody having the described characteristics of the variable domain in combination with one or more of the Isotypes are variants in which the Fc sequence has been altered to provide different effector functions. Includes editions.

[0015] For many applications of therapeutic antibodies, Fc-mediated effector functions are key to their mechanism of action. These Fc-mediated effector functions are not the only mechanism by which Rubbing may be harmful and pose a safety risk. To modify effector function, the Fc region can be genetically engineered to bind to FcgR or complement factors. This can be achieved by reducing the binding of FcgRI, FcgRIIa, FcgRIIIa, and FcgRIIIb) and inhibitory (FcgRIIb) FcgR or Binding of IgG to the first component of complement (C1q) is mediated by the hinge region and CH2 domain. The mutations are determined by the residues located in the domain. Mutations were introduced into IgG1, IgG2, and IgG4. The antibodies described herein are capable of reducing or silencing Fc function. These modifications may also be included.

[0016] In one embodiment, the antibody has the following properties: (a) reduced Fc activity when compared to the parent Fc. vector function, (b) FcgRI, FcgRIIa, FcgRIIb, and FcgRIIIb and / or reduced affinity for FcgRIIIa, (c) reduced affinity for FcgRI (d) decreased affinity for FcgRIIa; (e) decreased affinity for FcgRIIb (f) reduced affinity for FcgRIIIb; or (g) reduced affinity for Fc The Fc region has one or more of the following reduced affinity for gRIIIa.

[0017] In some embodiments, the antibody or antigen-binding fragment is an IgG or a derivative thereof, e.g. Examples are the IgG1, IgG2, IgG3, and IgG4 isotypes. In some embodiments having a 4 isotype, the antibody is K409R, S228P, L23 The antibodies described herein contain the IgA, IgE, and L235A substitutions in their Fc region. , and may include these modifications.

[0018] In some embodiments, the described antibodies have a titer of 5.9 nM as measured by ELISA. IC 50 can inhibit APRIL binding.

[0019] In some embodiments, the described antibodies bind to BCMA-positive multiple myeloma cell lines. .

[0020] In addition to the BCMA-specific antibodies and antigen-binding fragments described, the antibodies and Polynucleotide sequences capable of encoding the polypeptides and antigen-binding fragments are also provided. Also provided are vectors comprising the polynucleotides provided herein. Cells expressing BCMA-specific antibodies and antigen-binding fragments are also provided. Also described are cells capable of expressing the disclosed vectors. These cells can be mammalian cells, such as For example, 293F cells, CHO cells), insect cells (e.g., Sf7 cells), yeast cells, plant cells The antibodies described may also be hybridized to human cells, e.g., E. coli, or bacterial cells. It can be produced by doma cells.

[0021] Methods of using BCMA-specific antibodies Methods of using the described BCMA-specific antibodies or antigen-binding fragments are also disclosed. Particular antibodies for use in the methods described herein include those in Table 1. For example, these antibodies or antigen-binding fragments include those having the set of CDRs described. Fragment is a compound that treats cancer by inhibiting BCMA receptor interactions. That is, the antibody may be conjugated to a toxin and the toxin may be targeted to BCMA-expressing cancer. Furthermore, these antibodies or antigen-binding fragments can be used to target biological samples, e.g. To detect the presence of BCMA in blood or serum, a biological sample, e.g., blood or to quantify the amount of BCMA in serum, or to diagnose BCMA-expressing cancers. or to determine how to treat a subject suffering from a BCMA-expressing cancer in a subject. In some embodiments, the BCMA-expressing cancer may be useful for monitoring progression of the disease. The described method may be performed in a subject with a lymphoma, such as BCMA-expressing myeloma (MM). prior to receiving treatment for cancer, e.g., treatment with a multispecific antibody against BCMA and CD3 Further, the described methods may be used in treating a subject with a BCMA-expressing cancer, e.g. Following treatment with the multispecific antibodies against BCMA and CD3 described herein This may be done.

[0022] The described methods for detecting BCMA in a biological sample comprise subjecting the biological sample to a method comprising the steps of: Exposure to one or more of the BCMA-specific antibodies or antigen-binding fragments described This includes:

[0023] Also described are methods for diagnosing BCMA-expressing cancer in a subject, comprising: One or two of the BCMA-specific antibodies or antigen-binding fragments described herein. The method involves exposing a sample to more than 100 μg of BCMA, and quantitating the amount of BCMA present in the sample. and comparing the amount of BCMA present in the sample with a known standard or reference sample. and whether the subject's BCMA levels fall within the range of BCMA levels associated with cancer. and determining:

[0024] Also described herein are methods for monitoring BCMA-expressing cancer in a subject. The described method comprises treating a biological sample with a BCMA-specific antibody or antibody described herein. exposing the antibody or its antigen-binding fragment to one or more of the original binding fragments; quantitating the amount of BCMA present in the sample bound to the membrane; The amount of BCMA present in the subject is measured using a known standard or reference sample, or a sample previously obtained from the subject. comparing the amount of BCMA in a similar sample to either one of the samples; Based on differences in the amount of BCMA in the cells, subjects' BCMA levels may be associated with progression or regression of cancer. , or indicates no change.

[0025] Samples obtained from or derived from a subject include biological samples, e.g., urine, blood, , serum, plasma, saliva, ascites, circulating cells, non-tissue associated circulating tumor cells, tissue, surgical procedure Surgically resected tumor tissue, a biopsy, a fine needle aspirate sample, or a histological preparation.

[0026] The described BCMA-specific antibodies or antigen-binding fragments can be produced by any method described or by those skilled in the art. The antibodies may be labeled for use in other ways known to those skilled in the art, for example, by any of the methods described herein. The antibodies or antigen-binding fragments thereof may be radiolabeled, fluorescently labeled, epitope tagged, biotinylated, or Fluorescence, chromophore label, ECL label, enzyme, ruthenium, 111 In-DOTA, 111 In- Diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkali Phosphatase, and beta-galactosidase, or polyhistidine, or The area may be labeled with known similar such labels.

[0027] BCMA-specific antibody kit Kits comprising the disclosed BCMA-specific antibodies or antigen-binding fragments thereof are provided herein. The kits described herein are used to treat BCMA Using specific antibodies or antigen-binding fragments, or other methods known to those skilled in the art, In some embodiments, the described kits may include an antibody as described herein. or antigen-binding fragments thereof, for use in detecting the presence of BCMA in a biological sample. and a reagent for carrying out the method according to the present invention. One or more of the antibodies or antigen-binding fragments thereof and the antibody or antigen-binding fragments thereof that are not in use A container for containing the antibody or fragment and handling for use of the antibody or fragment. The antibody or fragment immobilized on a solid support as described herein is The antibody or fragment may include a detectably labeled form of the antibody or fragment.

[0028] BCMA×CD3 multispecific antibody Redirection of T lymphocytes to BCMA-expressing MM cells via the TCR / CD3 complex Direct induction of T cell proliferation offers an attractive alternative. The TCR / CD3 complex of T lymphocytes mediates the expression of CD4+ 3 labeled gamma (γ), delta (δ), epsilon (ε), zeta (ζ), and eta ( TCR alpha (α) / beta coexpressed on the cell surface with the invariant subunit of They consist of either TCR gamma (β) or TCR delta (δ) heterodimers. Human CD3ε is listed in UniProt P07766 (CD3E_HUMAN). The anti-CD3ε antibody described in the state of the art is SP34 (Yang SJ, T he Journal of Immunology(1986)137;1097~1 100) SP34 reacts with both primate and human CD3. Further anti-CD3 antibodies described in the state of the art are available from U. CHT-1 (see WO 2000041474). A further anti-CD3 antibody that may be used is BC-3 (Fred Hutchinson Cancer Center, er Research Institute;used in Phase I / II trials of GvHD,Anasetti et al.,Transpla Information 54:844 (1992)). SP34 is present only in the ε chain of CD3. Although it recognizes epitopes (Salmeron et al., (1991) J. Immunol. Nol. 147:3047), whereas UCHT-1 and BC-3 are expressed by both the ε and γ chains. SP34 recognizes an epitope provided by UCHT-1 and BC-3. The sequence of an antibody having the same sequence as antibody SP34 is disclosed in WO 2008 / 11996. No. 565, No. 2008119566, No. 2008119567, No. 20100 37836, 2010037837, and 2010037838. The heavy chain variable domain (VH) sequence of antibody SP34, which is 96% identical to that of antibody SP34, is described in U.S. Pat. 236308 (WO 2007042261).

[0029] Various bispecific antibodies against CD3 and BCMA are disclosed in WO 200711760 No. 0, No. 2009132058, No. 2012066058, No. 2012143 No. 498, No. 2013072406, No. 2013072415, and No. 201 However, there is no data describing its clinical application. No data is currently available.

[0030] As used herein, a recombinant multispecific antibody that binds BCMA and CD3 ("BCM The present invention relates to a multispecific antibody ("AxCD3 multispecific antibody") and a multispecific antigen-binding fragment thereof. In one embodiment, a recombinant antibody or antigen-binding fragment thereof that immunospecifically binds to BCMA is provided. Provide a segment.

[0031] In some embodiments, the BCMA specific arm of the multispecific antibody is Binds to cinnamon BCMA. In some embodiments, the BCMAxCD3 multispecific antibody or the BCMA-specific arm of the antigen-binding fragment binds to the extracellular domain of human BCMA. In a preferred embodiment, the BCMAxCD3 multispecific antibody or antigen-binding flag In some embodiments, the antibody is a bispecific antibody or antigen-binding fragment. a) the first heavy chain (HC1), b) the second heavy chain (HC2), c) the first light chain (LC1), and d) ) the second light chain (LC2), which, in combination with HC1 and LC1, is immunospecific for BCMA The first antigen-binding site binds to HC2, and HC2 and LC2 pair up to bind to CD3. A recombinant BCMA×CD3 bispecific antibody that forms a second antigen-binding site that specifically binds The antibody or a BCMAxCD3 bispecific binding fragment thereof is provided. In accordance with the present invention, recombinant cells expressing the antibodies or bispecific binding fragments are provided. In some embodiments, the BCMA binding arm (or "BCMAxCD3" multispecific antibody) of the BCMAxCD3 multispecific antibody is The "BCMA specificity arm" can be selected from any of the BCMA antibodies described herein (e.g., those listed in Table 1). The CDR sequences are derived from an antibody having the CDR sequences specified above.

[0032] In some embodiments, the B of a BCMAxCD3 multispecific antibody or antigen-binding fragment The CMA specificity arm is an IgG or a derivative thereof. BCMAxCD3 multispecific antibodies show at least Both are capable of binding to BCMA with a dissociation constant of 0.18 nM. The resulting BCMAxCD3 multispecific antibody is not agonistic. In some embodiments, The described BCMA×CD3 multispecific antibody inhibits NF-κB activation at concentrations below 10 nM does not change.

[0033] In some embodiments, the CD3 binding arm (or “C”) of a BCMAxCD3 multispecific antibody The D3 specificity arm is composed of mouse monoclonal antibody SP34, mouse IgG3 / lambda Obtained from Isotype. (KRAbhinandan and ACMar tin, 2008. Mol. Immunol. 45, 3832-3839). In one embodiment, the CD3 binding arm of the BCMAxCD3 multispecific antibody is selected from Table 2. It contains one heavy chain and one light chain.

[0034] [Table 2]

[0035] The IgG class has four isotypes in humans: IgG1, IgG2, and IgG3. These isotypes are divided into IgG1, IgG2, and IgG3. These isotypes differ in the amino acid sequence of the Fc region. They share over 95% homology with each other, but differ primarily in the amino acid composition and structure of the hinge region. The Fc region is responsible for effector functions, such as antibody-dependent cellular cytotoxicity (ADCC). and mediates complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody Fc receptors on the surface of immune effector cells, such as natural killer and macrophages. These immune effector cells also mediate phagocytosis or lysis of target cells. In CDC, antibodies induce the target cell to trigger the complement cascade on the cell surface. It kills by garging.

[0036] For many applications of therapeutic antibodies, Fc-mediated effector functions are key to their mechanism of action. These Fc-mediated effector functions are not the only mechanism by which Rubbing may be harmful and pose a safety risk. To modify effector function, the Fc region can be genetically engineered to bind to FcgR or complement factors. This can be achieved by reducing the binding of FcgRI, FcgRIIa, FcgRIIIa, and FcgRIIIb) and inhibitory (FcgRIIb) FcgR or Binding of IgG to the first component of complement (C1q) is mediated by the hinge region and CH2 domain. The mutations are determined by the residues located in the domain. Mutations were introduced into IgG1, IgG2, and IgG4. This reduces or silences Fc function.

[0037] In one embodiment, the antibody has the following properties: (a) reduced Fc activity when compared to the parent Fc. vector function, (b) FcgRI, FcgRIIa, FcgRIIb, and FcgRIIIb and / or reduced affinity for FcgRIIIa, (c) reduced affinity for FcgRI (d) decreased affinity for FcgRIIa; (e) decreased affinity for FcgRIIb (f) reduced affinity for FcgRIIIb; or (g) reduced affinity for Fc The Fc region has one or more of the following reduced affinity for gRIIIa.

[0038] In some embodiments, the CD3-specific antibody from which the CD3-specific arm of the multispecific antibody is derived In some embodiments, the antibody or antigen-binding fragment is an IgG or a derivative thereof. The CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the polyspecific antibody is derived. In some embodiments, the antibody is an IgG1 or a derivative thereof. The Fc region of the CD3-specific IgG1 antibody from which the above-mentioned can be obtained contains L234A, L In some embodiments, the CD3 specific antibody comprises the 235A, 235B, and F405L substitutions. The CD3-specific antibody or antigen-binding fragment from which the isomeric arm is derived may be IgG4 or In some embodiments, for example, the CD3-binding arm is a derivative of the CD3 specific The Fc region of the IgG4 antibody contains S228P, L234A, L235A, In some embodiments, the CD3 The CD3-specific antibody or antigen-binding fragment from which the specific arm is derived is then cultured in primary human T cells. and / or binds to CD3ε on primary cynomolgus monkey T cells. CD3-specific antibodies or antigen-binding fragments from which the CD3-specific arms of the specific antibodies are derived activates primary human CD4+ T cells and / or primary cynomolgus monkey CD4+ T cells.

[0039] In addition to the described BCMA×CD3 polyspecific antibodies, Polynucleotide sequences capable of encoding bispecific antibodies are also provided. In some embodiments, HC1, HC2, L of BCMA×CD3 bispecific antibody or bispecific binding fragment An isolated synthetic polynucleotide encoding LC1 or LC2 is provided. Vectors containing the polynucleotides provided herein are also provided, as are vectors containing the BCs provided herein. Cells expressing the MAxCD3 multispecific antibody are also provided. Also described are cells capable of expressing the . These cells include mammalian cells (e.g., 293F cells). , CHO cells), insect cells (e.g., Sf7 cells), yeast cells, plant cells, or bacterial cells. (e.g., E. coli). The described antibodies can also be produced by hybridoma cells. In some embodiments, BCMAxC can be produced by culturing the cells. Methods for producing D3 bispecific antibodies or bispecific binding fragments are provided. .

[0040] As used herein, a BCMAxCD3 multispecific antibody or antigen-binding fragment and a therapeutic and a pharma- ceutically acceptable carrier.

[0041] How to use BCMAxCD3 multispecific antibodies Described BCMAxCD3 multispecific antibodies and multispecific antigen-binding fragments thereof Also disclosed are methods of using the BCMAxCD3 multispecific antibodies and their The polyspecific antigen-binding fragment is for use in the treatment of BCMA-expressing cancer in a subject in need thereof. In some embodiments, the BCMA-expressing cancer is a cancer of the type 2 (e.g., multiple myeloma), which may be useful for treating It is lymphoma.

[0042] The described method of treating a BCMA-expressing cancer in a subject in need thereof includes The subject is administered a therapeutically effective amount of a described BCMA×CD3 multispecific antibody or a multiplicity thereof. In some embodiments, the subject is a mammal. In a preferred embodiment, the subject is a human, preferably a human. administering the D3 bispecific antibody or bispecific antigen-binding fragment to a patient in need thereof; For treating a subject having cancer by administration for a time sufficient to treat the cancer. A method is provided.

[0043] A therapeutically effective amount of BCMAxCD3 bilayer to inhibit the growth or proliferation of cancer cells. The administration of specific antibodies or bispecific antigen-binding fragments inhibits the growth of cancer cells. or inhibiting proliferation.

[0044] Also provided herein is a therapeutically effective amount of a compound that can be used to redirect T cells to a cancer. BCMA×CD3 bispecific antibody or bispecific binding fragment Also provided are methods for redirecting T cells to BCMA-expressing cancer cells.

[0045] BCMA×CD3 specific antibody kit Described herein are kits that include the disclosed BCMAxCD3 multispecific antibodies. The kits described herein can be used to produce BCMAxCD3 multiplex specific antibodies provided herein. Methods using heterologous antibodies or other methods known to those skilled in the art can be used. In one embodiment, the described kits include an antibody described herein and a method for treating BCMA-expressing cancer. and reagents for use in performing the method of the present invention. or one of the multispecific antibodies or multispecific antigen-binding fragments thereof described in one or more containers for holding the antibody or fragment when not in use, and and / or instructions for use of the antibodies or fragments as described herein. The antibody or fragment immobilized on a solid support and / or a detectably labeled antibody or fragment thereof The antibody may include any recognized form of antibody or fragment thereof. [Brief description of the drawings]

[0046] [Figure 1A] Vectors used to clone human BCMA (Figure 1A) and cynomolgus BCMA (Figure 1B). [Figure 1B] Vectors used to clone human BCMA (Figure 1A) and cynomolgus BCMA (Figure 1B). [Figure 2A] Epitope location of BCMB69 and interaction of BCMB69 with human BCMA. (Figure 2A) Overview of epitope location. BCMB69 binds to the concave surface of BCMA (black area). (Figure 2B) Interaction map showing direct contacts between BCMA and BCMB69. Residues from all CDRs except CDR-L1 contact BCMA. Van der Waals interactions are shown as dashed lines, hydrogen bonds are solid lines, and arrows indicate backbone hydrogen bonds and point to backbone atoms. BCMA residues that contact both BCMB69 and APRIL are boxed in black. A distance cutoff of 4 Å was used to identify contact residues (3.5 Å distance threshold for hydrogen bonds). (Figures 2C and 2D) Zoomed-in views of the main interactions of BCMA with the light chain (Figure 2C) and heavy chain (Figure 2D) of BCMB69. Hydrogen bonds are shown as dashed lines with distances in angstroms. [Figure 2B]Epitope location of BCMB69 and interaction of BCMB69 with human BCMA. (Figure 2A) Overview of epitope location. BCMB69 binds to the concave surface of BCMA (black area). (Figure 2B) Interaction map showing direct contacts between BCMA and BCMB69. Residues from all CDRs except CDR-L1 contact BCMA. Van der Waals interactions are shown as dashed lines, hydrogen bonds are solid lines, and arrows indicate backbone hydrogen bonds and point to backbone atoms. BCMA residues that contact both BCMB69 and APRIL are boxed in black. A distance cutoff of 4 Å was used to identify contact residues (3.5 Å distance threshold for hydrogen bonds). (Figure 2C and Figure 2D) Zoomed-in views of the main interactions of BCMA with the light chain (Figure 2C) and heavy chain (Figure 2D) of BCMB69. Hydrogen bonds are shown as dashed lines with distances in angstroms. [Figure 2C] Epitope location of BCMB69 and interaction of BCMB69 with human BCMA. (Figure 2A) Overview of epitope location. BCMB69 binds to the concave surface of BCMA (black area). (Figure 2B) Interaction map showing direct contacts between BCMA and BCMB69. Residues from all CDRs except CDR-L1 contact BCMA. Van der Waals interactions are shown as dashed lines, hydrogen bonds are solid lines, and arrows indicate backbone hydrogen bonds and point to backbone atoms. BCMA residues that contact both BCMB69 and APRIL are boxed in black. A distance cutoff of 4 Å was used to identify contact residues (3.5 Å distance threshold for hydrogen bonds). (Figure 2C and Figure 2D) Zoomed-in views of the main interactions of BCMA with the light chain (Figure 2C) and heavy chain (Figure 2D) of BCMB69. Hydrogen bonds are shown as dashed lines with distances in angstroms. [Figure 2D]Epitope location of BCMB69 and interaction of BCMB69 with human BCMA. (Figure 2A) Overview of epitope location. BCMB69 binds to the concave surface of BCMA (black area). (Figure 2B) Interaction map showing direct contacts between BCMA and BCMB69. Residues from all CDRs except CDR-L1 contact BCMA. Van der Waals interactions are shown as dashed lines, hydrogen bonds are solid lines, and arrows indicate backbone hydrogen bonds and point to backbone atoms. BCMA residues that contact both BCMB69 and APRIL are boxed in black. A distance cutoff of 4 Å was used to identify contact residues (3.5 Å distance threshold for hydrogen bonds). (Figure 2C and Figure 2D) Zoomed-in views of the main interactions of BCMA with the light chain (Figure 2C) and heavy chain (Figure 2D) of BCMB69. Hydrogen bonds are shown as dashed lines with distances in angstroms. [Diagram 3] Epitope and paratope residues of BCMB69. Epitope and paratope residues are shaded, CDR regions are underlined (Kabat definition), BCMA residues that differ from human are in bold italics. Only the BCMB69 Fab and extracellular BCMA sequences are shown. [Figure 4A] Clash areas between BCMB69 Fab and APRIL (Figure 4A) and between BCMB69 Fab and BAFF (Figure 4B). Structural overlay of the BCMA / BCMB69 complex to the BCMA / APRIL and BCMA / BAFF complexes showing the clash areas between the Fab and the ligand. The solvent accessible surface of BCMA is displayed. The Fab and ligand molecules are shown in grey and black, respectively. The overlay was obtained by superimposing equivalent BCMA Cα atoms in both complexes (RMSD of 0.9 Å for the APRIL complex and 1.2 Å for BAFF). [Figure 4B]Clash areas between BCMB69 Fab and APRIL (Figure 4A) and between BCMB69 Fab and BAFF (Figure 4B). Structural overlay of the BCMA / BCMB69 complex to the BCMA / APRIL and BCMA / BAFF complexes showing the clash areas between the Fab and the ligand. The solvent accessible surface of BCMA is displayed. The Fab and ligand molecules are shown in grey and black, respectively. The overlay was obtained by superimposing equivalent BCMA Cα atoms in both complexes (RMSD of 0.9 Å for the APRIL complex and 1.2 Å for BAFF). [Diagram 5] SPR data for BCMB72 demonstrates that the molecule has binding to human, cynomolgus, and mouse BCMA. The average KD for cynomolgus and mouse BCMA is approximately 36-fold and 402-fold, respectively, when compared to human BCMA. [Figure 6] Determination of EC50 for BCMB72 binding in BCMA+ cell lines. Cell lines were stained for BCMA with BCMB72. The geometric mean fluorescence intensity of BCMB72 binding to cells is shown. EC50 is indicated in the legend. Saturation was reached at a concentration of approximately 100 nM. Taking into account the mean fluorescence intensity, EC50 values ​​were obtained for U2932 (EC50=7.92 nM), MM1R (EC50=8.74 nM), H929 (EC50=14.7 nM), EJM (EC50=17.5 nM), and LP1 (EC50=22.3 nM) cells. Graphing and fitting of data was performed in GraphPad Prism 6 using nonlinear regression with variable slope (4 parameter) function. [Figure 7]Binding profile of BCMB72 in whole blood. Whole blood from three normal human donors was stained with monoclonal or polyclonal antibodies against BCMA or BCMB72. Gating analysis was performed to identify lymphocytes in the leukocyte population using standard cell-specific markers. Staining intensity of one representative donor is shown in the panel, with the solid black line being the antibody of interest and the dashed grey filled line being the corresponding isotype. No BCMA expression was observed on lymphocytes, monocytes, granulocytes, or plasmacytoid DCs in three normal donors. BCMB72 showed binding to CD3+ T cells in all three donors, but with different intensities between donors. BCMB72 did not bind to any other cell types tested in this assay. [Figure 8A] BCMB72-dependent T cell activation in the presence of various MM cell lines. H929 (Figure 9A), MM.1R (Figure 9B), RPMI 8226 (Figure 9C), U266 (Figure 9D), and Mv4-11 (Figure 9E) cells were subjected to the indicated antibodies for 48 hours in the presence of T cells from six normal donors (donor mean ± SEM shown) and Fc blocker (2 mg / mL). EC50 values ​​are shown in the graphs. Statistical analysis: In addition to the simple fact of model convergence, the width of the 95% confidence interval around the LogEC50 is taken into account to evaluate the adequacy of the fit. (Because of symmetry, we use the confidence interval around the LogEC50 and not the confidence interval around the EC50 itself.) An interval of less than + / - 2 (or the entire 95% confidence interval width of less than 4) is taken into account appropriately. [Figure 8B]BCMB72-dependent T cell activation in the presence of various MM cell lines. H929 (Figure 9A), MM.1R (Figure 9B), RPMI 8226 (Figure 9C), U266 (Figure 9D), and Mv4-11 (Figure 9E) cells were subjected to the indicated antibodies for 48 hours in the presence of T cells from six normal donors (donor mean ± SEM shown) and Fc blocker (2 mg / mL). EC50 values ​​are shown in the graphs. Statistical analysis: In addition to the simple fact of model convergence, the width of the 95% confidence interval around the LogEC50 is taken into account to evaluate the adequacy of the fit. (Because of symmetry, we use the confidence interval around the LogEC50 and not the confidence interval around the EC50 itself.) An interval of less than + / - 2 (or the entire 95% confidence interval width of less than 4) is taken into account appropriately. [Figure 8C] BCMB72-dependent T cell activation in the presence of various MM cell lines. H929 (Figure 9A), MM.1R (Figure 9B), RPMI 8226 (Figure 9C), U266 (Figure 9D), and Mv4-11 (Figure 9E) cells were subjected to the indicated antibodies for 48 hours in the presence of T cells from six normal donors (donor mean ± SEM shown) and Fc blocker (2 mg / mL). EC50 values ​​are shown in the graphs. Statistical analysis: In addition to the simple fact of model convergence, the width of the 95% confidence interval around the LogEC50 is taken into account to evaluate the adequacy of the fit. (Because of symmetry, we use the confidence interval around the LogEC50 and not the confidence interval around the EC50 itself.) An interval of less than + / - 2 (or the entire 95% confidence interval width of less than 4) is taken into account appropriately. [Figure 8D]BCMB72-dependent T cell activation in the presence of various MM cell lines. H929 (Figure 9A), MM.1R (Figure 9B), RPMI 8226 (Figure 9C), U266 (Figure 9D), and Mv4-11 (Figure 9E) cells were subjected to the indicated antibodies for 48 hours in the presence of T cells from six normal donors (donor mean ± SEM shown) and Fc blocker (2 mg / mL). EC50 values ​​are shown in the graphs. Statistical analysis: In addition to the simple fact of model convergence, the width of the 95% confidence interval around the LogEC50 is taken into account to evaluate the adequacy of the fit. (Because of symmetry, we use the confidence interval around the LogEC50 and not the confidence interval around the EC50 itself.) An interval of less than + / - 2 (or the entire 95% confidence interval width of less than 4) is taken into account appropriately. [Figure 8E] BCMB72-dependent T cell activation in the presence of various MM cell lines. H929 (Figure 9A), MM.1R (Figure 9B), RPMI 8226 (Figure 9C), U266 (Figure 9D), and Mv4-11 (Figure 9E) cells were subjected to the indicated antibodies for 48 hours in the presence of T cells from six normal donors (donor mean ± SEM shown) and Fc blocker (2 mg / mL). EC50 values ​​are shown in the graphs. Statistical analysis: In addition to the simple fact of model convergence, the width of the 95% confidence interval around the LogEC50 is taken into account to evaluate the adequacy of the fit. (Because of symmetry, we use the confidence interval around the LogEC50 and not the confidence interval around the EC50 itself.) An interval of less than + / - 2 (or the entire 95% confidence interval width of less than 4) is taken into account appropriately. [Figure 9A] Summary of EC50 and maximum T cell activation values ​​from two independent studies using T cells from multiple normal donors. Individual donor values ​​and donor averages are shown for each cell line and each study. No data = study not performed, no fit = software unable to generate curve, ~values ​​= approximate values ​​based on model extrapolation. [Figure 9B] Summary of EC50 and maximum T cell activation values ​​from two independent studies using T cells from multiple normal donors. Individual donor values ​​and donor averages are shown for each cell line and each study. No data = study not performed, no fit = software unable to generate curve, ~values ​​= approximate values ​​based on model extrapolation. [Figure 10A] T cell-mediated BCMB72-dependent cytotoxicity of various multiple myeloma cell lines. H929 (Figure 11A), MM.1R (Figure 11B), RPMI 8226 (Figure 11C), U266 (Figure 11D), and Mv4-11 (Figure 11E) cells were subjected to the indicated antibody concentrations for 48 hours in the presence of T cells from six normal donors (donor mean ± SEM shown) and Fc blocker (2 mg / mL). EC50 values ​​are shown in the graphs. Statistical analysis: In addition to the simple fact of model convergence, the width of the 95% confidence interval around the LogEC50 is taken into account to assess the adequacy of the fit. (Because of symmetry, we use the confidence interval around the LogEC50 and not the confidence interval around the EC50 itself.) An interval of less than + / - 2 (or the entire 95% confidence interval width of less than 4) is taken into account appropriately. [Figure 10B] T cell-mediated BCMB72-dependent cytotoxicity of various multiple myeloma cell lines. H929 (Figure 11A), MM.1R (Figure 11B), RPMI 8226 (Figure 11C), U266 (Figure 11D), and Mv4-11 (Figure 11E) cells were subjected to the indicated antibody concentrations for 48 hours in the presence of T cells from six normal donors (donor mean ± SEM shown) and Fc blocker (2 mg / mL). EC50 values ​​are shown in the graphs. Statistical analysis: In addition to the simple fact of model convergence, the width of the 95% confidence interval around the LogEC50 is taken into account to assess the adequacy of the fit. (Because of symmetry, we use the confidence interval around the LogEC50 and not the confidence interval around the EC50 itself.) An interval of less than + / - 2 (or the entire 95% confidence interval width of less than 4) is taken into account appropriately. [Figure 10C]T cell-mediated BCMB72-dependent cytotoxicity of various multiple myeloma cell lines. H929 (Figure 11A), MM.1R (Figure 11B), RPMI 8226 (Figure 11C), U266 (Figure 11D), and Mv4-11 (Figure 11E) cells were subjected to the indicated antibody concentrations for 48 hours in the presence of T cells from six normal donors (donor mean ± SEM shown) and Fc blocker (2 mg / mL). EC50 values ​​are shown in the graphs. Statistical analysis: In addition to the simple fact of model convergence, the width of the 95% confidence interval around the LogEC50 is taken into account to assess the adequacy of the fit. (Because of symmetry, we use the confidence interval around the LogEC50 and not the confidence interval around the EC50 itself.) An interval of less than + / - 2 (or the entire 95% confidence interval width of less than 4) is taken into account appropriately. [Figure 10D] T cell-mediated BCMB72-dependent cytotoxicity of various multiple myeloma cell lines. H929 (Figure 11A), MM.1R (Figure 11B), RPMI 8226 (Figure 11C), U266 (Figure 11D), and Mv4-11 (Figure 11E) cells were subjected to the indicated antibody concentrations for 48 hours in the presence of T cells from six normal donors (donor mean ± SEM shown) and Fc blocker (2 mg / mL). EC50 values ​​are shown in the graphs. Statistical analysis: In addition to the simple fact of model convergence, the width of the 95% confidence interval around the LogEC50 is taken into account to assess the adequacy of the fit. (Because of symmetry, we use the confidence interval around the LogEC50 and not the confidence interval around the EC50 itself.) An interval of less than + / - 2 (or the entire 95% confidence interval width of less than 4) is taken into account appropriately. [Figure 10E]T cell-mediated BCMB72-dependent cytotoxicity of various multiple myeloma cell lines. H929 (Figure 11A), MM.1R (Figure 11B), RPMI 8226 (Figure 11C), U266 (Figure 11D), and Mv4-11 (Figure 11E) cells were subjected to the indicated antibody concentrations for 48 hours in the presence of T cells from six normal donors (donor mean ± SEM shown) and Fc blocker (2 mg / mL). EC50 values ​​are shown in the graphs. Statistical analysis: In addition to the simple fact of model convergence, the width of the 95% confidence interval around the LogEC50 is taken into account to assess the adequacy of the fit. (Because of symmetry, we use the confidence interval around the LogEC50 and not the confidence interval around the EC50 itself.) An interval of less than + / - 2 (or the entire 95% confidence interval width of less than 4) is taken into account appropriately. [Figure 11A] Summary of EC50 and maximum lysis values ​​from two independent studies using T cells from multiple normal donors. Individual donor values ​​and donor averages are shown for each cell line and each study. No data = study not performed, no fit = software unable to generate curve, ~values ​​= approximate values ​​based on model extrapolation. [Figure 11B] Summary of EC50 and maximum lysis values ​​from two independent studies using T cells from multiple normal donors. Individual donor values ​​and donor averages are shown for each cell line and each study. No data = study not performed, no fit = software unable to generate curve, ~values ​​= approximate values ​​based on model extrapolation. [Figure 12A]Cytotoxicity and T cell activation in H929 cells. BCMAxCD3 bispecific antibodies (variant molecules of BCMB72) were tested in a T cell-mediated cytotoxicity assay. A BCMA positive cell line (H929) was incubated with various antibody concentrations in the presence of exogenous human T cells from normal donors (donor IDs: M5763 and M6576) for 48 hours. After 48 hours of incubation, cell killing was measured by a flow cytometry-based method (FACS) and reported as % cytotoxicity in FIG. 12A. FIG. 12B shows T cell activation as assessed by upregulation of CD25 on the T cell surface. In general, the data points fit closely along the generated fitted curve with little variability between T cell donors and replicates. [Figure 12B] Cytotoxicity and T cell activation in H929 cells. BCMAxCD3 bispecific antibodies (variant molecules of BCMB72) were tested in a T cell-mediated cytotoxicity assay. A BCMA positive cell line (H929) was incubated with various antibody concentrations in the presence of exogenous human T cells from normal donors (donor IDs: M5763 and M6576) for 48 hours. After 48 hours of incubation, cell killing was measured by a flow cytometry-based method (FACS) and reported as % cytotoxicity in FIG. 12A. FIG. 12B shows T cell activation as assessed by upregulation of CD25 on the T cell surface. In general, the data points fit closely along the generated fitted curve with little variability between T cell donors and replicates. [Figure 13] Summary of EC50 values ​​for BCMB72-mediated cytokine release. Supernatants of RPMI8226 cells obtained from cytotoxicity studies (see Example 12, Figure 8) were collected and analyzed for six different cytokine concentrations using a MSD-based multiplex assay. BCMB72 (BCMA x CD3) and control antibodies (BCMA x None and None x CD3) were used at various concentrations. [Figure 14A]A T cell-mediated BCMB72-dependent cytotoxicity assay was performed using the BCMA-positive H929 cell line. Cells were subjected to various concentrations of BCMB72 for 48 hours in the presence of T cells from multiple normal donors (representatively, three donors M7197, M5137, and M6457 are outlined) and Fc blocker (2 mg / mL). The effector / target (E / T) ratio was 5:1. Figure 14A shows the cytotoxicity, and Figure 14B on the right shows the T cell activation curves, which were similar between the various lots of BCMB72. [Figure 14B] A T cell-mediated BCMB72-dependent cytotoxicity assay was performed using the BCMA-positive H929 cell line. Cells were subjected to various concentrations of BCMB72 for 48 hours in the presence of T cells from multiple normal donors (representatively, three donors M7197, M5137, and M6457 are outlined) and Fc blocker (2 mg / mL). The effector / target (E / T) ratio was 5:1. Figure 14A shows the cytotoxicity, and Figure 14B on the right shows the T cell activation curves, which were similar between the various lots of BCMB72. [Figure 15] H929 cells were treated with BCMB72 (BCMA x CD3) and control antibodies (BCMA x None and None x CD3) for 30 minutes at the doses indicated on the X-axis of the graph above, and total protein was analyzed using Simple Western analysis following standard protocols as per the ProteinSimple user manual. Data was normalized using actin as a housekeeping gene, and ratios were plotted on the Y-axis. APRIL and BAFF induced phosphorylation of P38 as expected, and the antibodies had no stimulatory effect at any of the concentrations tested. [Figure 16A]HEK-NFkB cells expressing BCMA (Figure 16A, 16C, and 16E) or parental cells (Figure 16B, 16D, and 16F) were stimulated with TNFα and various concentrations of APRIL or BCMB72. Three time points were analyzed: 16 hours (Figure 16A and 16B), 24 hours (Figure 16C and 16D), and 48 hours (Figure 16E and 16F). TNFα induced NF-kB activation in both HEK-Nf-kB parental and HEK-NF-kB-BCMA cells, whereas induction by APRIL was only seen in the BCMA-specific cell line. BCMB72 had no effect in the parental cell line and showed activation only at high concentrations in BCMA expressing cells. [Figure 16B] HEK-NFkB cells expressing BCMA (Figure 16A, 16C, and 16E) or parental cells (Figure 16B, 16D, and 16F) were stimulated with TNFα and various concentrations of APRIL or BCMB72. Three time points were analyzed: 16 hours (Figure 16A and 16B), 24 hours (Figure 16C and 16D), and 48 hours (Figure 16E and 16F). TNFα induced NF-kB activation in both HEK-Nf-kB parental and HEK-NF-kB-BCMA cells, whereas induction by APRIL was only seen in the BCMA-specific cell line. BCMB72 had no effect in the parental cell line and showed activation only at high concentrations in BCMA expressing cells. [Figure 16C] HEK-NFkB cells expressing BCMA (Figure 16A, 16C, and 16E) or parental cells (Figure 16B, 16D, and 16F) were stimulated with TNFα and various concentrations of APRIL or BCMB72. Three time points were analyzed: 16 hours (Figure 16A and 16B), 24 hours (Figure 16C and 16D), and 48 hours (Figure 16E and 16F). TNFα induced NF-kB activation in both HEK-Nf-kB parental and HEK-NF-kB-BCMA cells, whereas induction by APRIL was only seen in the BCMA-specific cell line. BCMB72 had no effect in the parental cell line and showed activation only at high concentrations in BCMA expressing cells. [Figure 16D]HEK-NFkB cells expressing BCMA (Figure 16A, 16C, and 16E) or parental cells (Figure 16B, 16D, and 16F) were stimulated with TNFα and various concentrations of APRIL or BCMB72. Three time points were analyzed: 16 hours (Figure 16A and 16B), 24 hours (Figure 16C and 16D), and 48 hours (Figure 16E and 16F). TNFα induced NF-kB activation in both HEK-Nf-kB parental and HEK-NF-kB-BCMA cells, whereas induction by APRIL was only seen in the BCMA-specific cell line. BCMB72 had no effect in the parental cell line and showed activation only at high concentrations in BCMA expressing cells. [Figure 16E] HEK-NFkB cells expressing BCMA (Figure 16A, 16C, and 16E) or parental cells (Figure 16B, 16D, and 16F) were stimulated with TNFα and various concentrations of APRIL or BCMB72. Three time points were analyzed: 16 hours (Figure 16A and 16B), 24 hours (Figure 16C and 16D), and 48 hours (Figure 16E and 16F). TNFα induced NF-kB activation in both HEK-Nf-kB parental and HEK-NF-kB-BCMA cells, whereas induction by APRIL was only seen in the BCMA-specific cell line. BCMB72 had no effect in the parental cell line and showed activation only at high concentrations in BCMA expressing cells. [Figure 16F] HEK-NFkB cells expressing BCMA (Figure 16A, 16C, and 16E) or parental cells (Figure 16B, 16D, and 16F) were stimulated with TNFα and various concentrations of APRIL or BCMB72. Three time points were analyzed: 16 hours (Figure 16A and 16B), 24 hours (Figure 16C and 16D), and 48 hours (Figure 16E and 16F). TNFα induced NF-kB activation in both HEK-Nf-kB parental and HEK-NF-kB-BCMA cells, whereas induction by APRIL was only seen in the BCMA-specific cell line. BCMB72 had no effect in the parental cell line and showed activation only at high concentrations in BCMA expressing cells. [Figure 17A]T cells do not demonstrate sBCMA-mediated and BCMB72-dependent activation. BCMB72 (Figure 17A) and no xCD3 control antibody (Figure 17B) were titrated on T cells from two normal donors (M7077 and M5137) in the presence of various doses of soluble BCMA ECD. Data: mean ± SEM. [Figure 17B] T cells do not demonstrate sBCMA-mediated and BCMB72-dependent activation. BCMB72 (Figure 17A) and no xCD3 control antibody (Figure 17B) were titrated on T cells from two normal donors (M7077 and M5137) in the presence of various doses of soluble BCMA ECD. Data: mean ± SEM. [Figure 18A] Figure 18. Effect of soluble factors, sBCMA, APRIL, and BAFF, on T cell activation and T cell-mediated cytotoxicity of BCMB72 in H929 cells. Cells were subjected to a killing assay for 48 hours using donor T cells (M7077 & M6521) and BCMB72. Target cytotoxicity is shown in the left graph and T cell activation is shown in the right graph (n=2). EC50 values ​​for each treatment are shown in the legend. Cytotoxicity in the presence of sBCMA (Figure 18A), APRIL (Figure 18B), and BAFF (Figure 18C) is shown. T cell activation in the presence of sBCMA (Figure 18D), APRIL (Figure 18E), and BAFF (Figure 18F) is shown. Data: mean ± SEM. [Figure 18B] Figure 18. Effect of soluble factors, sBCMA, APRIL, and BAFF, on T cell activation and T cell-mediated cytotoxicity of BCMB72 in H929 cells. Cells were subjected to a killing assay for 48 hours using donor T cells (M7077 & M6521) and BCMB72. Target cytotoxicity is shown in the left graph and T cell activation is shown in the right graph (n=2). EC50 values ​​for each treatment are shown in the legend. Cytotoxicity in the presence of sBCMA (Figure 18A), APRIL (Figure 18B), and BAFF (Figure 18C) is shown. T cell activation in the presence of sBCMA (Figure 18D), APRIL (Figure 18E), and BAFF (Figure 18F) is shown. Data: mean ± SEM. [Figure 18C]Figure 18. Effect of soluble factors, sBCMA, APRIL, and BAFF, on T cell activation and T cell-mediated cytotoxicity of BCMB72 in H929 cells. Cells were subjected to a killing assay for 48 hours using donor T cells (M7077 & M6521) and BCMB72. Target cytotoxicity is shown in the left graph and T cell activation is shown in the right graph (n=2). EC50 values ​​for each treatment are shown in the legend. Cytotoxicity in the presence of sBCMA (Figure 18A), APRIL (Figure 18B), and BAFF (Figure 18C) is shown. T cell activation in the presence of sBCMA (Figure 18D), APRIL (Figure 18E), and BAFF (Figure 18F) is shown. Data: mean ± SEM. [Figure 18D] Figure 18. Effect of soluble factors, sBCMA, APRIL, and BAFF, on T cell activation and T cell-mediated cytotoxicity of BCMB72 in H929 cells. Cells were subjected to a killing assay for 48 hours using donor T cells (M7077 & M6521) and BCMB72. Target cytotoxicity is shown in the left graph and T cell activation is shown in the right graph (n=2). EC50 values ​​for each treatment are shown in the legend. Cytotoxicity in the presence of sBCMA (Figure 18A), APRIL (Figure 18B), and BAFF (Figure 18C) is shown. T cell activation in the presence of sBCMA (Figure 18D), APRIL (Figure 18E), and BAFF (Figure 18F) is shown. Data: mean ± SEM. [Figure 18E] Figure 18. Effect of soluble factors, sBCMA, APRIL, and BAFF, on T cell activation and T cell-mediated cytotoxicity of BCMB72 in H929 cells. Cells were subjected to a killing assay for 48 hours using donor T cells (M7077 & M6521) and BCMB72. Target cytotoxicity is shown in the left graph and T cell activation is shown in the right graph (n=2). EC50 values ​​for each treatment are shown in the legend. Cytotoxicity in the presence of sBCMA (Figure 18A), APRIL (Figure 18B), and BAFF (Figure 18C) is shown. T cell activation in the presence of sBCMA (Figure 18D), APRIL (Figure 18E), and BAFF (Figure 18F) is shown. Data: mean ± SEM. [Figure 18F]Figure 18. Effect of soluble factors, sBCMA, APRIL, and BAFF, on T cell activation and T cell-mediated cytotoxicity of BCMB72 in H929 cells. Cells were subjected to a killing assay for 48 hours using donor T cells (M7077 & M6521) and BCMB72. Target cytotoxicity is shown in the left graph and T cell activation is shown in the right graph (n=2). EC50 values ​​for each treatment are shown in the legend. Cytotoxicity in the presence of sBCMA (Figure 18A), APRIL (Figure 18B), and BAFF (Figure 18C) is shown. T cell activation in the presence of sBCMA (Figure 18D), APRIL (Figure 18E), and BAFF (Figure 18F) is shown. Data: mean ± SEM. [Figure 19A] Signals from two independent experiments were normalized to the maximum signal of BCMA-Fc binding to APRIL and BAFF in the absence of competing antibody. BCMA binding to APRIL (Figure 19A) and BAFF (Figure 19B) was plotted as a function of BCMB72 and control antibody (no x CD3) concentration. [Figure 19B] Signals from two independent experiments were normalized to the maximum signal of BCMA-Fc binding to APRIL and BAFF in the absence of competing antibody. BCMA binding to APRIL (Figure 19A) and BAFF (Figure 19B) was plotted as a function of BCMB72 and control antibody (no x CD3) concentration. [Figure 20A]Cytotoxicity of BCMB72 against human primary MM plasma cells. Frozen bone marrow-derived mononuclear cells obtained from five different patients (MM240BM (Figure 20A), MM259BM (Figure 20B), MM270BM (Figure 20C), MM276BM (Figure 20D), and MM277BM (Figure 20E)) were used to evaluate BCMB72 binding, plasma cell cytotoxicity (middle), and T cell activation (right) compared to IgG4 isotype (CNTO 9412, left panel) control. For cytotoxicity assays, T cells from M7077 normal healthy donor were exogenously added to patient BMMC samples and incubated for 48 hours with BCMB72 (BCMA x CD3), BC3B4 (BCMA x None), or CNTO7008 (None x CD3). BCMB72 bound to plasma cells in a dose-dependent manner for all donor samples, and the mean fluorescence intensity was recorded on the Y-axis. Note the loss of viable plasma cells (CD138+) and the concomitant upregulation of CD25 on T cells in response to BCMB72 treatment. EC50 values ​​for T cell activation are shown on the graph. [Figure 20B] Cytotoxicity of BCMB72 against human primary MM plasma cells. Frozen bone marrow-derived mononuclear cells obtained from five different patients (MM240BM (Figure 20A), MM259BM (Figure 20B), MM270BM (Figure 20C), MM276BM (Figure 20D), and MM277BM (Figure 20E)) were used to evaluate BCMB72 binding, plasma cell cytotoxicity (middle), and T cell activation (right) compared to IgG4 isotype (CNTO 9412, left panel) control. For cytotoxicity assays, T cells from M7077 normal healthy donor were exogenously added to patient BMMC samples and incubated for 48 hours with BCMB72 (BCMA x CD3), BC3B4 (BCMA x None), or CNTO7008 (None x CD3). BCMB72 bound to plasma cells in a dose-dependent manner for all donor samples, and the mean fluorescence intensity was recorded on the Y-axis. Note the loss of viable plasma cells (CD138+) and the concomitant upregulation of CD25 on T cells in response to BCMB72 treatment. EC50 values ​​for T cell activation are shown on the graph. [Figure 20C]Cytotoxicity of BCMB72 against human primary MM plasma cells. Frozen bone marrow-derived mononuclear cells obtained from five different patients (MM240BM (Figure 20A), MM259BM (Figure 20B), MM270BM (Figure 20C), MM276BM (Figure 20D), and MM277BM (Figure 20E)) were used to evaluate BCMB72 binding, plasma cell cytotoxicity (middle), and T cell activation (right) compared to IgG4 isotype (CNTO 9412, left panel) control. For cytotoxicity assays, T cells from M7077 normal healthy donor were exogenously added to patient BMMC samples and incubated for 48 hours with BCMB72 (BCMA x CD3), BC3B4 (BCMA x None), or CNTO7008 (None x CD3). BCMB72 bound to plasma cells in a dose-dependent manner for all donor samples, and the mean fluorescence intensity was recorded on the Y-axis. Note the loss of viable plasma cells (CD138+) and the concomitant upregulation of CD25 on T cells in response to BCMB72 treatment. EC50 values ​​for T cell activation are shown on the graph. [Figure 20D] Cytotoxicity of BCMB72 against human primary MM plasma cells. Frozen bone marrow-derived mononuclear cells obtained from five different patients (MM240BM (Figure 20A), MM259BM (Figure 20B), MM270BM (Figure 20C), MM276BM (Figure 20D), and MM277BM (Figure 20E)) were used to evaluate BCMB72 binding, plasma cell cytotoxicity (middle), and T cell activation (right) compared to IgG4 isotype (CNTO 9412, left panel) control. For cytotoxicity assays, T cells from M7077 normal healthy donor were exogenously added to patient BMMC samples and incubated for 48 hours with BCMB72 (BCMA x CD3), BC3B4 (BCMA x None), or CNTO7008 (None x CD3). BCMB72 bound to plasma cells in a dose-dependent manner for all donor samples, and the mean fluorescence intensity was recorded on the Y-axis. Note the loss of viable plasma cells (CD138+) and the concomitant upregulation of CD25 on T cells in response to BCMB72 treatment. EC50 values ​​for T cell activation are shown on the graph. [Figure 20E]Cytotoxicity of BCMB72 against human primary MM plasma cells. Frozen bone marrow-derived mononuclear cells obtained from five different patients (MM240BM (Figure 20A), MM259BM (Figure 20B), MM270BM (Figure 20C), MM276BM (Figure 20D), and MM277BM (Figure 20E)) were used to evaluate BCMB72 binding, plasma cell cytotoxicity (middle), and T cell activation (right) compared to IgG4 isotype (CNTO 9412, left panel) control. For cytotoxicity assays, T cells from M7077 normal healthy donor were exogenously added to patient BMMC samples and incubated for 48 hours with BCMB72 (BCMA x CD3), BC3B4 (BCMA x None), or CNTO7008 (None x CD3). BCMB72 bound to plasma cells in a dose-dependent manner for all donor samples, and the mean fluorescence intensity was recorded on the Y-axis. Note the loss of viable plasma cells (CD138+) and the concomitant upregulation of CD25 on T cells in response to BCMB72 treatment. EC50 values ​​for T cell activation are shown on the graph. [Figure 21] In vivo efficacy of BCMB72 in the H929 prophylactic model. [Figure 22] Serum soluble BCMA concentrations in H929 xenografted mice. Serum soluble BCMA concentrations were detected using a human BCMA ELISA kit (R&D Systems). Soluble BCMA concentrations were significantly lower in mice treated with 1 μg and 0.5 μg / mouse of BCMB72 compared to PBS controls, which correlates well with tumor burden in these animals. Lower doses of BCMB72 (0.1 μg / mouse) had no effect on sBCMA concentrations or tumor size. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] definition Various terms are used throughout this specification and claims to refer to aspects of this specification. Unless otherwise indicated, such terms are to be given their ordinary meaning in the art. Other specifically defined terms are provided herein. shall be construed in a manner consistent with the definition.

[0048] As used in this specification and the appended claims, the singular forms "a," "a "n" and "the" refer to plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes two or more cells. and the like.

[0049] As used herein, "about" when referring to a measurement, e.g., amount, duration, etc. The term is meant to encompass a maximum deviation of ±10% from the specified value. Such deviations are permissible for carrying out the disclosed methods. The quantities of ingredients, molecular weights, reaction conditions, and other properties used in the specification and claims are All numbers herein are understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated, the following specification and the appended claims The numerical parameters recited in ranges are intended to represent the desired properties sought to be obtained by the present invention. These are approximations that may vary depending on the scope of the claims. No attempt is made to limit the application of the principles herein, and each numerical parameter is reported to the number of significant digits. should be interpreted at least in part by taking into account the characters and applying normal rounding techniques. be.

[0050] While the numerical ranges and parameters setting forth the broad scope of the invention are approximations, specific examples may be used. The values ​​set forth herein are reported as precisely as possible; however, any values ​​may vary depending on the individual test measurements. These measurements inherently contain certain errors which necessarily result in the standard deviation found in the determination of the mean values.

[0051] "Isolated" means that a biological component (e.g., a nucleic acid, peptide, or protein) is Other biological components of the organism in which these components naturally occur, i.e., other chromosomal and extrachromosomal components. Substantially separated from DNA, RNA, and proteins and produced separately from other components "Isolated" means that the substance has been purified or separated from other components. The nucleic acids, peptides, and proteins used in this study were purified by standard purification methods. "Isolated" nucleic acids, peptides, and proteins are part of a composition. Such compositions may be part of the natural environment of a nucleic acid, peptide, or protein. The term also refers to recombinant expression in a host cell. This includes nucleic acids, peptides, and proteins prepared by the present invention, as well as chemically synthesized nucleic acids. As used herein, an "isolated" antibody or antigen-binding fragment is a fragment of a species. Antibodies or antigen-binding fragments that are substantially free of other antibodies or antigen-binding fragments having different antigen specificities. is intended to mean an antigen-binding fragment (e.g., a fragment that specifically binds BCMA) The isolated antibody is substantially free of antibodies that specifically bind to antigens other than BCMA. However, monoclonal antibodies that specifically bind to BCMA epitopes, isoforms, or variants are not The isolated antibodies may, for example, be directed against other related antigens from other species (e.g., BCMA interspecies host). It may have cross-reactivity to IgG4 (mologs).

[0052] "Polynucleotide" is used interchangeably with "nucleic acid molecule," "nucleotide," or "nucleic acid." Any polynucleotide, which may be unmodified RNA or DNA or modified RNA or DNA, may be used. "Polynucleotide" means a ribonucleotide or a polydeoxyribonucleotide. " includes single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single stranded RNA, DNA and dsRNA that are single-stranded and double-stranded, as well as mixtures of single- and double-stranded regions. Hybrid molecules containing RNA (single-stranded, or more typically, double-stranded, or single-stranded and In addition, "polypeptide" may be used in combination with "polypeptide" or "polypeptide" to obtain a double-stranded region. "Nucleotide" refers to a triple-stranded region containing RNA or DNA or both RNA and DNA. The term polynucleotide includes DNA or RNA, and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases, such as inosine. Various modifications may be made to DNA and RNA. "Derivatives" refer to chemical, enzymatic or metabolic modifications of polynucleotides typically found in nature. These include forms that have been identified as viral or cellular forms, as well as chemical forms that have characteristics of viral and cellular DNA and RNA. "Polynucleotide" also refers to a relatively short strand of nucleic acid (often an oligonucleotide). This also includes the endothelial cells (called chids).

[0053] The meaning of "substantially the same" may vary depending on the context in which the term is used. Natural sequence variations likely exist in the heavy and light chains and in the genes encoding them. Therefore, some level of variation may occur in the amino acid sequences or sequences described herein. is predicted to be found within genes encoding antibodies or antigen-binding fragments. However, this will have little effect on the intrinsic binding properties (e.g., specificity and affinity). Such predictions are due, in part, to the degeneracy of the genetic code and conservative amino acid sequences. The evolutionary success of sequence variations in the encoded proteins Thus, in the context of nucleic acid sequences, " "Substantially the same" means at least 65% identity between two or more sequences. More preferably, the term refers to at least 70% identity between two or more sequences, and more preferably, At least 75% identity, more preferably at least 80% identity, even more preferably Preferably, the sequence is at least 85% identical, more preferably at least 90% identical, and even more preferably at least 90% identical. or at least 91% identity, more preferably at least 92% identity, Preferably, at least 93% identity, more preferably at least 94% identity, More preferably, there is at least 95% identity, and more preferably, there is at least 96% identity. more preferably at least 97% identity, more preferably at least 98% It means identity, and more preferably at least 99% identity between the two sequences. The percent identity is calculated based on the number of gaps and the length of each gap shared by the sequences. The homology ratio is a function of the number of identical positions found in the sequence (i.e., percent homology = number of identical positions / number of positions). The same consideration is necessary to derive an optimal alignment of two sequences. The percent identity between two nucleotide or amino acid sequences can be determined, for example, by the method of E. Meyers and W.Miller,Comput.Appl.Biosci 4,11~17 (1988) algorithm, which is It is incorporated into the N program (version 2.0) and uses the PAM120 weighted residue table. Use a gap length penalty of 12 and a gap penalty of 4. In addition, The percent identity between the amino acid sequences of Determined using the algorithm of J. Mol. Biol. 48, 444-453 (1970) It may be determined.

[0054] It is possible to introduce a nucleotide sequence into a protein without having a substantial effect on the function of the protein. The degree of variation that can occur is much smaller than the degree of variation in nucleic acid sequences. This is because the same degeneracy principle does not apply to amino acid sequences. In the context of antigen-binding fragments, "substantially the same" means that the 90%, 91%, 92%, 93%, 94%, 95%, 96% for the bound fragments 97%, 98%, or 99% identity to the antibody or antigen-binding fragment. Other embodiments include antibodies and antigen-binding fragments that are significantly identical to the antibodies and antigen-binding fragments described herein. They do not share the same affinity but may have one or more CDRs or other sequences required to confer affinity. and a framework, scaffold, or other non-binding region incorporating the sequence of For such sequences described in the specification, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identity to a BCMA-specific antibody or an antigen-binding fragment.

[0055] A "vector" is a molecule that transmits another nucleic acid segment so as to bring about the replication or expression of the segment. A replicon, such as a plasmid, phage, cosmid or virus, into which a vector can be operatively inserted. This is about Luz.

[0056] A "clone" is a population of cells derived by mitosis from a single cell or common ancestor. A "cell line" is a cell that can be stably grown in vitro for many generations. In some examples provided herein, the cell is a clone of a primary cell that can be cultured by culturing the cell. Transformation occurs by transfection with DNA.

[0057] The terms "express" and "produce" are used interchangeably herein and refer to These terms include the transcription of a gene into RNA. These terms also encompass the translation of RNA into one or more polypeptides, all of which The expression of an antibody or an antigen-binding fragment thereof further includes natural post-transcriptional and post-translational modifications of the antibody. The expression or production may be within the cytoplasm of a cell or within the extracellular environment, e.g., the growth medium of a cell culture. stomach.

[0058] The term "treating" or "treatment" refers to the attenuation or amelioration of an injury, pathology, or condition. This refers to any success or sign of success in a medical condition, including remission, relief, reduction in symptoms, improvement in the condition, or making the treatment more tolerable to the patient; slowing the rate of degeneration or decline; improving the patient's physical or mental health; The objective or subjective parameters, such as the ability to treat or prolong survival, may be included. Treatment may be evaluated by objective or subjective parameters, including: These may include the results of a physical examination, a neurological examination, or a psychiatric evaluation.

[0059] An "effective amount" or "therapeutically effective amount" is a desired amount, at dosages and for periods of time required. A therapeutically effective amount of a BCMAxCD3 antibody means an amount effective to achieve a therapeutic result. The antibody that elicits the desired response in an individual is determined based on the individual's disease state, age, sex, and weight. A therapeutically effective amount may also vary according to factors such as the ability of the antibody or antibody portion to bind to the antibody. It is also an amount in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.

[0060] Unless otherwise specified, the term "antibody" refers to any immunoglobulin isotype (IgG, Ig IgA, IgE, IgM, IgD, and IgY) and various monomeric, polymeric, and textured Polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides Antibodies, such as chimeric and humanized antibodies, are specifically encompassed by the term "antibody." .

[0061] An "antigen-binding fragment" is any protein that can exhibit binding affinity for a particular antigen. The antigen-binding fragments can be isolated by any known method, for example by enzymatic cleavage. Some antigen-binding fragments are provided by peptide synthesis and recombinant techniques. The antibody fragments consist of a portion of an intact antibody that retains the antigen-binding specificity of the parent antibody molecule. For example, an antigen-binding fragment can be an antibody that is known to bind to a particular antigen. , at least one variable region (either a heavy chain or a light chain variable region), or one or two or more CDRs. Abodies and single chain molecules, as well as Fab, F(ab')2, Fc, Fabc, and Fv molecules. antibody, single chain (Sc) antibody, individual antibody light chains, individual antibody heavy chains, antibody chains or CDRs and other Chimeric fusions between the proteins, protein scaffolds, heavy chain monomers or is a dimer, a light chain monomer or dimer, a dimer consisting of one heavy chain and one light chain, VL , a monovalent fragment consisting of the VH, CL, and CH1 domains, International Publication No. 200705 9782, a monovalent antibody linked by a disulfide bond in the hinge region. A bivalent fragment containing two Fab fragments, V.sub.H and C.sub. An Fd fragment consisting essentially of the H1 domain, the VL and VH domains of one arm of an antibody. an Fv fragment consisting essentially of a domain; an Fv fragment consisting essentially of a VH domain; Also called the endothelial cell (Holt et al., Trends Biotechnol. 200 3 Nov., 21(11):484-90) dAb fragments (Ward et al. l., Nature 341, 544-546 (1989)), camel or nanobody Revets et al, Expert Opin Biol Ther.200 5 Jan., 5(1):111-24), and isolated complementarity determining regions (CDRs) are included. All antibody isotypes include, but are not limited to, antigen-binding fragments. In addition, antigen-binding fragments may be used to generate A non-binding domain that can conveniently encapsulate polypeptide segments in an orientation that confers affinity for an antigen. The antibody may include a protein framework, e.g., a protein scaffold. Antigen-binding fragments may be produced recombinantly or enzymatically from intact antibodies. "Antibody or antigen-binding fragment thereof" may be produced by electrical or chemical cleavage. The phrase refers to a given antigen-binding fragment that is a single or doublet of the antibodies mentioned in the phrase. The term "amino acid segment" can be used to mean the inclusion of the above amino acid segments.

[0062] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually refer to surface groupings of molecules, e.g., amino acids or sugar side chains. They usually have specific three-dimensional structural characteristics as well as specific charge characteristics. A synthetic epitope is one in which the binding to the former but not the latter is lost in the presence of denaturing solvents. Epitopes are distinguished by the amino acid residues that are directly involved in binding and the amino acid residues that are directly involved in binding. Other amino acid residues that do not bind to the peptide specifically may effectively block the peptide. The amino acid residues that are blocked or covered (i.e., the amino acid residues that are specifically bound to the antigen) within the footprint of the binding peptide).

[0063] When used in the context of an antibody or antibody fragment, "specific binding" or "Immunospecific binding" or its derivatives refers to binding to immunoglobulin genes or immunoglobulins. Contains a mixed population of molecules across domains encoded by fragments of genes It binds to one or more of the proteins of interest without preferentially binding to other molecules in the sample. Typically, the antibody binds to one or more epitopes using surface plasmon resonance (SPPR). Approximately 1 × 10 -8 K under M d in, Binds to the same antigen, i.e. "antigen-specific" antibodies (e.g., BCMA-specific antibodies) The phrase is intended to convey that the recited antibody specifically binds to the recited antigen. Taste.

[0064] As used herein, "K D The term "antibody-antigen interaction" refers to the dissociation of a particular antibody-antigen interaction. It stands for equilibrium constant.

[0065] The term "subject" refers to humans and non-human animals, including all vertebrates, e.g., mammals. and non-mammals, e.g., non-human primates, mice, rabbits, sheep, dogs, cats, horses, Cattle, chickens, amphibians, and reptiles. In many embodiments of the methods described, The subject is a human.

[0066] As used herein, the term "sample" refers to a similar sample isolated from a subject. Fluids, cells, or tissues (e.g., surgically removed tumor tissue, including fine needle aspirates) that Biopsy refers to a collection of fluids, cells, or tissues present within a subject. In one embodiment, the sample is a body fluid. Body fluids are typically liquid at physiological temperatures. They are present in, taken from, or released from a subject or biological source. , or otherwise extracted naturally occurring fluids. Certain body fluids obtained from localized areas and certain other body fluids may be administered to a subject or biological source. Examples of body fluids include blood, serum and plasma. Membrane fluid, plasma, lymph, urine, saliva, cyst fluid, tears, feces, sputum, mucous secretions of secretory tissues and organs, Vaginal secretions, ascites fluid, e.g., associated with non-solid tumors, pleural, pericardial, peritoneal, abdominal and other Examples of body fluids include fluids from body cavities of a subject, fluids collected by bronchial lavage, etc. Solutions in contact with biological sources, such as cell and organ culture media, including cell or organ conditioned media It may include culture medium, washings, etc. As used herein, the term "sample" , includes material removed from a subject or material present in a subject.

[0067] A "known standard" may be a solution having a known amount or concentration of BCMA. The solution may be a naturally occurring solution, e.g., a solution having early, intermediate, late, advanced, or static cancer. The solution may be a sample from a patient known to have a known amount of B The known standards described herein may be synthetic solutions such as buffered water with CMA. BCMA isolated from, recombinant or purified BCMA protein, or a combination thereof The associated BCMA concentration values ​​may also be included.

[0068] As used herein, the term "BCMA" means human B-cell maturation antigen. , BCMA, CD269, and TNFRSF17 (UniProt Q02223). It is well known that the tumor necrosis receptor superfamily is preferentially expressed in differentiated plasma cells. The extracellular domain of human BCMA consists of amino acids 1–5 of the UniProt sequence. 4 (or 5-51). As used herein, "anti-BCMA" refers to The term "antibody, anti-BCMA antibody" refers to an antibody that immunospecifically binds to BCMA. .

[0069] The term "CD3" refers to the human CD3 protein multisubunit complex. The CD3 protein multisubunit complex consists of six distinct polypeptide chains. These are the CD3 gamma chain (SwissProt P09693), the CD3 delta chain (SwissProt P04234), two CD3ε chains (SwissProt P0 7766), and one CD3 ζ chain homodimer (SwissProt 20963). It associates with the α and β chains of the T cell receptor. naturally expressed by cells (including T cells) or encoding those polypeptides Any gene or cDNA that can be expressed on a cell transfected with the gene or cDNA that corresponds to the This includes all CD3 variants, isoforms, and interspecies homologs.

[0070] A "BCMAxCD3 antibody" is a multispecific antibody, optionally a bispecific antibody, The bispecific antibody contains two different antigen-binding regions, one of which binds to an antigen. One antigen-binding region specifically binds to BCMA, and the other antigen-binding region specifically binds to CD3. A polyspecific antibody may be a bispecific antibody, a diabody, or a similar molecule (e.g., a diabody). For a description of the body, see, for example, PNAS USA 90(14), 6444-8(1 993). The bispecific antibodies, diabodies, etc. provided herein are In addition to being a portion of CMA, any suitable target may be bound. The term is understood to mean an antibody that has two antigen-binding regions defined by different antibody sequences. This can be understood as differential target binding, but not binding to different epitopes in one target. This includes binding sufficiently.

[0071] A "reference sample" is a sample that is compared against another sample, e.g., a test sample. A reference sample is a sample from which the characteristics of a test sample can be determined. It will have some characterized properties that will serve as a basis for comparison with pull. For example, the reference sample may be a benchmark for BCMA levels indicative of subjects with cancer. A reference sample can be analyzed in parallel with the test sample. Thus, in some cases, the reference sample may be a sample that specifically reflects a given condition. A predetermined value or range to indicate, for example, a BCMA level indicative of cancer in a subject. The term may also refer to a physiological state or pathology associated with, for example, a BCMA-expressing cancer. Although it is known that BCMA can be expressed in a wide range of tissues, a sample with an unknown amount of BCMA was used for comparison purposes. include.

[0072] When used in the context of progression of BCMA-expressing cancers, the term "progression" is more commonly used to describe This includes the change in cancer from a less severe to a more severe state. This includes the number or severity of tumors. This may include an increase in the degree of metastasis, the rate at which the cancer grows or proliferates, etc. Bowel cancer progression includes the progression of such cancers from less severe to more severe forms. Progression, e.g., from Stage I to Stage II, from Stage II to Stage III, etc. The lines are listed.

[0073] When used in the context of regression of BCMA-expressing cancer, the term "regression" is more commonly used. This includes the change in cancer from a severe to a less severe state. This includes the number or severity of tumors. It is believed that such a treatment may include a decrease in the degree of metastasis, the rate at which the cancer grows or proliferates, and the like. For example, "colon cancer regression" involves the progression of such cancer from a more severe to a less severe state. regression to a worse state, e.g. from stage III to stage II, from stage II to stage Examples of progression include Di I.

[0074] When used in the context of stable BCMA-expressing cancers, the term "stable" , the cancer is significantly more severe over a clinically significant period of time than is necessary to be considered a progressive or regressive cancer. The term is intended to describe a condition that does not change or has not changed in a minute.

[0075] The embodiments described herein relate to specific methods, reagents, compounds, compositions, or biological systems. It is not intended to be limiting and may of course vary.

[0076] BCMA-specific antibodies and antigen-binding fragments As used herein, a recombinant monoclonal antibody or antigen that specifically binds to BCMA The overall structure of an antibody molecule includes an antigen-binding domain. The domains include heavy and light chains and an Fc domain, which is involved in complement fixation and binding antibody receptors. It performs various functions including those of the body.

[0077] The BCMA-specific antibodies or antigen-binding fragments described are of all isotypes. IgA, IgD, IgE, IgG, and IgM, as well as four-chain immunoglobulin structures The described antibodies or antigen-binding fragments may also be used in combination with hens or chickens. IgY isotypes commonly found in the serum of butterflies and in egg yolks of hens and turkeys. This also includes Yip.

[0078] BCMA-specific antibodies and antigen-binding fragments can be produced by recombinant techniques from any species. For example, the antibody or antigen-binding fragment may be isolated from a mouse, rat, goat, horse, or , pig, cow, chicken, rabbit, camel, donkey, human, or chimeric versions thereof. For use in human administration, antibodies or antigen-binding fragments of non-human origin may be The antibody may be genetically or structurally modified so as to be substantially less antigenic upon administration to a human patient. It can be changed.

[0079] In some embodiments, the antibody or antigen-binding fragment is chimeric. When used herein, the term "chimeric" refers to a non-human mammalian, rodent, or reptilian antibody amine. having at least a portion of at least one variable domain derived from the amino acid sequence of The remaining portion of the antibody or antigen-binding fragment thereof is derived from a human. It means fragment.

[0080] In some embodiments, the antibody is a humanized antibody. A humanized antibody is an antibody that is synthesized from a non-human immunoglobulin. Chimeric immunoglobulins, their immunoglobulin chains, or or fragments (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding fragments of antibodies). For the most part, humanized antibodies have been designed to mimic the complementarity determining regions (CDRs) of the recipient. A non-human species (donor antibody) in which residues from the CDRs have the desired specificity, affinity, and capacity For example, human immunoglobulins in which residues from mouse, rat, or rabbit have been replaced. Generally, a humanized antibody contains at least one Typically, it will contain substantially all of two variable domains. or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin; Virtually all of the framework regions are identical to those of human immunoglobulin sequences. Humanized antibodies are antibodies that contain an immunoglobulin constant region (Fc), typically a human immunoglobulin. It may contain at least a portion of phosphorus.

[0081] The antibodies or antigen-binding fragments described herein may exist in a variety of forms, including, but not limited to, It will contain one or more of the antibody CDRs shown in Table 1.

[0082] Provided herein are recombinant antibodies and antigen-binding fragments that immunospecifically bind to BCMA. In some embodiments, a BCMA-specific antibody or antigen-binding fragment is The BCMA specific antibody exemplified herein is human IgG or a derivative thereof. or antigen-binding fragments are human, but the exemplified antibodies or antigen-binding fragments may be chimerized.

[0083] In some embodiments, the CDR1, CDR2 and / or CDR3 of any one of the antibodies listed in Table 1 are and a heavy chain comprising CDR3. In some embodiments, the CDR1 of any one of the antibodies listed in Table 1 is provided. A heavy chain comprising CDR1, CDR2, and CDR3, and any one of the CDRs of the antibodies listed in Table 1. and a light chain comprising DR1, CDR2, and CDR3. The original binding fragment is provided.

[0084] In some embodiments, the BCMA-specific antibodies and antigen-binding fragments have the sequence SEQ ID NO:4. heavy chain CDR1 comprising SEQ ID NO:5, heavy chain CDR2 comprising SEQ ID NO:6, In some embodiments, the BCMA-specific antibodies and antigen-binding fragments comprise SEQ ID NO: heavy chain CDR1 comprising SEQ ID NO:4, heavy chain CDR2 comprising SEQ ID NO:5, heavy chain CDR3 comprising SEQ ID NO:6 3. A light chain CDR1 comprising SEQ ID NO: 7, a light chain CDR2 comprising SEQ ID NO: 8, and a light chain CDR2 comprising SEQ ID NO: 9 The BCMA-specific antibody or antigen-binding fragment comprises a light chain CDR3 comprising a human fibronectin. The BCMA-specific antibody or antigen-binding fragment may comprise a framework sequence. , IC of at least 5.9 nM 50 In some embodiments, APRIL binding can be inhibited by BCMA-specific antibodies and antigen-binding fragments are substantially the same as SEQ ID NO: 10 or In some embodiments, the BCMA-specific antibody and the antigen-binding The fragment comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 10 and a heavy chain variable domain having SEQ ID NO: No. 11 and a light chain variable domain substantially the same or identical to that of No. 12. The heavy and light chain variable domains of the antibody are each an anti-BCMA arm. are suitable for inclusion in a bispecific construct.

[0085] In some embodiments, the BCMA-specific antibodies and antigen-binding fragments have the sequence SEQ ID NO:4. heavy chain CDR1 comprising SEQ ID NO:5, heavy chain CDR2 comprising SEQ ID NO:6, In some embodiments, the BCMA-specific antibodies and antigen-binding fragments comprise SEQ ID NO: heavy chain CDR1 comprising SEQ ID NO:7, heavy chain CDR2 comprising SEQ ID NO:5, heavy chain CDR3 comprising SEQ ID NO:6 3. A light chain CDR1 comprising SEQ ID NO:24, a light chain CDR2 comprising SEQ ID NO:25, and a light chain CDR3 comprising SEQ ID NO:26. 26. This BCMA-specific antibody or antigen-binding fragment comprises a light chain CDR3 comprising: In some embodiments, the BCMA-specific antibody and The antigen-binding fragment comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO:57. In some embodiments, the BCMA-specific antibodies and antigen-binding fragments include those represented by SEQ ID NO: 57 and a heavy chain variable domain substantially the same or identical to SEQ ID NO:28. The heavy chain variable domains of the antibodies discussed in this paragraph contain identical light chain variable domains. The light and dark chain variable domains are bispecific constructs, one arm of which is an anti-BCMA arm. Suitable for inclusion in

[0086] In some embodiments, the BCMA-specific antibodies and antigen-binding fragments have the sequence SEQ ID NO:7. heavy chain CDR1 comprising SEQ ID NO:5, heavy chain CDR2 comprising SEQ ID NO:6, In some embodiments, the BCMA-specific antibodies and antigen-binding fragments comprise SEQ ID NO: heavy chain CDR1 comprising SEQ ID NO:7, heavy chain CDR2 comprising SEQ ID NO:5, heavy chain CDR3 comprising SEQ ID NO:6 3. A light chain CDR1 comprising SEQ ID NO:24, a light chain CDR2 comprising SEQ ID NO:25, and a light chain CDR3 comprising SEQ ID NO:26. 26. This BCMA-specific antibody or antigen-binding fragment comprises a light chain CDR3 comprising: In some embodiments, the BCMA-specific antibody and The antigen-binding fragment has a heavy chain variable domain substantially the same as or identical to SEQ ID NO:34. In some embodiments, the BCMA-specific antibodies and antigen-binding fragments include those represented by SEQ ID NO: 34 and a heavy chain variable domain substantially the same or identical to SEQ ID NO:28. The heavy chain variable domains of the antibodies discussed in this paragraph contain identical light chain variable domains. The light and dark chain variable domains are bispecific constructs, one arm of which is an anti-BCMA arm. Suitable for inclusion in

[0087] In some embodiments, the BCMA-specific antibodies and antigen-binding fragments have the sequence SEQ ID NO:4. heavy chain CDR1 comprising SEQ ID NO:5, heavy chain CDR2 comprising SEQ ID NO:19 In some embodiments, the BCMA-specific antibodies and antigen-binding fragments include the sequence Heavy chain CDR1 comprising SEQ ID NO: 4, heavy chain CDR2 comprising SEQ ID NO: 5, heavy chain CDR3 comprising SEQ ID NO: 19 DR3, light chain CDR1 comprising SEQ ID NO:24, light chain CDR2 comprising SEQ ID NO:25, and the sequence The BCMA-specific antibody or antigen-binding fragment comprises a light chain CDR3 comprising SEQ ID NO: 26. may comprise human framework sequences. and the antigen-binding fragment has a heavy chain variable domain substantially the same or identical to SEQ ID NO:39. In some embodiments, the BCMA-specific antibodies and antigen-binding fragments include the sequence a heavy chain variable domain substantially the same as or identical to SEQ ID NO:39 and a heavy chain variable domain substantially the same as SEQ ID NO:28 The heavy chain variable domains of the antibodies discussed in this paragraph contain either or the same light chain variable domain. The main and light chain variable domains are arranged in a bispecific configuration, with one arm being an anti-BCMA arm. Suitable for inclusion in structures.

[0088] In some embodiments, the BCMA-specific antibodies and antigen-binding fragments have the sequence SEQ ID NO:4. heavy chain CDR1 comprising SEQ ID NO:8, heavy chain CDR2 comprising SEQ ID NO:6, and heavy chain CDR3 comprising SEQ ID NO:7. In some embodiments, the BCMA-specific antibodies and antigen-binding fragments comprise SEQ ID NO: heavy chain CDR1 comprising SEQ ID NO:4, heavy chain CDR2 comprising SEQ ID NO:8, heavy chain CDR3 comprising SEQ ID NO:6 3. A light chain CDR1 comprising SEQ ID NO:24, a light chain CDR2 comprising SEQ ID NO:25, and a light chain CDR3 comprising SEQ ID NO:26. 26. This BCMA-specific antibody or antigen-binding fragment comprises a light chain CDR3 comprising: In some embodiments, the BCMA-specific antibody and The antigen-binding fragment has a heavy chain variable domain substantially the same as or identical to SEQ ID NO:40. In some embodiments, the BCMA-specific antibodies and antigen-binding fragments include those represented by SEQ ID NO: 40 and a heavy chain variable domain substantially the same as or identical to SEQ ID NO:28. The heavy chain variable domains of the antibodies discussed in this paragraph contain identical light chain variable domains. The light and dark chain variable domains are bispecific constructs, one arm of which is an anti-BCMA arm. Suitable for inclusion in

[0089] In some embodiments, the BCMA-specific antibodies and antigen-binding fragments have the sequence of SEQ ID NO: 13. heavy chain CDR1 comprising SEQ ID NO:5, heavy chain CDR2 comprising SEQ ID NO:19 In some embodiments, the BCMA-specific antibodies and antigen-binding fragments comprise the sequence Heavy chain CDR1 comprising sequence number 13, heavy chain CDR2 comprising sequence number 5, heavy chain CDR3 comprising sequence number 19 light chain CDR3 comprising SEQ ID NO:24, light chain CDR2 comprising SEQ ID NO:25, and The BCMA-specific antibody or antigen-binding fragment comprises a light chain CDR3 comprising SEQ ID NO: 26. In some embodiments, the construct may comprise human framework sequences. The antibodies and antigen-binding fragments have a heavy chain variable domain substantially the same as or identical to SEQ ID NO:58. In some embodiments, the BCMA-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same as or identical to SEQ ID NO:58 and a heavy chain variable domain substantially the same as SEQ ID NO:28 The heavy chain variable domains of the antibodies discussed in this paragraph contain the same or identical light chain variable domains. The variable domain and the light chain variable domain are bispecific, with one arm being an anti-BCMA arm. Suitable for inclusion in the gender construct.

[0090] In some embodiments, the BCMA-specific antibodies and antigen-binding fragments have the sequence of SEQ ID NO: 13. heavy chain CDR1 comprising SEQ ID NO: 8, heavy chain CDR2 comprising SEQ ID NO: 19 In some embodiments, the BCMA-specific antibodies and antigen-binding fragments comprise the sequence Heavy chain CDR1 comprising sequence number 13, heavy chain CDR2 comprising sequence number 8, heavy chain CDR3 comprising sequence number 19 light chain CDR3 comprising SEQ ID NO:24, light chain CDR2 comprising SEQ ID NO:25, and The BCMA-specific antibody or antigen-binding fragment comprises a light chain CDR3 comprising SEQ ID NO: 26. In some embodiments, the construct may comprise human framework sequences. The antibodies and antigen-binding fragments have a heavy chain variable domain substantially the same as or identical to SEQ ID NO:43. In some embodiments, the BCMA-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same as or identical to SEQ ID NO:43 and a heavy chain variable domain substantially the same as SEQ ID NO:28 The heavy chain variable domains of the antibodies discussed in this paragraph contain the same or identical light chain variable domains. The variable domain and the light chain variable domain are bispecific, with one arm being an anti-BCMA arm. Suitable for inclusion in the gender construct.

[0091] In some embodiments, the antibody or antigen-binding fragment is an IgG or a derivative thereof, e.g. Examples are the IgG1, IgG2, IgG3, and IgG4 isotypes. In some embodiments, the antibody comprises an IgG1 Fc region (SEQ ID NO: 74). ).

[0092] SEQ ID NO:74 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

[0093] In some embodiments where the antibody is of the IgG4 isotype, the antibody is of the S228P, L2 It contains a L34A, and a L235A substitution in its Fc region (SEQ ID NO: 73).

[0094] SEQ ID NO:73 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK

[0095] The characteristics defined by the CDR and / or variable domain sequences discussed in the paragraph above Heteromeric antibodies may include these IgG Fc regions.

[0096] An isolated nucleic acid encoding an antibody or antigen-binding fragment that immunospecifically binds to BCMA. Synthetic polynucleotides that are suitable for use in the manufacture of the variable domain segments provided herein are also disclosed. The isolated polynucleotide capable of encoding the antibody can be used to produce an antibody or antigen-binding fragment. The vectors may be contained in the same or different vectors.

[0097] Polynucleotides encoding the recombinant antigen binding proteins are also within the scope of this disclosure. In some embodiments, the described polynucleotide (and the polypeptide thereof) The leader sequence may be any leader sequence known in the art. Leader sequences may include restriction sites or translation initiation sites. The following are possible examples, but are not limited to these.

[0098] The BCMA-specific antibodies or antigen-binding fragments described herein are The biological properties of the CMA-specific antibody or antigen-binding fragment (e.g., binding affinity or immunity) one or more amino acid substitutions, deletions, or additions that retain the immunoeffector activity of the polypeptide; In the context of the present invention, the following notes include variants having a mutation, unless otherwise stated. i) Substitution of an amino acid at a given position, e.g., K409 R. K409R means substitution of lysine at position 409 with arginine. ii) For specific variants, the specific three-letter or one-letter code is The lysine at position 409 is used to indicate the amino acid residue, including the suffix and X. The substitution of arginine for is designated K409R, or lysine at position 409. The substitution of any amino acid residue at position 409 is designated K409X. In the case of the deletion of the gene, the deletion is K409 * Those skilled in the art will recognize that one or can generate variants with multiple amino acid substitutions, deletions, or additions.

[0099] These variants include (a) variants in which one or more amino acid residues are conservative or non-conservative; (b) a variant in which one or more amino acids are replaced by a specific amino acid; (c) a variant in which one or more amino acids are added to or deleted from the polypeptide; (d) variants in which the above amino acids contain substituents; and (e) variants in which the polypeptide is a polypeptide of another peptide or polypeptide. peptides, e.g., variants fused to fusion partners, protein tags, or other chemical moieties. These can include those that provide useful properties for the polypeptide, e.g., for antibodies. Epitopes, polyhistidine sequences, biotin moieties, etc. may also be added. The antibodies or antigen-binding fragments selected herein may be either at conserved or non-conserved positions. including variants in which an amino acid residue from one species is replaced by the corresponding residue in another species. In other embodiments, the amino acid residues at the non-conserved positions may be either conserved or non-conserved residues. The methods for obtaining these variants include genetic (deletions, mutations, etc.), chemical These include chemical and enzymatic techniques known to those skilled in the art.

[0100] The BCMA-specific antibodies or antigen-binding fragments described herein comprise multiple antibody antigens. It is possible to embody isotypes such as IgM, IgD, IgG, IgA, and IgE. In some embodiments, the antibody isotype is IgG1, IgG2, IgG3, or is of IgG4 isotype, preferably of IgG1 or IgG4 isotype. The specificity of an antibody or antigen-binding fragment thereof is primarily determined by the amino acid sequence and arrangement of the CDRs. Therefore, the CDRs of a given isotype can change antigen specificity. Alternatively, the antigen specificity can be altered. A technique that allows hybridomas to switch from one antibody isotype to another (A Isotype switching has been established. is within the scope of the described antibodies or antigen-binding fragments.

[0101] The BCMA-specific antibodies or antigen-binding fragments described herein are IC of at least 5.9 nM for 50 The BCMA specific antibodies described or IC of antigen-binding fragment 50 can be prepared by various methods well known in the art, e.g. For example, it can be determined by ELISA-based methods or flow cytometry (FACS). IC by ELISA 50 The assay measures the presence and absence of BCMA-specific antibodies. Various concentrations of APRIL are used with BCMA-bound plates in the presence of BCMA antibodies that inhibit binding to BCMA have been shown to be effective in preventing APR as measured by ELISA. IL can be inhibited.

[0102] Also provided is a vector comprising the polynucleotide described herein. Thus, the vector may contain a sequence encoding a polypeptide of interest. Recombinant expression vectors that can be used to express the polypeptides of the present invention are contemplated as being within the scope of the present disclosure. Alternatively, the additional sequence may contain two or more additional sequences. The additional sequences may include, for example, a regulatory sequence (e.g., , promoters, enhancers), selection markers, and polyadenylation signals. Vectors for transforming a wide variety of host cells are well known. Plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial Chromosomes (BAC), yeast artificial chromosomes (YAC), and other bacteria, yeast, and viruses Examples of vectors include, but are not limited to,

[0103] Recombinant expression vectors within the scope of this description are operably linked to suitable regulatory elements. A synthetic, genetic or other method for the preparation of a recombinant protein encoding at least one recombinant protein capable of The regulatory elements include nucleic acid fragments derived from cDNA. The sequence includes the sequence encoding the appropriate mRNA ribosome binding site, and the sequence encoding the termination of transcription and translation. Expression vectors, particularly mammalian expression vectors, include sequences that control the expression of the Also, one or more non-transcriptional elements, e.g., an origin of replication, Suitable promoters and enhancers, other 5' or 3' flanking non-transcribed sequences, , 5' or 3' untranslated sequences (e.g., essential ribosome binding sites), polyadenylation sites, It may also contain splice donor and acceptor sites, or transcription termination sequences. An origin of replication that confers the ability to replicate in a vector may also be included.

[0104] Transcriptional and translational controls in expression vectors used to transform vertebrate cells The vector sequence can be provided by a viral source. An exemplary vector is the Okay Ama and Berg, 3 Mol. Cell. Biol. 280 (1983) It may be constructed as described above.

[0105] In some embodiments, the coding sequence for the antibody or antigen-binding fragment comprises a strong constitutive promoter. Promoters, such as those of the genes: hypoxanthine phosphoribosyltransferase (H PRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin It is placed under the control of the promoters for human creatine, human hemoglobin, human muscle creatine, etc. In addition, many viral promoters function constitutively in eukaryotic cells and may be used in the described embodiments. Such viral promoters include those derived from the cytomegalovirus (C MV) intermediate early promoter, early and late promoters of SV40, mouse mammary tumor virus (MMTV) promoter, Moloney leukemia virus, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and other retroviruses Viral long terminal repeats (LTRs) and herpes simplex virus thymidine kinase In one embodiment, the promoter includes, but is not limited to, a BCMA-specific promoter. The coding sequence for the antibody or antigen-binding fragment thereof may be driven by an attractive promoter, e.g. Lothionine promoter, tetracycline-inducible promoter, doxycycline-inducible promoter A promoter, one or more interferon stimulated response elements (ISREs), e.g. For example, protein kinase R, 2',5'-oligoadenylate synthetase, MX gene, It is placed under the control of a promoter containing ADAR1, etc.

[0106] The vectors described herein contain one or more internal ribosome entry sites (IRSs). Inclusion of an IRES sequence in a fusion vector may enhance the expression of some proteins. In some embodiments, the vector system may be one or more The gene will contain two or more polyadenylation sites (e.g., SV40). The vector may be located upstream or downstream of any of the nucleic acid sequences described above. The may be closely linked or may be spaced apart to provide optimal spacing for expression of the gene product. (i.e., by introducing "spacer" nucleotides between the ORFs). Regulatory elements, such as IR, may be located in the ribosome or in another manner. The ES motifs may also be positioned to provide optimal spacing for expression.

[0107] The vector may include a selection marker as known in the art. Positive and negative selection markers, e.g., antibiotic resistance genes (e.g., neomycin Resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline Phosphate resistance gene, penicillin resistance gene, puromycin resistance gene, blast cystine resistance gene), glutamate synthase gene, HSV for ganciclovir selection -TK, HSV-TK derivatives, or bacterial purine nucleosides for 6-methylpurine selection Phosphorylase gene (Gadi et al., 7 Gene Ther. 1738~ 1743 (2000). The cloning site is upstream of the nucleic acid sequence encoding the polypeptide of interest or the cloning site. Or it may be downstream.

[0108] The vectors described herein can be used to produce the described antibodies or antigen-binding fragments. A variety of cells can be transformed with genes encoding the can be used to generate BCMA-specific antibody or antigen-binding fragment producing cells. Thus, another embodiment is an antibody or antigen-binding fragment thereof that specifically binds to BCMA. Coated antibodies, e.g., antibodies or antigen-binding fragments as described and exemplified herein. The invention features a host cell transformed with a vector containing a nucleic acid sequence that encodes a nucleotide sequence encoding the nucleotide sequence of the present invention.

[0109] Numerous techniques are known in the art for introducing exogenous genes into cells. and for carrying out the described methods according to the various embodiments described and illustrated herein. Depending on the technique used, heterologous genes can be used to construct recombinant cells for the same purpose. The plasmid should be appropriately transformed into the host cell so that the heterologous gene sequence is not can be inherited and expressed by the progeny of the recipient cell and are essential for the growth and survival of the recipient cell The physical function of the organism is not affected. Techniques that can be used include chromosome transfer (e.g. , cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer), physical methods ( For example, transfection, spheroplast fusion, microinjection, Lectoporation, liposome carriers), viral vector introduction (e.g., recombinant DNA A virus, recombinant RNA virus, etc. (Cline, 29 Pharma C. Ther. 69-92 (1985)) are not limited thereto. Calcium phosphate precipitation of bacterial protoplasts and polyethylene glycol (PEG) ) Trigger fusion can also be used to transform cells.

[0110] For use in expressing the BCMA-specific antibodies or antigen-binding fragments described herein Suitable cells for the production of the present invention are preferably eukaryotic cells, more preferably plant, rodent or human cells. These cells include, for example, NSO, CHO, CHOK1, perC .6, Tk-ts13, BHK, HEK293 cells, COS-7, T98G, CV-1 / EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2 / 0 myeloma cells, and In addition, the expression of antibodies can be achieved by hybridization with a hybridoma. This may be accomplished using hybridoma cells. It is well established in the art.

[0111] Cells transformed with the expression vectors described herein can be used to express the Selected or screened for recombinant expression of the antibody or antigen-binding fragment. Recombinant positive cells may be characterized as cells that express the desired protein through protein modification or altered post-translational modifications. phenotype, e.g., high levels of expression, improved growth characteristics, or desirable biochemical properties. The subclones are expanded and screened for the ability to produce the desired protein. These phenotypes may be due to inherent properties of a given subclone or due to mutations. , chemicals, UV light, radiation, viruses, insertional mutagenesis, inhibition of DNA mismatch repair, or is obtained by a combination of such methods.

[0112] Methods of Using BCMA-Specific Antibodies for Treatment Provided herein are methods for administering BCMA-specific antibodies or antigen-binding fragments thereof for use in therapy. In particular, these antibodies or antigen-binding fragments are useful in treating cancer, e.g. These may be useful in treating BCMA-expressing cancers. Administering an antibody as described, e.g., a BCMA-specific antibody or antigen-binding fragment. For example, the use may involve the treatment of cancer by inhibiting the expression of BCMA receptors. It may be by inhibition using an interaction, i.e., an antibody is bound to the toxin, In some embodiments, the BCMA-expressing cancer is a multicenter, Antibodies for use in these methods include those for lymphomas such as myeloma (MM). , those described herein above, e.g., features listed in Table 1 (e.g., CDRs or variable domain sequence) and the characteristics listed in further discussion of these antibodies. A-specific antibodies or antigen-binding fragments.

[0113] In some embodiments described herein, the immune effector properties of the BCMA-specific antibody The activity can be improved or silenced by Fc modification using techniques known to those skilled in the art. For example, Fc effector functions, e.g., C1q binding, complement-dependent cell proliferation, toxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis Downregulation of cell surface receptors (e.g., B cell receptor, BCR) and / or can be controlled.

[0114] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a process that is used to treat antibodies that express Fc receptors (FcRs). Nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and Macrophages recognize the bound antibodies on target cells and subsequently induce lysis of the target cells. It refers to a cell-mediated reaction that causes

[0115] The ability of a monoclonal antibody to trigger ADCC can be modified by manipulating its oligosaccharide composition. Human IgG1 or IgG3 has an N-glycosylation at Asn297. Here, the majority of glycans are synthesized into the well-known biantennary G0, G0F, G1, G1F, G2 or G2F forms. Antibodies produced by non-genetically engineered CHO cells. The antibody typically has a glycan fucose content of at least about 85%. Removal of core fucose from the attached biantennary complex-type oligosaccharides resulted in improved F c. via gamma RIIIa binding, without altering antigen binding or CDC activity Such mAbs have a bi-branched complex type Fc oligonucleotide, which enhances the ADCC of the antibody. It has been reported that this results in the successful expression of relatively highly defucosylated antibodies bearing glycosylation Various methods, such as controlling the culture osmolarity (Konno et al., Cytotech nology 64:249-65,2012), and mutant CHO strain L as a host cell line Application of ec13 (Shields et al., J Biol Chem 277:2 6733~26740, 2002), and application of the mutant CHO strain EB66 as a host cell line (Olivier et al.,MAbs;2(4),2010, Epub ahea d of print, PMID:20562582), and rat high- Application of the bryomycin cell line YB2 / 0 (Shinkawa et al., J Biol Chem 278:3466~3473,2003), alpha.1,6-fucosyltra Introduction of small interfering RNA specific for the Mori transduction enzyme (FUT8) gene et al.,Biotechnol Bioeng 88:901~908,200 4) or beta-1,4-N-acetylglucosaminyltransferase III and Co-expression with alpha-mannosidase II or potent alpha-mannosidase I inhibitor The antibacterial agent kifunensine (Ferrara et al., J Biol Chem 2 81:5032~5036;2006, Ferrara et al.,Biotech nol Bioeng 93:851~861,2006;Xhou et al.,B This was achieved using the iotechnol Bioeng 99:652-65, 2008). It is possible.

[0116] In some embodiments described herein, the A elicited by the BCMA antibody DCC can also be improved by certain substitutions in the antibody Fc. Exemplary substitutions include, for example, , and amino acid positions 256, 290, as described in U.S. Pat. No. 6,737,056. 298, 312, 356, 330, 333, 334, 360, 378, or 430 (EU The substitutions are in the residue numbering according to the index.

[0117] How to Detect BCMA As used herein, a sample is a nucleic acid that is capable of binding to an antibody or antigen-binding fragment thereof as described herein. The present invention provides a method for detecting BCMA in a biological sample by contacting the sample with a As described herein, the sample may be urine, blood, serum, plasma, saliva, ascites, Circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissue (e.g., For example, from surgically removed tumor tissue, biopsies (including fine needle aspirations), histological preparations, etc. In some embodiments, the described methods can include subjecting the sample to a method as described herein. contacting the subject with any of the BCMA-specific antibodies or antigen-binding fragments thereof described herein. The method includes detecting BCMA in a biological sample by

[0118] In some embodiments, the sample is subjected to an antibody or antigen detection assay using a BCMA-specific antibody or antigen detection method as described herein. The sample may be contacted with more than one of the binding fragments. For example, the sample may be contacted with a first BCMA and then contacting the cells with a second BCMA-specific antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof may be contacted with a first antibody or antigen-binding fragment thereof. The binding fragment and the second antibody or antigen-binding fragment are the same antibody or antigen-binding fragment. In some embodiments, the first antibody or antigen-binding fragment thereof is not a Prior to contacting the sample, the surface, e.g., a multi-well plate, chip or similar In another embodiment, the first antibody or antigen-binding fragment thereof may be immobilized on a substrate. may not be fixed or attached at all prior to contacting with the sample.

[0119] The described BCMA-specific antibodies and antigen-binding fragments may be detectably labeled. In some embodiments, the labeled antibodies and antigen-binding fragments can be labeled according to the methods described herein. Detection of BCMA can be facilitated by the methods described herein. Many such labels are available to those of skill in the art. For example, suitable labels include radiolabels, fluorescent labels, epitope tags, and the like. , biotin, a chromophore label, an ECL label, or an enzyme. More specifically, the labels described include ruthenium, 111 I n-DOTA, 111 In-Diethylenetriaminepentaacetic acid (DTPA), Horseradish peroxide oxidase, alkaline phosphatase, and beta-galactosidase, polyhistidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazine dyes, Examples of the dyes include phenanthridine dyes, rhodamine dyes, and Alexafluor (registered trademark) dyes. can be done.

[0120] The described BCMA-specific antibodies and antigen-binding fragments are capable of detecting BCMA in biological samples. A variety of assays for detecting A may be used. Some suitable assays include Western blot analysis, radioimmunoassay, surface plasmon resonance, immunofluorescence assay, Immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry These include chemical methods, fluorescence activated cell sorting (FACS) or ELISA assays. should not be considered as being limited to

[0121] In some embodiments described herein, detection of BCMA-expressing cancer cells in a subject to determine that a subject may be treated with a therapeutic agent directed against BCMA It is possible.

[0122] BCMA is present at detectable levels in blood and serum samples. As used herein, the sample is subjected to a BCMA specific antibody or antigen-binding fragment thereof. In a sample obtained from blood, for example a serum sample, the A method for detecting BCMA is provided. A blood sample or a derivative thereof is diluted and The sample may be separated, fractionated, or otherwise processed to produce a sample on which the described methods may be performed. In some embodiments, BCMA can be administered in any manner known in the art. A number of assays can be used to detect in blood samples or their derivatives. For example, The assays include Western blot analysis, radioimmunoassay, and surface plasmon resonance. , immunofluorescence assay, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay These include immunohistochemistry, fluorescence activated cell sorting (FACS) or ELISA assays. Examples include, but are not limited to:

[0123] Methods for diagnosing cancer Provided herein are methods for diagnosing BCMA-expressing cancer in a subject. In some embodiments, the BCMA-expressing cancer includes lymphomas such as multiple myeloma (MM). In some embodiments, as described above, a biological sample, such as a blood sample or Detecting BCMA in a serum sample is diagnostic of cancer in the subject from whom the sample was obtained. Alternatively, in some embodiments, other samples, e.g., histological samples, may be used. Samples, fine needle aspirates, excised tumor tissue, circulating cells, and circulating tumor cells may also be used. It may be used to assess whether the subject from whom the sample was obtained has cancer. In this embodiment, the subject from whom the sample was obtained may already be known to have cancer. However, the type of cancer afflicting the subject may not yet be diagnosed or may be at the preliminary stage of diagnosis. Since BCMA is unknown by genetic testing, it can be detected by detecting it in biological samples obtained from subjects. For example, the subject may have cancer, and the cancer diagnosis may be possible or definitive. The cancer of interest may be known, but may not be known, or may be BCMA-positive. Whether or not it is expressed may be unknown.

[0124] In some embodiments, the methods described involve detecting a marker present in a biological sample obtained from a subject. The amount of BCMA is determined and the observed amount of BCMA is compared with that in the control or reference sample. Assess whether a subject is suffering from BCMA-expressing cancer by comparing with the amount of CMA In this case, the amount of BCMA in a sample obtained from a subject and a control or the amount of BCMA in the reference sample indicates that the subject is suffering from a BCMA-expressing cancer. In another embodiment, the observed level of the phenotype in a biological sample obtained from a subject is indicative of The amount of BCMA was determined based on levels of BCMA known to be associated with specific forms or stages of cancer. The results can be compared to the patient's cancer type or stage. In this embodiment, the amount of BCMA in a sample obtained from a subject is determined by isolating the sample against BCMA-immunospecific Heterologous binding antibodies or antigen-binding fragments thereof, such as those described herein, The presence of BCMA is assessed by contacting the patient with a MA-specific antibody. Samples included urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, tissue-associated free cells (i.e., isolated cells), tissues (e.g., surgically removed tumor tissues), , biopsy, including fine needle aspiration), histological preparations, etc. In some embodiments, BCMA-expressing cancers include hematological cancers such as multiple myeloma (MM). The subject is a human.

[0125] In some embodiments, the method of diagnosing a BCMA-expressing cancer comprises: CMA-specific antibodies or antigen-binding fragments thereof (e.g., the antibodies and fragments provided in Table 1) a fragment thereof, Thus, the amount of BCMA present in the bound sample can be quantified, and the amount of BCMA present in the sample can be comparing the amount of BCMA in the subject to a known standard or reference sample; and This will involve determining whether the level of BCMA in a patient is within the levels of BCMA associated with cancer. In a further embodiment, the diagnostic method is followed by the further step of administering or prescribing a cancer-specific treatment. In another embodiment, the diagnostic method communicates the results of the determination to facilitate cancer treatment. In some embodiments, the cancer specific treatment can be, for example, a method as described herein. The BCMA×CD3 multispecific antibodies described in the literature are directed against BCMA-expressing cancers. This may also be the case.

[0126] In some embodiments, the methods described include, in a blood or serum sample obtained from the subject, Determining the amount of BCMA present and comparing the observed amount of BCMA with a control or reference and comparing the amount of BCMA in a control sample to determine whether the subject is suffering from a BCMA-expressing cancer. In this case, the BC in a sample obtained from the subject is evaluated. The difference between the amount of BCMA and the amount of BCMA in the control or reference sample indicates that the subject has BCM. A expression is an indicator of suffering from cancer.

[0127] In some embodiments, the control or reference sample is a subject suffering from a BCMA-expressing cancer. In some embodiments, the control or reference sample may be obtained from a subject that does not have The control or reference sample may be obtained from a subject suffering from a BCMA-expressing cancer. In some embodiments, the control sample is obtained from a subject not suffering from a BCMA-expressing cancer. The amount of BCMA present in the test sample relative to that observed in the roll or reference sample. The observed increase in the amount of BCMA present indicates that the subject being evaluated is suffering from a BCMA-expressing cancer. The control samples were not suffering from BCMA-expressing cancer. In some embodiments, the same concentration is obtained from a control or reference sample. The observed difference in the amount of BCMA present in the test sample versus the amount of BCMA detected A decrease or similarity is indicative that the subject being evaluated is not afflicted with a BCMA-expressing cancer. The control or reference sample is obtained from a subject suffering from a BCMA-expressing cancer. In some embodiments, the amount of BCMA observed in a control or reference sample In contrast, the observed similarity in the amount of BCMA present in the test samples was evaluated. The presence of a BCMA-expressing antibody is indicative that the subject suffers from a BCMA-expressing cancer. In some embodiments, the control sample is obtained from a subject suffering from a BCMA-expressing cancer. The amount of BCMA present in the test sample relative to the amount observed in the test or reference sample The observed decrease in the amount of BCMA associated with the tumor indicates that the subject being evaluated is suffering from a BCMA-expressing cancer. This is an indicator that the

[0128] In some embodiments, the amount of BCMA in a sample obtained from a subject is determined by dividing the sample by B Antibodies or antigen-binding fragments thereof that specifically bind to CMA, such as those described herein The samples are evaluated for the presence of BCMA by contacting the samples with an antibody that has been The pools were collected from blood samples, serum samples, circulating cells, circulating tumor cells, and non-tissue associated cells. Cells (i.e., free cells), tissues (e.g., surgically removed tumor tissue, microneedle aspiration The antibody may be obtained from tissue samples (including biopsies), histological preparations, etc.

[0129] In various embodiments, the amount of BCMA is determined by isolating the sample with an antibody that specifically binds BCMA. or an antigen-binding fragment thereof. The sample is then subjected to two or more antibodies or antigen-binding fragments thereof that specifically bind to BCMA. In some embodiments, the sample may be contacted with a fragment that specifically binds to BCMA. and then contacting the cells with a first antibody or antigen-binding fragment thereof specific for BCMA. Alternatively, the antibody may be contacted with a second antibody or antigen-binding fragment thereof that binds to the antibody described herein. BCMA-specific antibodies or antigen-binding fragments such as those described above can be used in this capacity. May be used.

[0130] Various combinations of BCMA-specific antibodies and antigen-binding fragments were used to and providing a "first" and a "second" antibody or antigen-binding fragment for carrying out the diagnostic method. In some embodiments, the BCMA-expressing cancer is a cancer such as multiple myeloma (MM). Which includes lymphoma.

[0131] In certain embodiments, the amount of BCMA is measured by Western blot analysis, radioimmunoassay, or immunoassay. Immunofluorescence assay, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay For immunoassays, immunohistochemistry, fluorescence activated cell sorting (FACS) or ELISA assays is determined by.

[0132] In various embodiments of the described diagnostic methods, a control or reference sample is used. This sample should be tested for positive or negative results to ensure that the assay being used is functioning properly. It may be a negative assay control. For example, an assay control of this nature may be It is contemplated that the sample may be used generally in immunohistochemical assays. It may be a standardized reference for the amount of BCMA in biological samples from healthy subjects. In some embodiments, the observed BCMA levels in the tested subject are determined by BCMA expression. Compared to BCMA levels observed in samples from subjects known to have cancer In some embodiments, the control subject may be a subject suffering from a particular cancer of interest. In some embodiments, the control subject has an early stage cancer. It is known that the early stage cancer may be a BCMA-expressing cancer or a BCM In some embodiments, the control subject does not have to have an A-expressing cancer. and wherein the intermediate stage cancer may be a BCMA-expressing cancer, In some embodiments, the control subject may not have a BCMA-expressing cancer. It is known that the later stages of cancer may be BCMA-expressing or B The cancer does not have to be CMA-expressing.

[0133] Methods for monitoring cancer - Patent application Provided herein are methods for monitoring BCMA-expressing cancer in a subject. In some embodiments, the BCMA-expressing cancer is a lymphoma, such as multiple myeloma (MM). In some embodiments, the methods described include detecting a mutated nucleotide sequence present in a test sample obtained from a subject. To determine the amount of BCMA present in the tumor and to compare the amount of BCMA observed with that present in the tumor at earlier time points. By comparing the amount of BCMA in a biological sample obtained in a similar manner, BCMA This includes assessing whether the cancer is progressing, regressing, or remaining unchanged. If so, the difference between the amount of BCMA in the test sample and the amount of BCMA in the earlier sample It provides an indication of whether the cancer is progressing, regressing, or remaining unchanged. Containing increased amounts of BCMA relative to the amounts observed in earlier samples The test sample may be indicative of progression of a BCMA-expressing cancer. Conversely, earlier samples may be indicative of progression of a BCMA-expressing cancer. Test samples containing reduced amounts of BCMA relative to the amount observed for the BCMA Expression may indicate cancer regression.

[0134] Thus, the difference in BCMA abundance relative to that observed in earlier samples was Test samples with only minor differences show consistent pathology for BCMA-expressing cancers. In some embodiments, the amount of BCMA in a biological sample obtained from a subject can be determined by: The sample is subjected to detection of an antibody or antigen-binding fragment thereof that specifically binds BCMA, e.g. The presence of BCMA is assessed by contacting the subject with an antibody described herein. Samples evaluated include urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, Non-tissue-associated cells (i.e., free cells), tissue (e.g., surgically removed cells), The tumor tissue may be obtained from tissue samples (including tissue biopsies, including fine needle aspirations), histological preparations, etc. In embodiments, the subject is a human.

[0135] In some embodiments, the method of monitoring a BCMA-expressing cancer comprises: BCMA-specific antibodies or antigen-binding fragments thereof (e.g., the antibodies and fragments provided in Table 1). and determining whether BCMA is present in the sample. The amount of BCMA present in the sample was quantified at earlier time points in the same manner. The amount of BCMA determined to be present in similarly obtained biological samples from elephants was compared to that and to determine whether the subject's BCMA levels changed over time. , which would include an increased amount of BC relative to that observed in earlier samples. Test samples containing MA can indicate progression of cancer. Conversely, earlier samples Test samples containing reduced amounts of BCMA relative to the amounts observed for This could indicate regression of existing cancers. Test samples with only minor differences in the amount of BCMA relative to the amount of BCMA expression In some embodiments, the BCMA of the sample may be indicative of an unaltered pathology of the cancer. Levels were compared to those observed for samples assessed alone or at earlier time points. In addition, the CMA level may be compared to a known standard or reference sample. In some embodiments, the diagnostic method may be followed by the further step of administering a cancer-specific treatment. In this regard, the cancer-specific treatment may, for example, be a BCMAxCD3 multispecific antibody as described herein. The antibody may be directed against a BCMA-expressing cancer, such as the bladder.

[0136] In various embodiments, the amount of BCMA is determined by isolating the sample with an antibody that specifically binds BCMA. or an antigen-binding fragment thereof. The sample is then subjected to two or more antibodies or antigen-binding fragments thereof that specifically bind to BCMA. In some embodiments, the sample may be contacted with a fragment that specifically binds to BCMA. and then contacting the cells with a first antibody or antigen-binding fragment thereof specific for BCMA. Alternatively, the antibody may be contacted with a second antibody or antigen-binding fragment thereof that binds to the antibody described herein. Antibodies such as those described above may be used in this capacity.

[0137] Various combinations of antibodies and antigen-binding fragments described in Table 1 were used to "First" and "second" antibodies or antigen-binding fragments for carrying out the monitoring method described above. In some embodiments, the BCMA-expressing cancer is acute myeloid leukemia ( This includes blood cancers such as AML.

[0138] In certain embodiments, the amount of BCMA is measured by Western blot analysis, radioimmunoassay, or immunoassay. Immunofluorescence assay, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay For immunoassays, immunohistochemistry, fluorescence activated cell sorting (FACS) or ELISA assays is determined by.

[0139] Kits for detecting BCMA Provided herein is a kit for detecting BCMA in a biological sample. These kits include any of the BCMA-specific antibodies or antigen-binding fragments thereof described herein. The kit includes one or more of the components listed above and instructions for use of the kit.

[0140] The provided BCMA-specific antibodies or antigen-binding fragments may be in solution. It may be freeze-dried or affixed to a substrate, carrier, or plate. or may be detectably labeled.

[0141] The described kits may also include additional components useful for carrying out the methods described herein. By way of example, the kit may include means for obtaining a sample from a subject, a control A sample or reference sample, for example a subject with a slowly progressing cancer and / or A sample from a subject without cancer, one or more sample compartments, and / or Instructions for carrying out the methods of the invention, as well as tissue-specific controls or standards It may also include.

[0142] The means for determining the level of BCMA can be, for example, The instructions may further include buffers or other reagents for use in the assay. For example, printed instructions for performing the assay and / or methods for assessing BCMA expression levels. It can be an instruction manual for

[0143] The described kits may also include a means for isolating a sample from a subject. These means may be one or more of the instruments or reagents that may be used to obtain fluid or tissue from a subject. The means for obtaining a sample from a subject may also include a blood sample. Preferably, the kit comprises a means for isolating blood components, e.g., serum, from the blood. is designed for use in human subjects.

[0144] multispecific antibodies The binding domain of the anti-BCMA antibodies described herein is intended to be contemplated as a binding domain for antibodies that express BCMA on their surface. As noted above, expression of BCMA can be indicative of cancer cells. More specific targeting to a particular subset of cells can be achieved by using bispecific molecules, e.g., BCM By generating an antibody or antibody fragment that binds to A and another target (such as CD3) This can be achieved by combining a first domain that binds to BCMA and a second domain that binds to another target antigen. This can be achieved by creating a molecule that contains two binding regions. The antigen-binding region binds to the specific target. The binding region may take any form that allows for target recognition, for example, the binding region may be a heavy chain variable domain. In, Fv (combination of heavy chain variable domain and light chain variable domain), fibronectin Type III domain-based binding domains (e.g., derived from fibronectin or Based on the consensus of type III domain from bronectin or from tenascin, or tenascin-derived type III domain consensus, e.g., Janss Centyrin molecules available from En Biotech, Inc., e.g., WO 02 / 06333; see also US Pat. Nos. 2010 / 051274 and 2010 / 093627 Thus, a BCMA antibody may be capable of binding to both BCMA and another antigen. Bispecific molecules are provided that contain two different antigen-binding regions.

[0145] Some multispecific antibodies described herein bind to BCMA and CD3, respectively. In a preferred embodiment, the antibody comprises two different antigen-binding regions that bind to BCMA and CD3. Multispecific antibodies (BCMA x CD3 multispecific antibodies) and their multispecific antigen binding In some embodiments, the BCMAxCD3 multispecific antibody comprises: A first helical fragment that pairs to form a first antigen-binding site that immunospecifically binds to BCMA. The antibody has a first hematopoietic chain (HC1) and a first light chain (LC1), and a second antigen-binding domain that immunospecifically binds to CD3. a second heavy chain (HC2) and a second light chain (LC2) that pair to form a binding site; In a preferred embodiment, the BCMAxCD3 multispecific antibody is immunospecific for CD3. A first heavy chain (HC1) and a first heavy chain (HC2) that pair to form a first antigen-binding site that binds to The BCMA-specific arm contains the light chain of the BCMA antibody (LC1) and the second arm contains the BCMA-specific arm that immunospecifically binds to BCMA. A second heavy chain (HC2) and a second light chain (LC2) pair to form an antigen-binding site of In some embodiments, the bispecific antibody comprises a CD3 specific arm comprising a CD3 specific antibody and a CD4 specific arm comprising a CD4 specific antibody. Bispecific antibodies of the invention include antibodies having a full-length antibody structure. "Full-length antibody" means an antibody having two full-length heavy antibody chains and two full-length light antibody chains. A full-length antibody heavy chain (HC) comprises a heavy chain variable domain and a constant domain, VH, CH1, CH2. A full-length antibody heavy chain (LC) comprises a light chain variable domain and a constant domain, V A full length antibody contains a C-terminal lysine residue at either one or both heavy chains. The term "Fab arm" or "half molecule" refers to a Fab arm that is specific for an antigen. In some embodiments, the term "antigen binding domain" refers to a pair of heavy and light chains that specifically bind to each other. One of these is non-antibody-based binding domains, e.g., based on fibronectin type III domains. A binding domain based on the ribozyme, such as centyrin.

[0146] The BCMA binding arms of the multispecific antibodies provided herein are Some exemplary such BCMA binding arms may be derived from either the BCMA-binding or heterologous antibodies. In one embodiment, the first antigen-binding region that binds to BCMA is an antibody clone listed in Table 1. Such BCMA binding fragments include heavy chain CDR1, CDR2, and CDR3 derived from the BCMA antibody. In some exemplary embodiments of the hybridoma, the first antigen-binding region that binds to BCMA is The heavy chain CDR1, CDR2, and CDR3 obtained from the antibody clone described in 1 and the light chain CDR1, CDR2, and CDR3 obtained from the antibody clone described in 1 are A portion of such a BCMA-binding arm comprises CDR1, CDR2, and CDR3. In an exemplary embodiment, the first antigen-binding region that binds BCMA is selected from clone BCMB6 9, BCMB117, BCMB123, BCMB128, BCMB129, BCMB17 6, or BCMB177. In some exemplary embodiments of the CMA-binding arm, a first antigen-binding region that binds BCMA The regions are clones BCMB69, BCMB117, BCMB123, BCMB128, BC The heavy chain CDR1, CDR2, and CDR3 of BCMB129, BCMB176, or BCMB177 R3 and light chain CDR1, CDR2, and CDR3. In some exemplary embodiments of the present invention, the first antigen-binding region that binds to BCMA is one of the antigen-binding domains listed in Table 1. Such BCMA-binding antibodies include heavy chain variable domains derived from the antibody clones described herein. In some exemplary embodiments of the antibody, the first antigen-binding region that binds to BCMA is selected from the group consisting of those listed in Table 1. The antibody clones described herein contain heavy and light chain variable domains. In some exemplary embodiments of the BCMA binding arm, such as The antigen-binding region is cloned as follows: BCMB69, BCMB117, BCMB123, and BCMB1 28, BCMB129, BCMB176, or BCMB177 In some exemplary embodiments of such a BCMA binding arm, the first The antigen-binding region of 1 was identified by clones BCMB69, BCMB117, BCMB123, and BCM The heavy chain variable domain of B128, BCMB129, BCMB176, or BCMB177 and a light chain variable domain.

[0147] Table 3 shows one heavy and light chain pair specific for BCMA and another heavy and light chain pair specific for CD3. Provide a list of BCMAxCD3 bispecific antibodies with chain pairs and list the specific antibody IDs. The following describes the antigen-specific antibody arms used to generate the described embodiments: do.

[0148] [Table 3]

[0149] In some embodiments of the bispecific antibody, the BCMA binding arm is cynomolgus BCMA , and preferably also to its extracellular domain.

[0150] In some embodiments, the BCMA binding arm of the multispecific antibody is IgG or a derivative thereof. , for example, IgG1, IgG2, IgG3, and IgG4 isotypes. In some embodiments, the A binding arm has an IgG4 isotype, and the BCMA binding arm contains S228P, L234A, and L235A substitutions in its Fc region.

[0151] In some embodiments of the bispecific antibody, the second antigen-binding arm binds to human CD3. In some preferred embodiments, the CD3 specificity of the BCMAxCD3 bispecific antibody is The antibody binds to and activates human primary T cells and / or cynomolgus monkey primary T cells. In some embodiments, the CD3 binding arm is derived from a CD3ε specific antibody. In some embodiments, the CD3 binding arm binds to an epitope at the N-terminus. In some embodiments, the doublet contacts an epitope comprising the six N-terminal amino acids of D3ε. The CD3-specific binding arm of the specific antibody is the mouse monoclonal antibody SP34, mouse I In some embodiments, the CD3 binding arm is derived from a gG3 / lambda isotype. The CD3 binding arm contains the CDRs of antibody SP34. -7 Below M, e.g. For example, 1×10 -7 M or less, 5×10 -8 M or less, 1×10 -8 M or less, 5×10 -9 M or less, or 1×10 -9 It can bind to CD3 with an affinity of less than or equal to M. The arms may be humanized versions of the mouse monoclonal antibody SP34. The high frequency affinity (HFA) is used to humanize anti-CD3 antibodies that provide a CD3-specific arm. In some embodiments of the bispecific antibody, the CD3 binding arm may be selected from the group consisting of In another embodiment of the bispecific antibody, the antibody comprises a heavy chain and a light chain pair selected from the group consisting of CD3-binding The arms consist of heavy chain CDR1, CDR2, and CDR3, and light chain CDR1 as shown in Table 2. For example, the bispecific antibodies described herein include The heavy and light chain CDR sequences of some embodiments of the CD3 binding arm have the amino acid sequences May include: Hc CDR1, SEQ ID NO:59; Hc CDR2, SEQ ID NO:60; Hc CDR3, SEQ ID NO:61 R3, SEQ ID NO:61; Lc CDR1, SEQ ID NO:62; Lc CDR2, SEQ ID NO:63; and Lc CDR3, SEQ ID NO:64.

[0152] In some embodiments, the CD3 binding arm is an IgG or a derivative thereof. In the embodiment, the CD3 binding arm is an IgG1, IgG2, IgG3, or IgG4. In some embodiments, the CD3 binding arm has an IgG4 isotype. The system harbors S228P, L234A, L235A, F405L, and R409K substitutions. In some embodiments, the antibody or antigen-binding fragment comprises a primary human In some embodiments, the antibody or antigen-binding fragment binds to CD3ε on mouse T cells. In some embodiments, the antibody or anti- The original binding fragment binds to CD3ε on primary human and cynomolgus monkey T cells. In this embodiment, the antibody or antigen-binding fragment activates primary human CD4+ T cells. In some embodiments, the antibody or antigen-binding fragment is derived from primary cynomolgus monkey CD4+ Activates T cells.

[0153] In some embodiments, the antibody clones BCMB69, BCMB117, BCMB123, B comprising a heavy chain of BCMB128, BCMB129, BCMB176, or BCMB177 A BCMAxCD3 bispecific antibody having a CMA-binding arm is provided. In this study, antibody clones BCMB69, BCMB117, BCMB123, and BCMB128 were used. BCMA, including the heavy and light chains of BCMB129, BCMB176, or BCMB177. In some embodiments, a BCMAxCD3 bispecific antibody having a binding arm is provided. , BCMA×CD3 with a CD3-binding arm comprising the heavy chain of antibody clone CD3B219 Bispecific antibodies are provided. In some embodiments, the heavy chain of antibody clone CD3B219 and a CD3-binding arm comprising a light chain. In some embodiments, the antibody clones BCMB69, BCMB117, BCMB123, B comprising a heavy chain of BCMB128, BCMB129, BCMB176, or BCMB177 It has a CMA-binding arm and a CD3-binding arm containing the heavy chain of the antibody clone CD3B219. In some embodiments, the antibody clone comprises a BCMAxCD3 bispecific antibody. BCMB69, BCMB117, BCMB123, BCMB128, BCMB129, and a BCMA-binding arm comprising the heavy and light chains of BCMB176 or BCMB177, and an antibody. and a CD3 binding arm comprising the heavy and light chains of clone CD3B219. CD3 bispecific antibodies are provided.

[0154] Exemplary BCMAxCD3 bispecific antibodies are provided in Table 9.

[0155] Bispecific antibodies in various formats have been described, see Chames and Baty(2009)Curr Opin Drug Disc Dev 12:276 was recently reviewed in.

[0156] In some embodiments, the bispecific antibodies of the invention include those described herein. Diabodies, crossbodies, or bispecific antibodies obtained by controlled Fab arm exchange It is a sexual antibody.

[0157] In some embodiments, the bispecific antibody comprises complementary CH3 domains that heterodimerize. IgG-like molecule with a recombinant IgG-like dual targeting molecule (two aspects of this molecule are Each of the antibodies contains at least two Fab fragments or portions of Fab fragments. IgG fusion molecules (full-length IgG antibodies with extra Fab fragments or Fab fragments), Fc fusion molecules (single chain Fv molecules or stabilized dimers), Fab fusion fragments, in which the abodies are fused to a heavy chain constant domain, an Fc region or a portion thereof; Fab fragments (different Fab fragments fused to each other), ScFv- and diabodies - Base and heavy chain antibodies (e.g. domain antibodies, nanobodies) (different single chain Fv molecules or Different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) fused to each other or to another protein or carrier molecule).

[0158] In some embodiments, the IgG-like molecule having a complementary CH3 domain molecule includes a Trio mab / Quadroma(Trion Pharma / Fresenius Biot ech), Knobs-into-Holes (Genentech), CrossMA bs (Roche) and electrostatically matched antibodies (Amgen), LUZ-Y (Gene ntech), Strand Exchange Engineering Domain Body (SEEDbody) (EMD Se rono), Biclonic (Merus), and DuoBody® (G enmab A / S).

[0159] In some embodiments, the recombinant IgG-like dual targeting molecule comprises a Dual Targeting ting(DT)-Ig (GSK / Domantis), Two-in-one An tibody (Genentech), Cross-linked Mabs (Karm anos Cancer Center), mAb2 (F-Star), and CovX- body (CovX / Pfizer) are examples.

[0160] In some embodiments, the IgG fusion molecule comprises a Dual Variable Domain. n(DVD)-Ig(Abbott), IgG-like Bispecific(In nClone / Eli Lilly), Ts2Ab (MedImmune / AZ), and BsAb (Zymogenetics), HERCULES (Biogen Idec) , as well as TvAb (Roche).

[0161] In some embodiments, Fc fusion molecules include ScFv / Fc Fusions (Acad emic Institution), SCORPION (Emergent BioS olutions / Trubion,Zymogenetics / BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics), and Dual(ScFv).sub.2-Fab(Nat ional Research Center for Antibody Medicine ine--China).

[0162] In some embodiments, the Fab fusion bispecific antibody includes F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab(Genentech ), Dock-and-Lock (DNL) (ImmunoMedics), Bival ent Bispecific (Biotecnol), and Fab-Fv (UCB-C For ScFv-, diabody-based and domain antibodies, is Bispecific T Cell Engager (BITE) (Microm et), Tandem Diabody(Tandab)(Affimed), Dual Affinity Retargeting Technology (DART)(M acroGenics), Single-chain Diabody (Academi c), TCR-like Antibodies (AIT, ReceptorLogic s), Human Serum Albumin ScFv Fusion(Merri mack), and COMBODY (Epigen Biotech), a dual targeting nanoparticle Dual targeting nanobody (Ablynx), a dual targeting heavy chain only Domain antibodies (dual targeting heavy chain only domain antibodies) are Not limited to these.

[0163] The full-length bispecific antibodies of the present invention can be prepared, for example, by the synthesis of Fab arms between two monospecific bivalent antibodies. exchange (or half molecule exchange) in a cell-free environment in vitro or co-expression The preferred method is to use two antibody half molecules with different specificities, either To achieve efficient heterodimer formation, we introduced substitutions at the heavy chain CH3 interface of each half molecule. The Fab arm exchange reaction may be carried out by disulfide bond isomerization and CH The result is the dissociation-association of the three domains. The resulting free cysteine ​​of one of the parent monospecific antibodies is converted to a second It forms an intra-heavy chain disulfide bond with the cysteine ​​residue of the parent monospecific antibody molecule, and at the same time, The CH3 domain of the Fab arm is released and reformed by dissociation-association. The domain may be engineered to favor heterodimerization over homodimerization. Each of the antibodies targets a different epitope, i.e., an epitope on BCMA and an epitope on CD3. It is a bispecific antibody that has two Fab arms or half molecules that bind to a specific antigen.

[0164] As used herein, "homodimerization" refers to a process in which two or more dimers have the same CH3 amino acid sequence. As used herein, "homodimer" refers to the interaction of two heavy chains. The term "antibody having two heavy chains with the CH3 amino acid sequence of

[0165] As used herein, "heterodimerization" refers to the process of forming heterodimers by associating non-identical CH3 amino acid sequences with each other. As used herein, "heterodimer" refers to the interaction of two heavy chains having a heterodimer structure. refers to an antibody having two heavy chains that have non-identical CH3 amino acid sequences.

[0166] "Knobs-in-holes" strategies (see, e.g., WO 2006 / 028936 (i) may be used to generate full-length bispecific antibodies. Selected amino acids that form the interface of the CH3 domain in promote heterodimer formation To achieve this, the small An amino acid having a side chain (hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen. The amino acids with large side chains (knobs) are then attached to the heavy chains of the antibody that specifically binds to the second antigen. After co-expression of the two antibodies, the heterodimer is formed by introducing a "hole" into the heavy chain. The knob and hole are formed as a result of preferential interaction between the heavy chain and the "knob". Exemplary CH3 substitution pairs forming W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T3 66W / T394S, F405W / T394S, and T366W / T366S_L368 A_Y407V (altered position in the first CH3 domain of the first heavy chain / second heavy chain (expressed as the position of the modification in the second CH3 domain of the chain).

[0167] Other strategies, such as combining positively charged residues on one CH3 surface and Heavy chain heterodimerization using electrostatic interactions by replacing negatively charged residues in The promotion of somatization is described in U.S. Patent Application Publication No. 2010 / 0015133 and U.S. Patent Application Publication No. 2010 / 0015213. No. 2010 / 028637, or U.S. Pat. It may also be used as described in U.S. Patent Application Publication No. 2011 / 0123532. In brief, heterodimerization is described in U.S. Patent Application Publication No. 2012 / 0149876 or U.S. Pat. As described in Patent Application Publication No. 2013 / 0195849, the following substitutions: L351Y_ F405A Y407V / T394W, T366I_K392M_T394W / F405 A_Y407V, T366L_K392M_T394W / F405A_Y407V, L3 51Y_Y407A / T366A_K409F, L351Y_Y407A / T366V K409F Y407A / T366A_K409F, or T350V_L351Y_F4 05A Y407V / T350V_T366L_K392L_T394W (first heavy chain Modification position in the first CH3 domain / modification in the second CH3 domain of the second heavy chain This may be facilitated by the use of a displacement (expressed as a displacement).

[0168] In addition to the methods described above, the bispecific antibodies of the invention can be prepared according to the methods described in WO 2011 / 1317 According to the method described in No. 46, two single-specific Asymmetric mutations were introduced into the CH3 region of the isomeric homodimeric antibody to isomerize the disulfide bonds. The bispecific heterodimer is generated from two parent monospecific homodimeric antibodies under reducing conditions that allow In this method, a first monospecific bivalent an antibody (e.g., an anti-BCMA antibody) and a second monospecific bivalent antibody (e.g., an anti-CD3 antibody) has specific substitutions in the CH3 domain that promote heterodimer stability These antibodies are engineered to have disulfide bond isomerization sites in the hinge region. The Fab fragments are incubated together under reducing conditions sufficient to induce Fab fragmentation. Bispecific antibodies are generated by group exchange. Incubation conditions are optimally Reducing conditions may be returned. An exemplary reducing agent that may be used is 2-mercaptoethyl Amine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE ), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cis tetrahydrofuran, and beta-mercaptoethanol, preferably 2-mercaptoethylamine , dithiothreitol, and tris(2-carboxyethyl)phosphine For example, at a temperature of at least 20° C., at least 25 m In the presence of M2-MEA or at least 0.5 mM dithiothreitol, pH 5-8, e.g., pH 7.0 or pH 7.4, and incubation for at least 90 minutes. Option may be used.

[0169] In addition to the described BCMA×CD3 polyspecific antibodies, Polynucleotide sequences capable of encoding the bispecific antibodies are also provided. Vectors containing the nucleotides provided herein are also provided, as are BCMAxCD3 vectors provided herein. Cells expressing the multispecific antibodies are also provided. These cells include mammalian cells (e.g., 293F cells, CHO cells, etc.). ), insect cells (e.g., Sf7 cells), yeast cells, plant cells, or bacterial cells (e.g., E The antibodies described may also be produced by hybridoma cells. can be done.

[0170] Therapeutic compositions and treatments using multispecific antibodies and multispecific antigen-binding fragments thereof How to place The BCMA bispecific antibodies discussed above, such as the BCMAxCD antibodies discussed above. 3 Bispecific antibodies are useful in therapy. In particular, BCMA bispecific antibodies are useful in treating cancer. Also referred to herein is a therapeutically effective amount of the compounds described herein. and a pharma- ceutical acceptable carrier. and a therapeutic composition for treating a hyperproliferative disorder in a mammal, comprising: In a preferred embodiment, the multispecific antibody is a BCMAxCD3 More preferably, the present invention provides a multispecific antibody or a multispecific antigen-binding fragment thereof. A BCMAxCD3 bispecific antibody or a BCMAxCD3 bispecific antibody thereof as described in the specification. In one embodiment, the pharmaceutical composition is a specific antigen-binding fragment of a BCMA-expressing It is used to treat cancer, and BCMA-expressing cancers include BCMA-positive cancers such as multiple myeloma (MM). These include BCMA-expressing B-cell cancers, as well as other cancers currently determined to express BCMA. Cancer, including but not limited to the specific cancers discussed above. Particular bispecific antibodies that may be used to treat hematological cancers include the antibody BCMB 69, BCMB117, BCMB123, BCMB128, BCMB129, BCMB1 76, BCMB177, or CD3B219. One example of a bispecific antibody useful for treating hematological cancers, including certain cancers of the BCMB The number is 72.

[0171] The pharmaceutical compositions provided herein comprise: a) an effective amount of a multispecific antibody or antibody fragment of the invention; b) a pharma- ceutically acceptable carrier, which may be inert or physiologically active. In a preferred embodiment, the multispecific antibody comprises a BCM as described herein. A×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof, or a BCMAxCD3 bispecific antibody or its BCMAxCD3 derivative as described herein. As used herein, "pharmaceutical" refers to a CD3 bispecific antigen-binding fragment. The term "acceptable carrier" refers to any and all solvents, dispersion media, and / or other media that are physiologically compatible. Suitable carriers, diluents, and / or media, coatings, antibacterial and antifungal agents, etc. Examples of excipients include water, saline, phosphate buffered saline, dextrose, glycerol, Examples of the solvent include one or more of ethanol, ethanol, etc., and any combination thereof. In many cases, isotonic agents, for example, sugars, polyalcohols, or sodium chloride, are used. In particular, relevant examples of suitable carriers include: (1) Darbe Phosphate-buffered saline, pH 7.4, 1 mg / mL to 25 mg / mL human serum albumin (2) 0.9% saline (0.9% w / v sodium chloride) with or without albumin; (NaCl), and (3) 5% (w / v) dextrose; antioxidants, e.g. For example, it may also contain tryptamine and a stabilizer, such as Tween 20®. good.

[0172] The compositions herein may also contain additional therapeutic agents, depending on the particular disorder being treated. Preferably, the multispecific antibody or antibody fragment and the auxiliary active compound are combined. The compounds may have complementary activities that do not adversely affect each other. The further therapeutic agent may be cytarabine, anthracycline, histamine dihydrochloride, or indomethacin. Turleukin 2. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic agent.

[0173] The compositions of the present invention may be in a variety of forms. These include, for example, liquid, semi-solid, and solid forms. The preferred form depends on the intended mode of administration and therapeutic application. Typically preferred compositions are in the form of an injectable or infusible solution. is parenteral (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous). In a preferred embodiment, The compositions of the present invention can be administered intravenously, either as a bolus or by continuous infusion over a period of time. In another preferred embodiment, the composition exerts both a local and a systemic therapeutic effect. In order to administer the drug intramuscularly, subcutaneously, intra-articularly, intra-synovially, intratumorally, peritumorally, intralesionally, or intralesionally, It is administered by the perilesional route.

[0174] Sterile compositions for parenteral administration include those containing an antibody, antibody fragment, or antibody conjugate of the invention. The ester is incorporated in a suitable solvent in the required amount and subsequently separated by microfiltration. The solvent or medium may be water, physiological saline, phosphate buffered saline, etc. Saline, dextrose, glycerol, ethanol, etc., and combinations thereof In many cases, isotonic agents, for example, sugars, polyalcohols, or It may be preferable to include sodium chloride in the composition. These compositions may also contain the adjuvant In particular, the composition may contain wetting agents, isotonicity adjusting agents, emulsifying agents, dispersing agents, and stabilizing agents. The sterile composition to be administered may also be dissolved in sterile water or any other injectable sterile medium at the time of use. Alternatively, it may be prepared in the form of a sterile solid composition.

[0175] The multispecific antibodies or antibody fragments may also be administered orally. The composition may be in the form of tablets, pills, powders (gelatin capsules, sachets), or granules. In these compositions, the active ingredient according to the invention may be one or two The above inert diluents, for example, starch, cellulose, sucrose, lactose, or The compositions are mixed with silica under a stream of argon. For example, one or more lubricants, such as magnesium stearate or talc, Colours, coatings (sugar-coated tablets) or glazes may also be included.

[0176] For liquid compositions for oral administration, an inert diluent such as water, ethanol, glycerol, Medicinal acceptable solutions, suspensions, including ethanol, vegetable oil, or paraffin oil; Emulsions, syrups, and elixirs may also be used. These compositions contain no more than a diluent. External substances, such as wetting agents, sweeteners, thickeners, flavoring agents, or stabilizing products, may also be included.

[0177] The dosage depends on the desired effect, the duration of the treatment, and the route of administration used. Generally, the daily oral dose for adults is 5 mg to 1000 mg. The unit dose is The dosage ranges from 1 mg to 250 mg of the substance. Generally, a physician will determine the appropriate dosage for the patient. This will depend on the subject's age, weight, and any other factors specific to the subject.

[0178] Also, as used herein, the term "BCMA+" refers to a BCMA-specific antibody that binds to BCMA and recruits T cells to produce BCMA+ Multispecific antibodies capable of killing cells (i.e., redirecting T cells) are By administering this to a patient in need thereof, a method for killing BCMA+ cells is provided. Any of the multispecific antibodies or antibody fragments of the invention may be used therapeutically. For example, in one embodiment, the BCMAxCD3 multispecific antibody BCMB72 is It may be used therapeutically to treat cancer in a subject.

[0179] In a preferred embodiment, the multispecific antibody or antibody fragment of the invention is In a more preferred embodiment, the method of the present invention is used to treat a hyperproliferative disorder in a mammal. One of the pharmaceutical compositions disclosed above containing a multispecific antibody or antibody fragment. In one embodiment, the method is used to treat a hyperproliferative disorder in a mammal. The disorder is cancer. In particular, the cancer is a BCMA-expressing cancer, and These include BCMA-expressing B-cell cancers such as multiple myeloma (MM) and These include, but are not limited to, other cancers currently identified as having In a preferred embodiment, the multispecific antibody is a BCMAxCD antibody as described herein. 3. A multispecific antibody or a multispecific antigen-binding fragment thereof, more preferably The BCMAxCD3 bispecific antibody or its BCMAxCD3 derivatives as described herein. A polyspecific antigen-binding fragment.

[0180] Therefore, the pharmaceutical composition of the present invention is useful for treating or preventing various cancers, and Examples of cancers include, but are not limited to, BCMA-expressing cancers. Cancers include BCMA-expressing B-cell cancers such as acute multiple myeloma (MM) and BCMA These include, but are not limited to, other cancers currently identified as expressing cannot be used).

[0181] Also provided herein is a method for inhibiting the growth of a selected cell population, comprising: In one embodiment, BCMA-expressing target cells or tissues containing such target cells are treated with an effective amount of a multimeric antibody of the present invention. Bispecific antibodies or antibody fragments, alone or in combination with other cytotoxic or therapeutic agents in combination with, or in the presence of peripheral blood mononuclear cells (PBMCs). Further provided is a method comprising: A BCMA×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein. fragment, more preferably a BCMAxCD3 bilayer as described herein. The antibody is a BCMAxCD3 bispecific antibody or a BCMAxCD3 bispecific antigen-binding fragment thereof. In embodiments, the additional therapeutic agent is cytarabine, anthracycline, histamine dihydrochloride or interleukin 2. In a preferred embodiment, the further therapeutic agent is a chemotherapeutic agent Methods for inhibiting the growth of selected cell populations include in vitro, in vivo, or may be performed ex vivo.

[0182] Examples of in vitro uses include transplantation into the same patient to kill diseased or malignant cells. Prior processing of autologous bone marrow to kill competent T cells and prevent graft-versus-host disease (GVHD) Treatment of bone marrow prior to transplantation to kill all cells except for the desired variant that does not express the target antigen Examples of such methods include treatment of cell cultures to destroy or kill mutants expressing unwanted antigens. Non-clinical in vitro conditions of use are readily determined by one of skill in the art.

[0183] An example of a clinical ex vivo use is the extraction of tumor cells from bone marrow prior to autologous transplantation in cancer treatment. The treatment can be performed as follows: Bone marrow is extracted from the patient or other individual. and then incubated in serum-containing medium supplemented with a cytotoxic agent of the present invention. The concentration is in the range of about 10 μM to 1 μM at about 37° C. for about 30 minutes to about 48 hours. The exact conditions of concentration and time of incubation, i.e., dosage, can be determined by one skilled in the art. After incubation, the bone marrow cells are washed with serum-containing medium and incubated with a known The cells are then returned to the patient by iv infusion according to the method. Collection of bone marrow and reconstitution of processed cells in patients undergoing ablative chemotherapy or total body irradiation Between injections and times, the processed bone marrow cells are stored in liquid nitrogen using standard medical equipment. It is stored frozen.

[0184] For clinical in vivo use, a therapeutically effective amount of a multispecific antibody or antigen binding The fragment is administered to a subject in need thereof. For example, a BCMAxCD3 multispecific The present invention relates to a method for the treatment of B12-related myelopathy in a subject in need thereof. In some embodiments, the BCMA-expressing cancer may be useful for treating multiple myeloma. In a preferred embodiment, the multispecific antibody is a B-cell cancer, such as myeloma (MM). a BCMAxCD3 multispecific antibody or a multispecific antigen thereof as described herein binding fragment, more preferably BCMAxCD as described herein. 3. A bispecific antibody or a BCMA×CD3 bispecific antigen-binding fragment thereof. In some embodiments, the subject is a mammal, preferably a human. The polyspecific antibody or multispecific antigen-binding fragment is administered in a sterile tested solution. It will be possible.

[0185] The dosage regimen in the above methods of treatment and use is determined in accordance with the optimal desired response (e.g., a therapeutic response). For example, a single bolus may be administered, or several separate doses may be administered. Doses may be administered over time or may be adjusted as indicated by the exigencies of the therapeutic situation. Parenteral compositions have the advantages of ease of administration and uniformity of dosage. It may also be formulated into an administration form for sex.

[0186] Efficient administration and dosing regimens of multispecific antibodies and fragments are dependent on the disease or condition being treated. It depends on the condition and may be determined by one of ordinary skill in the art. An exemplary, non-limiting range is about 0.001 to 10 mg / kg, for example, about 0.001 to 5 mg / kg, for example, about 0.001 to 2 mg / kg, for example, about 0.001 to 1 mg / kg, for example, about 0.001, about 0.01, about 0.1, about 1, or about 10 mg / kg. do.

[0187] A physician or veterinarian having ordinary skill in the art will be able to determine the required pharmaceutical composition. The effective amount can be readily determined and prescribed, for example by a physician or veterinarian, to determine the desired treatment. Multispecific peptides utilized in pharmaceutical compositions at levels less than those required to achieve a therapeutic effect. Therapeutic agents should be started with a dose of the active antibody or fragment and gradually increased until the desired effect is achieved. In general, a suitable daily dose of the bispecific antibody of the invention will be Administration will be that amount of compound that is the lowest dose effective to produce the desired effect. Orally, for example, intravenously, intramuscularly, or subcutaneously. or fragments in mg / m 2 may be administered by injection in weekly doses calculated at Such doses can be determined, for example, according to the following: dose (mg / kg) x 70:1.8, as described above. Such administration can be based on the mg / kg dose provided in, for example, 1 to 8 mg / kg. The administration may be repeated once, for example, 3 to 5 times. The administration may be repeated every 2 to 24 hours, for example, 2 to 12 hours. In one embodiment, the multispecific The antibody or fragment may be administered for an extended period of time, e.g., 24 hours, to reduce toxic side effects. The drug may be administered by slow continuous infusion over a period of time.

[0188] In one embodiment, the multispecific antibody or fragment is given once a week. In the case of a combination of drugs, the drug may be administered in up to eight, e.g., four to six, weekly doses calculated as fixed doses. Such a schedule may be repeated once or twice as needed, for example after 6 or 12 months. Such a fixed dose may be, for example, as provided above. The dose can be calculated based on the mg / kg dose, assuming a body weight of 70 kg. For example, a biological sample may be collected and the BCMA antigen-binding domain of a multispecific antibody of the invention may be detected. The administration of bispecific antibodies of the invention can be achieved by using targeting anti-idiotypic antibodies. It may be determined or adjusted by measuring the amount in the blood at the time of administration.

[0189] In one embodiment, the multispecific antibody or fragment is administered as a maintenance treatment, e.g., for 6 months. During the above period, the drug may be administered once a week, etc.

[0190] The multispecific antibodies or fragments may also reduce the risk of cancer progression and may be useful in treating cancer progression. delay the onset of events in the course of cancer and / or reduce the risk of recurrence when cancer goes into remission It may also be administered prophylactically to reduce

[0191] The multispecific antibodies and fragments thereof described herein may also be used in combination therapy. It may also be administered in combination with other therapeutic agents relevant to the disease or condition being treated. Thus, in one embodiment, the antibody-containing drug may be one or two In some embodiments, the compound is intended for combination with one or more other therapeutic agents, e.g., chemotherapeutic agents. In some embodiments, the other therapeutic agent is cytarabine, anthracycline, histamine dihydrochloride, or The combination may be administered simultaneously, in any order, and / or in any combination with interleukin 2. For simultaneous administration, the agents may be administered in one composition or in a single composition, as appropriate. They may also be administered as separate compositions.

[0192] In one embodiment, the method comprises treating a disorder involving cells expressing BCMA in a subject. The method comprises administering to a subject in need thereof a therapeutically effective amount of a compound as described herein. The multispecific antibody or fragment thereof, e.g., a BCMAxCD3 bispecific antibody, In one embodiment, the method comprises administering to a subject a cancer The present invention relates to a method for treating or preventing an infection comprising administering to a subject in need thereof a therapeutically effective amount of and the multispecific antibodies or fragments described herein, e.g., BCMAxCD3 antibodies. The method includes administering to the body and administering radiation therapy, which may include: It may also include the associated administration of radiation or a radiopharmaceutical to the patient provided. The source may be either external or internal to the patient being treated (radiation treatments may be performed, e.g., For example, this may be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT). Radioactive elements that may be used to carry out such methods include, for example, radium, cesium, Mu-137, Iridium-192, Americium-241, Gold-198, Cobalt-57 , copper-67, technetium-99, iodine-123, iodine-131, and indium- 111 is an example.

[0193] kit Also provided herein are, for example, the multispecific antibodies described or their antigen-binding fragments. and instructions for using the antibodies or fragments to kill specific cell types. Also provided is a kit comprising the multispecific antibody and a description thereof. The present invention relates to a BCMA×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described in the above document. and more preferably a BCMAxCD3 bispecific fragment as described herein. The antibody is a BCMAxCD3 bispecific antigen-binding fragment thereof. The present invention relates to a method for producing a multispecific antibody or an antigen-binding fragment thereof, comprising administering the antibody or antigen-binding fragment to a subject in vitro, in vivo, or ex vivo. It may also include instructions for use in vivo.

[0194] Typically, the kit comprises a compartment containing a multispecific antibody or an antigen-binding fragment thereof. The multispecific antibodies or antigen-binding fragments thereof may be in a lyophilized or liquid state. The kit may be in the form of a paper bag, or in any other form amendable for inclusion in a kit. It may contain additional elements required to carry out the methods described in the instructions. The components may be used, for example, in sterile solutions for reconstituting lyophilized powders, multiple doses prior to administration to a patient, Additional agents for combination with specific antibodies or antigen-binding fragments thereof, and multiple specific It is a tool to aid in the administration of heterologous antibodies or antigen-binding fragments thereof to patients.

[0195] Diagnostic Use The multispecific antibodies and fragments described herein may also be used for diagnostic purposes. Thus, the present invention also includes multispecific antibodies or fragments as defined herein. Diagnostic compositions and uses thereof are also provided. In a preferred embodiment, the multispecific antibody comprises: A BCMAxCD3 multispecific antibody or its multispecific antigen binding agent as described herein. and more preferably a BCMAxCD3 combination fragment as described herein. A bispecific antibody or a BCMAxCD3 bispecific antigen-binding fragment thereof. In embodiments, the present invention provides a bispecific BCMAxCD3 antibody and an antibody against BCMA. and one or more reagents for detecting the binding of The present invention provides a kit for detecting the presence of a fluorescent tag, an enzyme tag, or other detectable Reagents may also include secondary or tertiary antibodies or reagents for enzymatic reactions. In this case, the enzymatic reaction produces a product that can be visualized. For example, the multispecific antibodies or antigen-binding fragments thereof described herein may be radiolabeled. Identification, fluorescent label, epitope tag, biotin, chromophore label, ECL label, enzyme, ruthenium , 111 In-DOTA, 111 In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase, and beta-galactosidase, or polyhistidine, or similar such tags known in the art. It may be recognized.

[0196] EXEMPLARY EMBODIMENTS OF THE DISCLOSED SUBJECT MATTER In order to provide a better and more complete description of the subject matter herein, this section provides A list of exemplary embodiments is provided.

[0197] Enumeration of embodiments: 1. A recombinant antibody, or antigen-binding fragment thereof, that immunospecifically binds to BCMA. Thus, an antibody has a heavy chain and a light chain, the heavy chain being a. Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:4, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:6 CDR3, b. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:4, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:6; c. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:7, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:6; d. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:4, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 19; e. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:4, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:6; f. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 13, the amino acid sequence of SEQ ID NO: 5 and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 19; g. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:13, the amino acid sequence of SEQ ID NO:8 and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 19. , a recombinant antibody, or an antigen-binding fragment thereof. 2. The antibody comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 25 a light chain CDR2 having the amino acid sequence of SEQ ID NO:26; The antibody, or antigen-binding fragment thereof, of embodiment 1, further comprising: 3. The heavy chain of the antibody of (a) comprises the amino acid sequence of SEQ ID NO: 27, and the heavy chain of the antibody of (b) (f) comprises the amino acid sequence of SEQ ID NO: 57, and the heavy chain of the antibody of (f) comprises the amino acid sequence of SEQ ID NO: 34. the heavy chain of the antibody (k) comprises the amino acid sequence of SEQ ID NO: 39; and the antibody (l) comprises the heavy chain of the antibody (k) comprises the amino acid sequence of SEQ ID NO: 39. The heavy chain of the antibody of (m) comprises the amino acid sequence of SEQ ID NO: 40, and the heavy chain of the antibody of (m) comprises the amino acid sequence of SEQ ID NO: 58. or the heavy chain of the antibody (n) comprises the amino acid sequence of SEQ ID NO: 43. 2. The antibody or antigen-binding fragment of embodiment 1. 4. The antibody of embodiment 2 or 3, wherein the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 28. or antigen-binding fragment. 5. The antibody or antigen-binding fragment thereof binds to the extracellular domain of human BCMA 5. The antibody or antigen-binding fragment according to any one of embodiments 1 to 4. 6. The antibody or antigen-binding fragment is a human antibody or antigen-binding fragment. The antibody or antigen-binding fragment according to any one of embodiments 1 to 5. 7. The antigen-binding fragment is a Fab fragment, a Fab2 fragment, or a single The antigen-binding fragment of any one of embodiments 1 to 6, which is a chain antibody. 8. The antibody or antigen-binding fragment thereof inhibits the interaction of BCMA and APRIL. The antibody or antigen-binding fragment according to any one of embodiments 1 to 7. 9. The antibody or antigen-binding fragment binds to BCMA and IC of approximately 5.9 nM for APRIL interaction 50 9. The antibody of embodiment 8, which exhibits or an antigen-binding fragment. 10. Any of embodiments 1 to 9, wherein the antibody or antigen-binding fragment thereof is an IgG. The antibody or antigen-binding fragment described in claim 1. 11. The antibody or antibody according to any one of embodiments 1 to 10, which is an IgG4 isotype. is the antigen-binding fragment. 12. IgG4 contains an S228P substitution, an L234A substitution, and an L23 The antibody of embodiment 11, having a 5A substitution. 13. The antibody or antigen-binding fragment thereof immunospecifically binds to human BCMA and inhibits inflammatory bowel disease. An antibody according to any one of embodiments 1 to 12, which cross-reacts with chinese monkey BCMA; Antigen-binding fragment. 14. The antibody or antigen-binding fragment thereof binds to BCMA on the surface of human myeloma cells. The antibody or antigen-binding fragment of any one of embodiments 1 to 13, 15. The antibody or antigen-binding fragment thereof binds to BCM on the surface of human multiple myeloma cells. The antibody or antigen-binding fragment according to any one of embodiments 1 to 14, which binds to A. . 16. Expressing the antibody or antigen-binding fragment according to any one of embodiments 1 to 15. Recombinant cells. 17. The cell of embodiment 16, wherein the cell is a hybridoma. 18. The cell of embodiment 16, wherein the antibody is recombinantly produced. 19. Recombinant BCMAxCD3 bispecific antibody or BCMAxCD3 bispecific binding site A combination fragment, a) the first heavy chain (HC1); b) the second heavy chain (HC2); c) the first light chain (LC1); and d) a second light chain (LC2); HC1 binds to LC1, and HC2 binds to LC2, and they bind immunospecifically to CD3. To form a first antigen-binding site, HC1 is composed of SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:70. LC1 includes sequence number 61, and LC2 includes sequence number 62, sequence number 63, and sequence number 64; To form a second antigen-binding site that immunospecifically binds to BCMA, HC2 has the sequence SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and LC2 includes SEQ ID NO:24, SEQ ID NO:25, and and SEQ ID NO: 26. 3. Bispecific Binding Fragments. 20. HC1 comprising SEQ ID NO: 55, LC1 comprising SEQ ID NO: 56, and LC2 comprising SEQ ID NO: 65 and LC2 comprising a) SEQ ID NO: 66 or b) SEQ ID NO: 76. 20. A recombinant BCMAxCD3 bispecific antibody or fragment thereof according to 19. 21. The antibody or bispecific binding fragment according to embodiment 20, wherein the antibody or bispecific binding fragment is an IgG. CMAxCD3 bispecific antibody or bispecific binding fragment. 22. The antibody or bispecific binding fragment is of the IgG4 isotype, an embodiment 22. The BCMAxCD3 bispecific antibody or bispecific antibody according to any one of claims 19, 20 or 21. Specific binding fragments. 23. The antibody or bispecific binding fragment has a specific binding activity as measured by surface plasmon resonance. and immunospecifically binds to human BCMA with an affinity of at least 0.22 nM. 23. A BCMAxCD3 bispecific antibody or bispecific binding fragment according to 19 to 22. 24. The antibody or bispecific binding fragment thereof binds to BCM on the surface of human myeloma cells. 24. The BCMA×CD3 bispecific antibody or bispecific Heteromer bond fragments. 25. The antibody or bispecific binding fragment thereof is a marker for the surface of human multiple myeloma cells. A BCMAxCD3 bispecific antibody according to any one of embodiments 19 to 24, which binds to BCMA, or Bispecific binding fragments. 26. The antibody or bispecific binding fragment has an EC 50 In 26. The BCMA×CD3 antibody of any one of embodiments 19 to 25, which induces human T cell activation in vitro. Bispecific antibodies or bispecific binding fragments. 27. The antibody or bispecific binding fragment has an EC 50 In The method according to any one of embodiments 19 to 26, which induces T cell-dependent cytotoxicity of BCMA-expressing cells in vitro. The BCMAxCD3 bispecific antibody or bispecific binding fragment thereof. 28. The antibody or bispecific binding fragment is not a BCMA agonist, BCMA×CD3 bispecific antibody or bispecific binding fragment according to any one of aspects 19 to 27. . 29. The antibody or bispecific binding fragment inhibits NF-κB activity at a concentration of less than 10 nM. 29. The BCMA×CD3 bispecific antibody or bispecific antibody according to any one of embodiments 19 to 28, Multispecific binding fragments. 30. The antibody or bispecific binding fragment according to any one of embodiments 19 to 29. A recombinant cell expressing a 31. The cell of embodiment 30, wherein the cell is a hybridoma. 32. A method for treating a subject having cancer, comprising any one of embodiments 19 to 29. Treatment of the BCMAxCD3 bispecific antibody or bispecific binding fragment according to any one of claims 1 to 4. and administering to a subject in need thereof a therapeutically effective amount for a period of time sufficient to treat the cancer. A method comprising: 33. A method for inhibiting the growth or proliferation of cancer cells, comprising the steps of any one of embodiments 19 to 29. BCMAxCD3 bispecific antibody or bispecific binding fragment according to any one of claims 1 to 4 to inhibit the growth or proliferation of cancer cells. 34. A method for redirecting T cells to BCMA-expressing cancer cells, comprising the steps of: 30. The BCMA×CD3 bispecific antibody or bispecific binding fragment according to any one of claims 29 to 30. and redirecting T cells to the cancer. Law. 35. The method of embodiment 32, 33, or 34, wherein the cancer is a blood cancer. 36. The method of embodiment 35, wherein the hematological cancer is a BCMA-expressing B-cell cancer. 37. The method of embodiment 36, wherein the BCMA-expressing B-cell cancer is multiple myeloma. 38. The method of embodiment 32, further comprising administering a second therapeutic agent. 39. The method of embodiment 38, wherein the second therapeutic agent is a chemotherapeutic agent or a targeted anti-cancer therapeutic agent. Method of posting. 40. The chemotherapy agent is cytarabine, anthracycline, histamine dihydrochloride, or isotonic acid. 40. The method of embodiment 39, wherein the antibody is interleukin 2. 41. The BCMAxCD3 bispecific antibody according to any one of embodiments 19 to 29 or A pharmaceutical composition comprising a bispecific binding fragment and a pharma- ceutically acceptable carrier. 42. By culturing the cells according to any one of embodiments 30 to 31, The BCMA×CD3 bispecific antibody or bispecific binding molecule according to any one of aspects 19 to 29. A method for generating a combined fragment. 43. The BCMA×CD3 bispecific antibody according to any one of embodiments 19 to 29 or a single sequence encoding the bispecific binding fragment HC1, HC2, LC1, or LC2; Isolated synthetic polynucleotides. 44. A BCMAxCD3 bispecific antibody as defined in any one of embodiments 19 to 29. Heterologous antibodies or bispecific binding fragments, and / or polyclonal antibodies as defined in claim 44. A kit comprising a oligonucleotide and packaging therefor. EXAMPLES

[0198] The following examples supplement the above disclosure and provide a better understanding of the subject matter described herein. These examples are provided for illustrative purposes only and should not be construed as limiting the subject matter described. The examples and embodiments described herein are for illustrative purposes only and are not intended to be limiting. In view of the above, various modifications and variations will be apparent to those skilled in the art. The examples and embodiments described are within the true scope of the present invention, and are not intended to be limiting unless otherwise specified. It is understood that this can be done without

[0199] Example 1: Materials BCMA ECD molecule Recombinant human (h)BCMA-Fc fusion protein (catalog #193-BC-050, hBCMA (corresponding to amino acids 1 to 54 of SEQ ID NO: 1) and recombinant mouse (m)BCMA MA-Fc fusion protein (catalog #593-BC-050, mBCMA (SEQ ID NO:2 ) was obtained from R&D Systems. The macaque BCMA protein was produced using gene synthesis technology (U.S. Patent No. 6,670,127, All proteins were prepared from cDNA obtained from the human IgG1 gene (No. 6,521,427). The resulting mixture was then tested for endotoxin and biotinylated for phage panning studies. These materials were also used for binding and affinity measurements.

[0200] Soluble human BCMA was purchased from AB Biosciences (catalog no. P011Xp, The material was obtained from the supplier (Port No. 033-013) and used for characteristic testing.

[0201] APRIL, BAFF, BAFF-R, and TACI molecules Soluble hAPRIL (Cat. #DY884), hBAFF (Cat. #2149-B F), hBAFF-R (catalog #1162-BR, amino acids 7 to 71 of hBAFF-R) hTAC1 (corresponding to amino acids 2-166 of TACI) and hTAC2 (corresponding to amino acids 2-166 of TACI) Systems. BAFF-R and TAC1 were biotinylated for SPR studies. It has become.

[0202] Generation of BCMA cell lines Vectors providing human BCMA (Figure 1A) and cynomolgus BCMA (Figure 1B) were HEK293 expi cells were transiently transfected using standard methods. Transfected 293F adherent cells were selected for stable plasmid integration. Single cells were then sorted and BCMA surface receptor expression was assayed using anti-human BCMA-PE Quantification was performed by FACS using labeled antibodies (R&D Systems FAB193P). Ta.

[0203] Example 2: Isolation of human BCMA monoclonal antibody expressing hybridomas Human immunoglobulin transgenic rat line (OmniRat®, OM Human BCMA monoclonal antibody-expressing hybridoma cells were cultured using a ELISA kit (T, Inc.). OmniRat® binds to the entire human IgL locus (Jκ-Cκ binding site). The chimeric human / rat IgH Rat C H 22 human V in their natural configuration bound to the locus H Segment, all human D and J H (For example, Osborn, et al. (2013) J Immunol 190(4):1481-1490). Rats showed reduced expression of rat IgM or κ and expressed the transduced human IgM in response to immunization. Heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human Ig G monoclonal antibody; preparation and use of OmniRat®; and The genomic modifications produced by such rats are described in WO 14 / 093908 (Bru (Gehmann et al.).

[0204] When immunized with recombinant human BCMA (rhBCMA), this transgenic The mice produce human IgG antibodies specific for human BCMA.

[0205] The immunization schedule was as follows: six rats were immunized with hBCMA-Fc fusion. After 21 days of immunoregulation, spleens and lymph nodes were harvested from the immunized rats. A total of four hybridoma libraries were generated using the method. The libraries were titrated and subjected to EL mAbs were identified that showed binding to biotinylated hBCMA by ISA. The antibodies were captured on MSD Streptavidin plates. Hybridomas that showed specific binding to human BCMA and cynomolgus monkey BCMA were then isolated. The supernatant was sequenced, cloned, expressed and converted to both human IgG1 and IgG4. .

[0206] Example 3: Purification of BCMA antibodies BCMA antibodies in the clarified culture supernatant were analyzed using MabSelect SuRe Protein The antibody was captured by in A resin and eluted with 100 mM sodium acetate (pH 3.5). The fractions containing were pooled and immediately neutralized with 2.5 M Tris HCl (pH 7.2). The buffer was then exchanged into 1x D-PBS or other desired buffer if specified. Protein concentration was determined by measuring OD280 with a NanoDrop spectrophotometer and the absorbance The purity and homogeneity of the antibody were determined by SDS-PAGE and SE-HPLC. If the monomer content was less than 95% by SE-HPLC, the Superde A SEC polishing step using x 200 was performed.

[0207] Example 4: Properties of BCMA antibody cell binding to BCMA Engineered BCMA-expressing cells and cancer cell lines U2392, EJM, MMIR, and U26 6. Binding of BCMA antibodies to OPM2 and RPMI-18226 was examined using MSD ( The antibodies were evaluated using the esoscale cell binding assay and flow cytometry. The goal of the screening assay is to identify antibodies that bind to cells expressing BCMA. and to identify cross-reactivity with cells expressing cynomolgus monkey BCMA. there were.

[0208] For the MSD cell binding assay, cells were fixed and BCMA antibody samples were up- Briefly, expression supernatants of purified BCMA antibodies were normalized to 10 μg / mL. 5000 cells per well were plated in a 384-well plate (MA6000, cat.L The cells were seeded onto 21XB, MSD (21XB, MSD) and allowed to attach for 2 hours. The cells were then resuspended in 20% F in PBS. The plate was blocked with PBS (Gibco) for 15 minutes. Then, the antibody supernatant was added and incubated at RT for 1 The cells were washed three times with PBS and then incubated with a ruthenium-labeled secondary antibody (Ja ckson Immuno Research) was added at 1 μg / mL and incubated at room temperature. Incubated for 1 hour. Then a further washing step was applied, followed by 3 washes per well. Add 5 μL of MSD Read Buffer T (without detergent) and mix for 30 min. The plates were then incubated for 1 min. Data were normalized to controls and analyzed using GraphPad Primer. Graphs were generated using sm Version 5. Positive binders were plotted against the background. Hits were determined to have signals 3-fold higher than the control. The assay was For each iteration, the top binder was selected for further development.

[0209] For flow cytometry, cells were incubated with a viability dye and incubated at 100 ,000 cells were added to a U-bottom plate and centrifuged to pellet the cells. After the incubation period, the cells were pelleted and incubated for 1 h at 37 °C. The cells were then incubated with 100 mL of AlexaFluor 647-labeled secondary antibody specific for the antibody. The cells were pelleted and washed seven times. The cells were resuspended in buffer and analyzed using a FACS CantoII. FSC-A vs. SSC-A for singlet and viability dyes, SSC-A vs. SSC The geoMFI values ​​of the live cell population were graphed and, if possible, That is, if the curve is sufficiently S-shaped, EC 50 was used to calculate the value.

[0210] Inhibition of APRIL ligand binding The BCMA antibody panel was screened in an APRIL binding competition ELISA. Soluble human April was purchased from R&D systems (catalog #DY884). The ability of anti-BCMA antibodies to inhibit binding of APRIL to immobilized BCMA was assessed.

[0211] Briefly, 96-well clear Maxisorb plates were filled with 0.5 μg of PBS-prepared 100 μL of 100 / mL BCMA-ECD and incubated overnight at room temperature. Wash the plate with ELISA wash buffer containing 0.05% Tween-20 in PBS. (R&D Systems, Cat #WA126) three times, followed by 1% in PBS Reagent Diluent containing BSA5 (R&D Systems, catalog Competitive binding was inhibited with 300 μL / well of BCMA antibody (#DY995). The mixture was added to the plate in a volume of 100 μl and incubated for 30 min before adding APRIL. After 30 min, 1 ng of APRIL was added per well and the plate was incubated overnight at 4°C. Unbound APRIL was washed off with ELISA wash buffer, and bound biotin was incubated. Tylated APRIL was detected using SA-HRP conjugate at an optical density of 450 nM.

[0212] Example 5: Hit evaluation and selection After characterization testing is completed, the antibody obtained from the M2 hybridoma has the following characteristics: (named BCMB69). Binds to recombinant human BCMA Binds to recombinant cynomolgus BCMA - Weak binding to mouse BCMA HEK-expressed human BCMA and HEK-expressed human BCMA as measured by flow cytometry Binds to both cynomolgus BCMA BCMA (U2392, EJM, MMIR, U266, OPM2, and RPMI-1 Binds to human cancer lines expressing ·I C 50 = 5.9nM inhibits APRIL binding As a result, to generate the BCMAxCD3 bispecific antibody, BCMB69 (Table 4 and Table 5) were expressed and purified.

[0213] [Table 4]

[0214] [Table 5]

[0215] Example 6: Crystal structure of anti-BCMA Fab Characterize antibody / antigen interactions in atomic detail to better understand the mechanism of antibody action and One anti-BCMA antibody (BCMB69) to support any necessary antibody engineering trials The crystal structure of was determined in the free Fab form as well as when bound to human BCMA.

[0216] material His-tagged BCMA Fab (SEQ ID NOs: 75 and 76, hereafter simply BCMB6 9 Fab) was expressed in HEK293 cells and analyzed by affinity and size exclusion chromatography. Fab was purified using a PBS. Fab was diluted in 130 mM NaCl, 20 mM MES (pH 7 .4) obtained during.

[0217] Human BCMA extracellular domain with a C-terminal His tag (residues 5 to 51 of SEQ ID NO: 1, hereinafter The BCMA gene (hereafter referred to simply as BCMA) was expressed using a baculovirus system and subjected to affinity and size exclusion. The protein was purified by chromatography in 50 mM NaCl, 20 mM T ris (pH 8).

[0218] Crystallization BCMA / BCMB69 Fab complex BCMA and BCMB69 Fab were incubated at 4°C for approximately 3.8:1 molar ratio (BCMA in excess). The mixture was mixed for 16 hours, while the buffer was exchanged into 20 mM Hepes (pH 7.5). The Fab / antigen complex was then prepared by the following procedure. Then, the Fab / antigen complex was diluted with 20 mM Hepes (pH 7.5) The complex was purified by Mono S 5 / 50 column using a gradient of 51 to 63 mM NaCl in Crystals suitable for X-ray diffraction were isolated using microseeding. Using the sitting drop vapor diffusion method at 20 °C, 25% PEG 3 kDa, 0 Obtained from 0.2M MgCl2, 0.1M Mes (pH 6.5).

[0219] BCMB69 Fab The BCMB69 Fab was concentrated to 9 mg / mL without further purification. The crystals were dissolved in 2M (NH4)2SO4 at 20°C using the sitting drop vapor diffusion method. 4, obtained from 5% MPD, 0.1 M Mes (pH 6.5).

[0220] X-ray data collection and structure determination For X-ray data collection, the crystals were incubated in a 30-mL flask with the corresponding mother liquor supplemented with 20% glycerol. The BCMA was immersed in lyoprotectant solution for a few seconds and then flash frozen in liquid nitrogen. X-ray diffraction data for the / BCMB69 complex was obtained from the Canadian Light Source. Rayonix 300HS CCD at beamline CMCF-08ID of e(CLS) The X-ray data for free BCMB69 Fab was collected using the Argonne detector. e National Laboratory's Advanced Photon So Dectris Pilatus 6M at beamline 17-ID of urce (APS) Diffraction data were collected using a Pixel Array detector. Processed (Otwinowski, Z. & Minor, W. (1997). ssing of X-ray diffraction data collection d in oscillation mode.Methods in Enzymol ogy 276:307~326.).

[0221] The structure was solved by molecular replacement (MR) using Phaser (Read, RJ (2001).Pushing the boundaries of molecu lar replacement with maximum likelihood. Acta Crystallogr D Biol Crystallogr 57:1 373-82) In the case of the free Fab structure, the search model for MR is the anti-influenza hemagglutinin (IFN-α) complex. Rutinine 5j8 Fab (PDB code: 4M5Y). BCMA / Fab complex In this case, the search model for MR was based on the crystal structure of BCMA (PDB code: 1XU2) and BC The structure was refined using PHENIX (Adams ,PD,Gopal,K.,Grosse-Kunstleve,RW,Hun g, LW, Ioerger, TR, McCoy, AJ, Moriarty, NW, Pai, RK, Read, RJ, Romo, TD, Sacche ttini,JC,Sauter,NK,Storoni,LC& Ter williger, TC (2004).Recent developments in the PHENIX software for automated cry stallographic structure determination.J Synchrotron Radiat 11:53~5.) Modeled using COOT The model was adjusted (Emsley P. & Cowtan, K. (2004). Coot: Model building tools for molecular graph ics.Acta Crystallogr.D60:2126~2132). All others Crystallographic calculations were performed using the CCP4 suite of programs (Collabora tive Computational Project Number 4,1994 ). All molecular graphics were generated using PyMol (DeLano, W. (2011) 02).The PyMOL molecular graphics system. Palo Alto, CA, USA; Delano Scientific).

[0222] Data statistics for both the BCMB69 free Fab structure and the complex are shown in Table 6.

[0223] [Table 6]

[0224] Epitopes, Paratopes, and Interactions BCMB69 binds to residues within the β-hairpin region of BCMA (residues Y13-H19) and Residues within the helix-loop-helix region (residues L26, R27, and N31-L35) ) (Figures 3 and 4). The gap is approximately 830 Å on BCMA. 2 The ligand-binding DXL motif (β-heterozygosity Residues D15-L18 within the type I turn of the apin, which is a complementarity determining region (C DR) into a shallow cavity. Leucine 17 at the tip of the DXL turn is completely buried within the antibody cavity and has extensive interactions with BCMB69. The key epitope residue is Arg27, which is 10 - on helix h1, heavy chain C It forms several hydrogen-bonding contacts with DR.

[0225] The BCMB69 paratope is composed of residues from all CDRs except CDR-L1 (Figures 2 and 3). The heavy chain has twice as many contacts with BCMA as the light chain. The small side chain at the tip of the H3 loop (102-GAVAG-106) (SEQ ID NO: 77) , which aids in the insertion of CDR-H3 into BCMA and achieving extensive antibody / antigen contact (CDR -H3 forms 40% of all contacts). The BCMB69 CDRs are the BCMA chair The CDR-L2 (residues Y48, D52, P54, S5 5), CDR-H1 (residues G32-Y34), and CDR-H3 (D101, A103, V104, Y110) is a "sheet" formed by the h1 helix and the h1h2 loop. ", but CDR-L3 (residues W90, S92, D95), CDR-H1 (F 35), CDR-H2(Y54, Y60), and CDR-H3(H100, G102, A 103, A105) interact with the "back" formed by the β-hairpin of BCMA. The only epitope residue in the "leg of the chair" (h2 helix), Leu35, , which has van der Waals contacts with residue D52 of CDR-L2.

[0226] BCMA has a small (approximately 50 residues) and compact extracellular domain. There are limited surfaces available for antibodies or ligands to bind to BCMA. Most of the 9 epitope residues are found in APRIL (12 of 14 epitope residues) and These are the residues that also bind to BAFF (9 of 14 residues). In the case of the ligand APRIL, the only epitope residues that are not shared are F14 and S16. In BAFF (Fig. 2B), the non-shared residues are F14, L26, T32, P33, and and L35. The DXL loop is buried by both the ligand and BCMB69.

[0227] Proposed mechanism of action of BCMB69 BCMB69 is a candidate for redirecting T cells against MM cancer cells. Cancer cell killing mediated by CMB69 × anti-CD3 bispecific antibody was The structure and location of the epitope are not expected to weaken it. The position allows the BCMB69 Fab arm to bind to membrane-bound BCMA, while the other Fab arm The antibody is still bound to CD3 in the T cell membrane.

[0228] BCMB69 also mediates cytotoxicity in plasma cells by steric occlusion and direct competition for the BCMA binding site. It can also disrupt the APRIL and BAFF signaling pathways of BCMA / BC Overlay of MB69 structure onto BCMA / APRIL and BCMA / BAFF structures (L iu, Y., Hong, X., Kappler, J., Jiang, L., Zhang, R., Xu, L., Pan, C.H., Martin, W.E., Murphy, R.C. .,Shu, H. B., Dai, S. & Zhang, G. (2003).Nature 423:49~56;Hymowitz,SG,Patel,DR,Wall weber, H.J.A., Runyon, S., Yan, M., Yin, J., Shr. iver, S.K., Gordon, N.C., Pan, B., Skelton, N.J. ., Kelley, RF & Starovasnik, MA (2005).J. Biol. Chem. 280:7218-7227.) , showing the clash region between BAFF (FIG. 2B and FIGS. 4A and 4B), and BCMA antibody APRIL and BAFF inhibit the binding of TACI and natural ligands simultaneously. They can signal using other receptors, such as BAFF-R, and BCMA knockouts Thus, AP induction by BCMA blockade is not possible. Inhibition of RIL and BAFF activity may not be critically toxic for MM patients.

[0229] Example 7: Structure-based design of BCMB69 mutants Computational assessment of post-translational modification motifs and aggregation risk of unbound BCMB69 variable domains. There may be a medium risk of isomerization of the D101-G102 residues (CDR-H3) and an aggregation risk. 486 Å in the CDR region 2 The most exposed hydrophobic residues in the patch are The residues are I58 (CDR-H2), F35 (CDR-H1), and V104 (CDR-H3). ) (V104 was related to the Fv homology model but not to the Fab crystal structure. To eliminate the risk of isomerization and aggregation in the BCMB69 variable domain, A variety of mutations were rationally designed (Table 7).

[0230] [Table 7]

[0231] The CDR sequences and VH and VL sequences of the structure-based BCMB69 variants are shown in Tables 8 and 9, respectively. The results are shown in Table 9.

[0232] [Table 8]

[0233] [Table 9-1]

[0234] [Table 9-2]

[0235] [Table 9-3]

[0236] Therefore, in addition to BCMB69, 28 mutants were expressed and analyzed as described in Example 3. and purified to BCMA-expressing cells by flow cytometry as described in Example 4. Seven of the 28 mutants were characterized for their binding to BCMA-expressing cells. Since the antibody bound to the BCMA-CD3 cells, we proceeded to generate a BCMA x CD3 bispecific panel. .

[0237] Example 8: IgG4 S228P, L234A, L235A bispecific format Preparation of BCMA and CD3 antibodies in The BCMA antibody contains the Fc substitutions S228P, L234A, and L235A (EU index The antibody was expressed as an IgG4 having the sequence numbering (SEQ ID NO: 55 and SEQ ID NO: 56). A monospecific anti-CD3 antibody, CD3B19, has also been produced, which contains heavy and light chains having the sequences of Successful.

[0238] Protein A column (HiTrap MabSelect SuRe column) Monospecific antibodies were purified using standard methods such as those described above. After elution, the pool was washed with D-PBS, Dialyzed into pH 7.2.

[0239] The bispecific BCMAxCD3 antibody was prepared using the methods described in (WO 2011 / 131746). In vitro Fab arm exchange (as described above) was performed with monospecific CD3 mAb and monospecific Briefly, PBS (p Approximately 1-20 mg / mL of anti-BCMA / anti-CD3 antibody (1:1 molar ratio) in 100 mL of 10 ... (Or in some cases, a 6% excess of one parent antibody and a sharp depletion of the other). and 75 mM 2-mercaptoethanolamine (2-MEA) were mixed and incubated at 31°C for 5 h. The mixture was then incubated for 2 h and then dialyzed, diafiltered, tangent filtered, and purified using standard methods. 2-MEA was removed by flow filtration and / or roll cell filtration. ) by either HPLC or hydrophobic interaction chromatography (HIC) HPLC. , and analyze the formation of bispecific BCMAxCD3 antibodies. If desired, CMAxCD3 antibodies were polished by preparative CEX or HIC to remove residual parental antibodies .

[0240] The heavy and light chains of a representative BCMAxCD3 bispecific antibody are shown in Table 10 below. MB178 was poorly expressed when combined with the CD3 arm, resulting in further characterization. No attachment was made.

[0241] [Table 10-1]

[0242] [Table 10-2]

[0243] [Table 10-3]

[0244] [Table 10-4]

[0245] [Table 10-5]

[0246] [Table 10-6]

[0247] [Table 10-7]

[0248] Example 9: Determining BCMA affinity of BCMA antibodies and BCMAxCD3 bispecific antibodies Surface Plasmon Resonance (SPR) is used to generate CD3 bispecific antibodies The affinity values ​​of the BCMA antibodies to human BCMA were measured. The protocol followed for SPR was The protocol was the same as that described in Example 4. The results shown in Table 11 were consistent across all samples. The results show that the parent mAb (BCMB69) bound to the monomeric BCMA antigen with varying affinities. ) had a binding affinity of ∼1.4 nM. BCMB117 and BCMB128 were Although it had affinity within the range of BCMB69, BCMB123, BCMB129, and BCMB17 6 and BCMB177 had relatively weak affinity due to their fast off-rates (3–15 To assess the reproducibility of the data, all samples were run at least three times and The deviation was recorded.

[0249] [Table 11]

[0250] We also used SPR to identify BCMA×CD3 antibodies against human and cynomolgus BCMA. The affinity values ​​of the polyspecific antibodies were also measured. The results in Table 12 show that all samples had various affinities. BC3B7 and BC3B9 bind to the Fc-BCMA antigen. A was within the range of BCMB72, whereas the remaining bispecific antibodies were It had 2-3 times weaker affinity than B72. BC3B7 and BC3B9 had 2-3 times stronger affinity than BCMB72 (K D The remaining mAbs showed similar binding to BCMB72. Maintained (K D To assess the reproducibility of the data, all samples were Samples were performed at least three times and the standard deviation was recorded.

[0251] [Table 12]

[0252] Fc-fused BCMA protein of anti-BCMA x CD3 bispecific antibody (BCMB72) The binding affinity to human, cynomolgus monkey, and mouse was measured using Biacore T200 s Surface plasmon resonance (SPR) using the GE Healthcare, NJ ) was measured.

[0253] Streptavidin-derivatized sensor chip (GE Healthcare, Prod # BR-1005-31) were loaded onto flow cells 2, 3, and 4, respectively, with biotinylated Fc- Immobilization was performed using fusion human, cynomolgus monkey, or mouse BCMA (BCMA immobilization concentration was 1 2-16 response units (RU); Fc-BCMA protein: human (R&D System ms; Prod# 193-FC), cynomolgus monkey (in-house; Cat# BCMW6.0 01) and mouse (R&D Systems; Prod# 593-BC) in-house. Flow cell 1 had no protein immobilized on it and was used as a reference surface. 2 h in running buffer (PBS pH 7.4, 0.005% P20, 3 mM EDTA) Binding kinetics studies were performed at 5°C. BCMB72 was diluted 5-fold starting at 100 nM. These solutions were diluted to 0.16 nM in running buffer at 50 μL / min for 5 min. The mixture was injected for 15 min (association phase) and dissociation was monitored for 15 min by running the running buffer. A short injection of glycine (pH 1.5) and running buffer at 100 μL / min The chip surface was regenerated. Binding kinetics analysis of the interaction of BCMB72 with Fc-BCMA was calculated by subtracting the curve generated by the buffer injection from the reference subtraction curve of the analyte injection. The overall kinetic fitting of the sensorgrams was performed using , Two-State binding Model using Biacore T 200 Evaluation Software(GE Healthcare,NJ Results obtained from the two-state binding model for different BCMA species The binding affinity results were calculated using the first complex (K D1 ) and the final complex (K D ) (Figure 5).

[0254] Example 10: BCMA in the presence of immortalized cell lines in a multiple myeloma background ×Target-specific T cell activation and cytotoxicity of CD3 antibody T cell activation mediated by BCMAxCD3 antibodies was evaluated. CMB72 (BCMA x CD3) and control antibodies (BCMA x none and none x CD3) was diluted to 800 μg / mL in PBS. A titration was prepared by serial dilution. The last column was left with PBS only (solvent control). roll).

[0255] Supplemented with GlutaMAX, 10% FBS, and 25 mM HEPES, antibiotics The target cells were cultured in RPMI 1640 medium (culture medium) without IgG. ) target cells were counted and 10 million cells were centrifuged at 1350 rpm for 3 min. The supernatant was then discarded. CellTrace FCSE proliferation dye was diluted with 18 μL of sterile DMSO. Reconstitute and dilute 1 µL of this solution in 10 mL of sterile PBS. The cells were then resuspended in 100% CFSE dilution and incubated for 8 minutes at room temperature, covered from direct light. After incubation, 1 mL of HI FBS was added to the cell suspension to quench excess CFSE. The cells were washed twice with RPMI-1640 containing 10% FBS. After reconstitution with RPMI, cells were counted and cell viability was recorded in a summary chart. ×10^5 / mL and incubated at 37°C until use.

[0256] Pan-T cells from normal donors were thawed in a 37°C water bath, and the contents of the frozen vials were then transferred to a 50 ml The cells were then transferred to a 1 L conical vial and reconstituted with 15 mL of cold medium. The cells were then incubated at 4°C for 1 Centrifuge at 350 rpm for 3 min. Discard the supernatant and resuspend the cell pellet in 5-10 mL of medium. The T cells were counted and viability was recorded. The cells were then diluted in culture medium at 1.1×10^6 / mL.

[0257] 2×10^5 target cells were added to wells of a 96-well U-bottom plate, followed by Fc-blocking. Rocker was added (to a final concentration of 2 mg / mL). All cell lines were incubated at room temperature for 10 min. The Fc receptor activity was blocked by incubating the cells with 1×10^5 T cells. After mixing the target and T cells, 20 μL of BCM was added. AxCD3 antibody dilutions were added to each well. The plates were incubated at 37°C with 5% CO2 for 4 h. Incubated for 8 hours.

[0258] After 2 days, the plate was centrifuged at 1350 rpm for 3 min at 4° C. and 100 μL of The supernatant was transferred to a separate plate and stored at -80°C for cytokine release assays. Wash with 0.1 µL of PBS and add 50 µL of Near-Infrared Live / Dead dye (1:200 dilution) Anti-CD25 PE antibody (1:50 dilution) was added to the wells of the 50-well plate and incubated for 20 min at room temperature. The cells were then washed once with 200 μL of FACS buffer and finally with 150 μL of FACS buffer. The target cytotoxicity (target %) and T cell activation CD25+ (viable The percentage of remaining T cells was measured using FACSCanto II and FlowJo 7.6. The cells were analyzed. Graphing and fitting of the data was performed using the least squares method with variable slope. Nonlinear regression with (4 parameter) function using GraphPad Prism 6 So I went.

[0259] FIG. 8 shows that BC, as assessed by upregulation of CD25 on the surface of T cells, We show that MB72 consistently promotes target-specific T cell activation. The antibody was used to prevent Fc receptor-dependent binding of the antibody to the target cells. The results fit closely to the fitted curve and showed little variability between T cell donors. .BCMA + In cells, 45-85% of maximum activation was achieved, and BCMA - 4-10 in cells % (equivalent to background concentration). EC obtained from two independent studies 50 and maximum T cell activation values ​​are summarized in FIG.

[0260] Figure 10 shows that BCMB72 is a BCMA + Consistently showed strong cytotoxicity against cell lines Using Fc blockers, we demonstrated that BCMB72 binds to target cells in a Fc receptor-dependent manner. In general, the data points lined up closely along the fitted curve, and the T-strips were There was little variability between cell donors. + Maximum lysis of cells: 62-97% Achieved BCMA - The percentage of patients with T cells from multiple normal donors ranged from 4% to 18%. EC obtained from two independent studies using 50 and maximum lysis values ​​are summarized in FIG.

[0261] The other six BCMA × CD3 antibodies had maximum cytotoxicity of 83–93% (Figure 12A) and BCMA using T cells from two different donors + 74-83% of T cells in H929 cells These six BCMAxCD3 antibody molecules showed a 0.04 activation. EC in the range of ~0.09 nM50 values ​​and are effective in killing BCMA+ target cells.

[0262] Example 11: Binding efficiency of BCMB72 in BCMA+ cell lines EC of BCMB72 binding to various BCMA+ cell lines of malignant background 50 value Briefly, the bispecific antibody BCMB72 (BCMA × CD3) was administered in PBS Serially diluted 3-fold in PBS in a 96-well U-bottom plate The last column was left with PBS only (solvent control). .

[0263] Supplemented with GlutaMAX, 10% FBS, and 25 mM HEPES, antibiotics H929 target cells were cultured in RPMI 1640 medium (culture medium) without IgG. For B, the density and viability of the target cells were measured, and the cells were then incubated at 4°C for 100 The cell pellet was then washed with 10 mL of PBS and centrifuged at 100 rpm for 5 min. Centrifugation was performed at 1000 rpm for 5 minutes. 5 Resuspend cells in PBS at 100 cells / mL The cells were then suspended in water and aliquoted into a 96-well U-bottom plate at 90 μL per well. 10 μL / well of the CMB72 dilution was dispensed. The plate was incubated at 4°C in the dark for 1 hour. The mixture was incubated, then centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The plate was washed twice with 200 μL of FACS buffer. Dissolve the labeled secondary antibody in FACS buffer at 1:25 and add 50 μL of this mixture to the corresponding The samples were incubated at 4°C for 20 min and then FACS analyzed as above. Wash with buffer and prepare for analysis on the FACSCanto II with 150 μL of FACS buffer. The data for BCMB72 binding were reconstructed using FlowJo 7.6. Analyze, graph and fit the data using a least squares method with a variable slope function. The data were analyzed using nonlinear regression with GraphPad Prism 6.

[0264] As can be seen in Figure 6, BCMB72 binds to all BCMA+ cell lines tested. EC binding to H929 cells 50 is 14.7 nM, and MM.1 9.74 nM for R cells, 17.5 nM for EJM cells, and 17.5 nM for LP1 cells. The concentrations were 22.3 nM for the IL-14 receptor agonist and 7.92 nM for the U-2932 cells.

[0265] Example 12: Expression of BCMA and B in whole blood from normal human donors ex vivo CMB72 binding analysis BCMA expression and BCMB72 binding in leukocytes were examined ex vivo in three normal human The assay was performed in whole blood from healthy donors. Briefly, fresh peripheral blood from normal human donors was used. Blood was stored in heparin-coated tubes prior to testing. Blood was then transferred in 100 μL aliquots to 96-well U-bottom plates. The staining antibodies were added to the master mix as indicated in the study summary. A master mix was prepared containing an antibody against BCMA (i.e., BCMB72) and After 30 minutes of incubation at room temperature, the plate with blood was The pellet was centrifuged at 1350 rpm for 3 min at 4°C. The supernatant plasma was discarded and the pellet was The red blood cells were subjected to lysis in successive washes with a 5 minute incubation between each wash. After incubation, the pellet was washed once with PBS and then stained with 1:200 Live / Dead near-infrared dye. and 1:50 anti-IgG4 PE (only for wells with BCMB72). The plates were further incubated at room temperature for 15 minutes. The pool was washed with 200 μL of FACS buffer and finally analyzed on the LSRFortessa. Each well was reconstituted with 150 μL of FACS buffer for 10 min. Approximately 100,000 events were recorded. Analyses were performed using FlowJo 7.6.

[0266] As shown in Figure 7, no BCMA expression was observed in lymphocytes, monocytes, and lymphocytes in three normal donors. BCMB72 was observed in granulocytes or plasmacytoid DCs in all three donors. BCMB72 showed binding to 3+ T cells, but the strength varied between donors. It did not bind to any other cell types tested in the assay.

[0267] Example 13: Effect of BCMB72 on cytokine profile The cytokine profile in the supernatants obtained from the T cell-mediated killing assay was CMB72 and a control antibody were used to evaluate T cell-mediated The cells were seeded as in the cytotoxicity assay (see Example 10). After incubation, cell supernatants were harvested and analyzed using an MSD-based ELISA for different (10 / 30) Plex) cytokines were measured. Cytokine concentrations are expressed as pg / mL and data are Graphing and fitting of nonlinear regression with variable slope (four parameter) function The analysis was performed in GraphPad Prism 6 using RP with six T cell donors. EC of six cytokines obtained from MI8226 cell line50 The values ​​are shown in Figure 13. indicates significant cytokine release resulting from T cell activation. , low / absent cytokine release was observed (data not shown).

[0268] Example 14: HEK and CHO-produced T cells in T cell activation and T cell-mediated target cell killing Functionality comparison of live (transient & stable cell line) BCMB72 Bispecific antibodies produced in different cells and under different expression systems have different activities. Therefore, HEK cells (transient expression) or CHO cells (transient or stable expression) The in vitro efficacy of BCMB72 produced by the ELISA (expression) was evaluated.

[0269] BCMB72 was diluted to 800 μg / mL in PBS. Prepare titrations in either 3- or 4-fold serial dilutions in PBS in well U-bottom plates The last column was left with PBS only (solvent control).

[0270] Supplemented with GlutaMAX, 10% FBS, and 25 mM HEPES, antibiotics H929 target cells were cultured in RPMI 1640 medium (culture medium) without On day 1, cells were counted and 10 million cells were centrifuged at 1350 rpm for 3 min. The supernatant was discarded. CellTrace FCSE proliferation dye was diluted with 18 μL of sterile DMSO. Reconstitute and dilute 1 µL of this solution into 10 mL of sterile PBS. H929 cell pellet Resuspend the cells in 1 mL of CFSE dilution and incubate for 8 minutes at room temperature, covered from direct light. After incubation, 1 mL of HI FBS was added to the cell suspension to remove excess CFS. The E was quenched. The cells were washed twice with 1640 RPMI with 10% FBS. After reconstitution with 10 mL of RPMI, cells were counted and cell viability was recorded in a summary chart. was diluted to the indicated concentrations and incubated at 37°C until use.

[0271] T cells from normal donors were thawed in a 37°C water bath and the contents of the vial were then transferred to a 50 mL conical The cells were transferred to a vial and reconstituted with 15 mL of cold medium. The cells were then incubated at 4° C. for 1350 Centrifuge at 400 rpm for 3 min. Discard the supernatant and reconstitute the cell pellet in 5–10 mL of medium. T cells were counted and reconstituted in media at the appropriate concentration (see summary tables for each study).

[0272] H929 cells were added to the wells, followed by T cells (effector:target in a 5:1 ratio). In this set of experiments, no Fc blockers were used. Targets and T cells were mixed. Afterwards, 20 μL of BCMB72 dilution was added to each well. The plate was incubated at 37° C. Incubated at 5% CO2 for 48 hours.

[0273] After 2 days, the plate containing the cells was centrifuged and the supernatant was either discarded or assayed for cytokine release. The cells were washed with 200 μL of PBS and incubated in 50 μL of NIR Live / D ead dye (1:200 dilution) and anti-CD25 PE antibody (1:50 dilution) at room temperature. Incubate for 20 min. Then wash the cells once with 200 μL of FACS buffer. Finally, the cells were reconstituted in 150 μL of FACS buffer. Flow cytometry was performed using a CT II to measure target cytotoxicity (% on target) and T Cell activation CD25+ (% viable T cells) was analyzed using FlowJo 7.6. Graphing and fitting of the data was performed using a variable slope (four parameter) function and the least squares method. The data were analyzed using GraphPad Prism 6 using nonlinear regression.

[0274] As can be seen in FIG. 14, BCMB72 produced in HEK cells and CHO cells The BCMB72 induced cytotoxicity against target cells in a T cell redirection assay. The mAb performed virtually identically in terms of efficacy and stimulation of T cells. % T cell activation was generally achieved. Mean EC 50 Values ​​are from antibodies produced in HEK cells. BCMB72 produced in CHO cells was 0.42 nM. The mean EC for T cell activation was ~0.47 nM. 50 Values ​​are for B produced in HEK cells. 0.28 nM for CMB72 and 0.37–0 for BCMB72 produced in CHO cells. Comparative analysis using Student's T-test revealed that the EC 50 There was no statistical significance between the values.

[0275] Example 15: P38 signaling activation by BCMB72 Both BAFF and APRIL bind to two receptors: BCMA (B cell maturation antigen, TNF-. F 17) and TACI (transmembrane activator and CAML interactor, TNFRSF 1 BCMA binding activates the JNK and p38 MAPK signaling pathways. BCMA×CD3 bispecific antibody BCMB72 targets BCMA and activates This study included two parts: 1. APRIL Changes in P38a MAPK in H929 or MM1.R cells after BAFF treatment 2. Develop a simple Western analysis assay to monitor the newly developed Using this assay, we determined whether BCMB72 had any agonistic effect towards BCMA. Find out whether or not to do so.

[0276] Cell treatment H929 or MM1.R cells were incubated at 37°C in the presence of 5% CO2 for 24 h before treatment. The cells were seeded at 1.5e6 / mL in serum-free RPMI medium for 1 h. On the day of treatment, the cells were spun down. The cells were incubated at 4°C for 1 h and resuspended at 1.5e6 / mL in serum-free RPMI. The cells were aliquoted into 10 tubes, 5 mL per tube. The cells in each tube were diluted to 1000 ng / mL. APRIL (R&D Systems, Catalog #5860-AP-010) or 10 00ng / mL BAFF (R&D Systems, catalog #2149-BF-01 0) for 0, 5, 15, 30, and 60 minutes at 37°C in 5% CO After incubation, cells were immediately pelleted and cell lysates were The BCMB72 agonist activity assay was performed using a 100% PBS-free 100% PBS buffer. The H929 cell treatment groups are listed in Table 13. The BCMB72 agonist assay was performed twice. went.

[0277] [Table 13]

[0278] Preparation of cell lysates for Simple Western analysis Cells were lysed in RIPA buffer containing phosphatase and protease inhibitors. Protein concentrations were determined using the BioRad DC Protein Assay (BioRad # 500-0112) and bovine serum albumin standard, SpectraMax Plus s 384 microplate reader (Molecular Devices, Sun Measurements were performed at a 30-well plateau in New York, Calif., USA.

[0279] Simple Western analysis Simple Western Analysis, ProteinSimple User Manual Wes-Rabbit (12-230KDa) Master kit (Pro Briefly, cell lysates were Mix the samples with the master mix and add 1x sample buffer, 1x fluorescent molecular weight marker and 40 mM dithiothreitol (DTT) final concentrations, then heated at 95°C for 5 minutes. Heat. Sample, blocking reagent, primary antibody fluorophore-p38 MAPK (ThermoFis her:VWR# MA5-15182) or Actin-beta(Cell Sig naling, #8457S), HRP-conjugated secondary antibody, chemiluminescent substrate, and separation and accumulation Dispense the layer matrix into designated wells in a Simple Wes microplate; After the plate was read, the separation electrophoresis and immunodetection steps were carried out in a capillary system. During electrophoresis, proteins are separated by a stacking and separation matrix. The molecules are separated based on their molecular weight by a photocatalytic filter and trapped on the capillary wall using a proprietary light-activated capture chemistry. The primary antibody was diluted in Antibody Diluent II (ProteinSimple #042-2 The target protein was incubated with primary antibody for 60 minutes and then with HRP. Immunodetection was performed using conjugated secondary antibodies. Simon-simple Western The analysis was performed at room temperature and the default settings of the instrument were used. Digital images were analyzed using the Compass software. The protein was analyzed using ProteinSimple software and quantitative data was obtained. Data were recorded as molecular weight, signal / peak intensity, and peak area.

[0280] result A 15 minute incubation endpoint was used based on information obtained from a time course study. We performed a BCMB72 agonist assay in H929 cells. The normalized values ​​obtained from the two assays were compared with the human beta-actin signal. The average p38 MAPK signal is shown in Figure 15. The BCMB72 agonist assay BCMB72 does not have agonistic activity towards BCMA in H929 cells. This demonstrated that:

[0281] Example 16: NFκB signaling by BCMB72 BCMA is a surface receptor that can initiate NF-κB signaling in response to endogenous ligands. The effect of BCMA-bound BCMB72 on NF-κB pathway activation was BCMA expressing alkaline phosphatase (SEAP) under the NFκB promoter was assessed using a reporter cell line.

[0282] Cells were cultured in DMEM medium (culture medium) supplemented with GlutaMAX and 10% FBS. The night before the experiment, the cells were trypsinized (0.25% trypsin pre-warmed to 37°C). The cells were then harvested by incubation in a centrifuge for 5 min and washed with 30 mL of culture medium. The cells were then incubated at 4°C for 1 Centrifuge at 000 rpm for 5 min and resuspend in serum-free DMEM (containing GlutaMAX). The cells were reconstituted at 2.5×10^5 cells / mL. 5×10^4 cells were plated in a 96-well flat-bottom plate. The plate was then added to wells and incubated at 37°C for 16 hours.

[0283] The next morning, various stimulatory agents (TNFα, APRIL, BCMB72) were added to the corresponding wells. (see test plate map) and the plates were incubated at 37°C for an additional 16, 24, or 48 hours. These represent early, middle, and late signaling time points, respectively. After each time point, 10 μL of conditioned culture medium was collected from the wells and analyzed using the SEAP kit (Cayman et al., 2011). The plates were then transferred to a 96-well solid plate provided in the US Pat. No. 600272 and covered with a lid. AP standard was prepared by dissolving the bulk standard (5 U / mL) in serum-free DMEM (GlutaMAX A 1:10 dilution was then made with 1:2 serial dilutions. The range of the concentration is 50-0.78 mU / mL. The plate with the samples was incubated at 65°C for 30 minutes. The endogenous alkaline phosphatase was inactivated by incubation for 1 h. The expressed SEAP is stable under these incubation conditions. After incubation with 50 μL of 10 μL of standard dilutions were added to the appropriate wells. Substrate solution was added to all wells and the samples were vortexed briefly to distribute the solution into the wells. Incubate the samples for 20-30 minutes and then use the PerkinElmer EnVision Chemiluminescence was assessed using the 2104 Multilabel Reader. Luminescence readings were converted to activity unit concentrations based on a standard curve and values ​​were analyzed using Microsoft Excel. Analyzed in xcel 2010 and imported into Graph Prism 6 for graphical analysis did.

[0284] Figure 16 shows that APRIL was able to stimulate BCMA even at a low concentration of 0.46 nM. Overall, BCMB72 inhibited the NF-κB pathway in BCMA-transduced cells at concentrations below 10 nM. At high BCMB72 concentrations (44–133 nM), A degree of BCMB72-dependent activation was observed.

[0285] Example 17: Role of the extracellular domain of BCMA in T cell activation in the absence of target cells Effects of external additives The extracellular domain (ECD) of BCMA can form trimers in solution. A number of bispecific antibodies have been shown to bind BCMA ECD trimers and cross-linked TCR complexes in the absence of target cells. This may in turn activate T cells in a target-independent manner. This study demonstrated that the ECD of exogenously added BCMA inhibits CD2 T cells without interacting with target cells. We investigated whether 5 expression levels could induce T cell activation.

[0286] BCMB72 (BCMA × CD3) and control (no × CD3) were incubated at 800 The doses were serially diluted 3-fold in PBS in a 96-well U-bottom plate. The setup was adjusted so that the last column was left with PBS only (solvent control).

[0287] Soluble BCMA ECD (sBCMA) at 36 μg / mL (6.67 μM) in PBS The titration was prepared in a 96-well U-bottom plate by serial 3-fold dilutions in PBS. The top well was left with PBS only (vehicle control).

[0288] Pan-T cells from normal donors were thawed in a 37°C water bath, and the contents of the frozen vials were then transferred to a 50 ml The cells were then transferred to a 1 L conical vial and reconstituted with 30 mL of cold medium. The cells were then incubated at 4°C for 1 Centrifuge at 350 rpm for 3 min. Discard the supernatant and reconstitute the cell pellet in 10 mL of medium. T cells were counted and viability was recorded. The cells were then diluted to 0.525×10^6 in culture medium. / mL.

[0289] 1×10^5 T cells (190 μL) were added to the wells, followed by 5 μL of sBCMA The dilutions and 5 μL of BCMB72 dilutions were added. The plates were incubated at 37° C. with 5% CO The mixture was incubated at 2 for 48 hours.

[0290] After 2 days, the plates were centrifuged at 1500 rpm for 3 minutes at 4° C. and the supernatant was discarded. Wash the cell pellet with 200 μL of PBS and add 50 μL of near-infrared Live / Dead dye. (1:200 dilution) and anti-CD25 PE antibody (1:50 dilution) for 20 min at room temperature. After incubation, the cells were washed once with 200 μL of FACS buffer and then Reconstituted in 150 μL of FACS buffer. T cell activation CD25+ (% viable T cells) Analyze cells using FACSCanto II and FlowJo 7.6 for The data were graphed and fitted using a nonlinear least squares fitting method. Regression was performed in GraphPad Prism 6 using

[0291] T cells from normal donors demonstrate sBCMA ECD-mediated activation in the presence of BCMB72 At high concentrations (>40 nM) of BCMB72, sBCMA-independent A weak activation of a low percentage of T cells (10–15%) was observed (Figure 17).

[0292] Example 18: Soluble BCMA in T cell activation and BCMB72-dependent cytotoxicity The influence of ECD, APRIL, and BAFF Soluble BCMA ECD can act as a sink for BCMA×CD3 antibodies, but APR IL and BAFF are competitive inhibitors of the interaction of BCMA×CD3 antibodies with surface receptors. Possible. Soluble BC in the in vitro cytotoxicity of BCMB72-dependent cell killing The effects of MA ECD and the endogenous ligands APRIL and BAFF were investigated in the fixed cell line H92 9 and normal donor M7077-derived pan-T cells were assessed in a T cell redirection assay did.

[0293] BCMB72 was diluted to 800 μg / mL in PBS. The titration was prepared by serially diluting the solution 3-fold with PBS. The last column was left with PBS only. Soluble BCMA ECD was diluted to 9 μg / mL and and BAFF were diluted to 10 μg / mL. A titration of both reagents was prepared by two-fold serial dilution.

[0294] Supplemented with GlutaMAX, 10% FBS, and 25 mM HEPES, antibiotics H929 target cells were cultured in RPMI 1640 medium (culture medium) without On day 1, count the target cells and centrifuge 10 million cells at 1350 rpm for 3 minutes. After separation, the supernatant was discarded. CellTrace FCSE proliferation dye was added to 18 μL of sterile D Reconstitute with MSO and dilute 1 µL of this solution into 10 mL of sterile PBS. Cell pellet Resuspend the cells in 1 mL of CFSE dilution and incubate for 8 minutes at room temperature, covered from direct light. After incubation, 1 mL of HI FBS was added to the cell suspension to remove excess CFS. The E was quenched. The cells were washed twice with RPMI-1640 containing 10% FBS. After reconstitution with 10 mL of RPMI, cells were counted and cell viability was recorded in a summary chart. was diluted to 2.2×10^5 / mL and incubated at 37°C until use.

[0295] Pan-T cells from normal donors were thawed in a 37°C water bath, and the contents of the frozen vials were then transferred to a 50 ml The cells were then transferred to a 1 L conical vial and reconstituted with 30 mL of cold medium. The cells were then incubated at 4°C for 1 Centrifuge at 350 rpm for 3 min. Discard the supernatant and reconstitute the cell pellet in 10 mL of medium. T cells were counted and viability recorded. The cells were then diluted in culture medium at a concentration of 1.1×10^6 / m Reconstructed into L.

[0296] 2×10^5 H929 cells were added to the wells of a 96-well U-bottom plate. In our experiments, incubation with Fc blockers was not necessary. T cells were added to the wells (effector:target ratio of 5:1). Target and T cells were mixed. Afterwards, 20 μL of either sBCMA, APRIL, or BAFF was added to the wells. 5 μL of antibody dilution was then added. The plates were incubated at 37° C. with 5% CO2 for 48 hours. Incubated.

[0297] After 2 days, the plates were centrifuged at 1500 rpm for 3 minutes at 4° C. and the supernatant was discarded. Wash the cells with 200 μL of PBS and add 50 μL of near-infrared Live / Dead dye (1:2 Incubate with anti-CD25 PE antibody (1:00 dilution) and anti-CD25 PE antibody (1:50 dilution) for 20 minutes at room temperature. The cells were then washed once with 200 μL of FACS buffer and finally with 150 μL of The target cytotoxicity (% on target) and T cell activation CD2 For 5+ (% viable T cells), FACSCanto II and FlowJo 7.6 were used. The cells were analyzed using the least squares method for graphing and fitting the data. Nonlinear regression with variable slope (4 parameter) function using GraphPad Pri Went with sm6.

[0298] BCMB72 exerts cytotoxicity on H929 cells in the presence of soluble BCMA ECD BCMA ECD at high doses (>160 nM) inhibited EC 50 Very slight shadow T cell activation was similarly affected (see Figures 18A and 18D). APRIL is an EC inhibitor of cytotoxicity and T cell activation. 50 The value was 6 at the high dose (46 nM). 2-fold increase in the agonist concentration, whereas the lower dose had minimal effect on the assay (Figure 18B and 18E). Maximum killing was unaffected by sBCMA or APRIL. In contrast, exogenous BAFF had no effect on BCMB72-mediated cytotoxicity at concentrations below 51 nM. In all cases, the T cell activation was as expected. This correlated well with the wound data (see Figure 18F).

[0299] Example 19: BCMB72, APRIL, and IL-16 in Binding to BCMA in Vitro and BAFF conflict Two TNF ligands, APRIL and BAFF, bind to BCMA and mediate signal transduction The extracellular domain of BCMA can trigger a cascade that leads to cell survival and proliferation. , a short nucleotide sequence that binds to these two ligands, as well as the antibodies raised against this motif. The 54 amino acid fragment is shown here. We evaluated the competitiveness of the

[0300] The assay was set up in an ELISA-based format. In preparing the competitive assay BCMA-Fc was labeled with MSD SulfoTag. Reconstitute the ial in 500 µL of PBS to 0.1 mg / mL (3.125 µM monomer). 150 nmoles of NHS-sulfoTag was dissolved in 50 μL of water to obtain a 3 mM solution. For the 10x excess labeling reaction, add 5.2 μL of 3 mM NHS-SulfoT Ag (15.6 nmoles) was added to 500 uL of BCMA-Fc (1.56 nmoles monomer). The reaction was allowed to proceed in the dark at room temperature for 2 hours. 50 μL of 1 M Tris was added to remove unreacted The NHS was quenched. Excess Sulfotag and Tris were washed with 2 ml of PBS-equilibrated PBS. The final volume was removed by buffer exchange on a 100 mL 7k MWCO Zeba spin column. The volume was ~630 μL, therefore the final SulfoTag-BCMA-Fc was 2.5 μL. Used as M.

[0301] For the competitive assay, anti-BAFF (100 μg) and anti-APRIL (100 μg) Reconstituted in 200 μL of PBS to obtain a stock solution of 0.5 mg / mL. Anti-APRIL and anti-BAFF were each diluted in 2.97 mL of PBS to a concentration of 2 μg / m To each well of a 96-well MSD high binding plate, 25 μL of 2 μg / m L anti-APRIL was added to each well of a second 96-well MSD high binding plate. 1 μL of 2 μg / mL anti-BAFF was added. The plate was kept overnight at 4°C to allow antibody fixation. The contents of the anti-APRIL and anti-BAFF coated plates were discarded and 3 100 μL of SuperBlock was added. After 1 hour of inhibition at room temperature, the plates were washed with PBS- The plate was washed three times with T. 10 μg of each of recombinant APRIL and BAFF was added to 100 μL of PBS. Resuspended to obtain a 0.1 mg / mL solution. 2 μg of each of APRIL and BAFF in 3 mL To prepare a 1 / mL solution, add 60 μL of freshly reconstituted protein to 2.94 mL of SuperBl 25 μL of 2 μg / mL APRIL was diluted with anti-APRIL To each well of the IL-coated plate, add 25 μL of 2 μg / mL BAFF as anti-BAFF antibody. After capture for 1 hour at room temperature, the plate was washed three times with PBS-T. Wash. 500 μg of anti-BCMA (R&D Sys Mab193) in 1 mL of PBS to obtain a stock solution of 0.5 mg / mL (3.3 μM). Anti-BCMA Mab193 , BCMB72.004, and control antibody (no × CD3) were used in SuperBlock Mix 100 μL of antibody with 200 μL of SuperBlock. A 3-fold serial dilution series of 11pt was prepared by adding 6mL of 30nM SulfoTa g-BCMA-FC was prepared by dissolving 72 μL of the above protein in 5.928 mL of SuperBlo ck. Add 25 μL of each antibody from step 11 to the following Add to each well of the APRIL / BAFF capture plate according to the plate map. Add 1 μL of 30 nM SulfoTag-BCMA-Fc to each well on both plates. After 1 hour at room temperature, the plate was washed three times with PBS-T. 150 μL of 1×MSD Add Read Buffer T to each well and scan the plate with a Sector 6000 imager. The test was repeated exactly as above to obtain a second independent set of results.

[0302] As seen in Figure 19, when incubated with increasing amounts of BCMB72, BCMA-Fc protein was not observed in the control antibody (no CD3) but was The binding of the β-lactam to APRIL and BAFF was prevented. Two independent repeated tests were consistent.

[0303] Example 20: Binding of bone marrow CD138-positive cells from multiple myeloma patients to BCMB72 and and cytotoxicity To evaluate the ability of BCMB72 in primary samples from multiple myeloma patients, Using frozen bone marrow samples from five patients with multiple myeloma and T cells from healthy donors The antibody was tested in a cytotoxic killing assay using a mAb. Antibody binding and T cell activation were also measured. did.

[0304] BCMB72 binding assay 100 μL of cell suspension was dispensed per well in a 96-well U-bottom plate and then Next, 95 μL of medium was taken. Then, 5 μL of serial dilutions of BCMB72 or control were added. was added to the wells and the plate was incubated for 1 hour at 4°C. After staining, the cells were The cells were centrifuged at 100 rpm for 3 min and washed once with 200 μL of PBS. After separating and discarding the supernatant, the pellet was soaked in 50 μL of near-infrared Live / Dead dye (1:20 0 dilution), anti-human IgG4 Fc PE antibody (1:50 dilution), anti-CD138 (MI 15 1:50 dilution and DL-101 1:50 dilution) and incubated in a dark room. The cells were then centrifuged and transferred to 200 μL of FACS buffer. The cells were washed with 100 μL of FACS buffer and finally reconstituted with 150 μL of FACS buffer. The binding strength of BCMB72 in NC was examined using FACSCanto II and FlowJo. The samples were analyzed using 7.6. Data fitting was performed using the least squares method. Using nonlinear regression with a variable slope (4 parameters) function, GraphPad Pro It was done at ism 6.

[0305] T cell redirection assay 1×10^5 target cells were added to wells of a 96-well U-bottom plate, followed by 1×1 0^5 T cells were added (approximate effector:target ratio of 5:1, but bone marrow derived After mixing the target and T cells, the plasma cell count in the mast cells was set at 20% on average. 1 μL of BCMB72 dilution was added to each well. The plate was incubated at 37° C. with 5% CO2 The mixture was incubated at RT for 48 hours.

[0306] After 2 days, the plates were centrifuged and the supernatant was discarded. The cells were washed with 200 μL of PBS. 50 μL of PBS with Near-Infrared Live / Dead dye (1:200 dilution), anti-CD 138 (1:50 dilution in MI15 and 1:50 dilution in DL-101), anti-TCR α Anti-CD25 PE (1:50 dilution) and anti-CD25 β (1:50 dilution) were incubated at room temperature for 20 min. After incubation, the cells were washed once with 200 μL of FACS buffer and then Reconstituted in 150 μL of FACS buffer. Plasma cytotoxicity (% of killed CD138+ cells) ) and T cell activation CD25+ (% viable T cells) were measured using FACSCanto II and Cells were analyzed using FlowJo 7.6 and FlowJo 7.6. Data graphing and fitting were performed. The regression was performed using a nonlinear regression with a variable slope (four parameters) function using the least squares method. This was done using GraphPad Prism 6.

[0307] result Figure 20 shows CD138 + BCMB72 was 48% positive for plasma cell loss. It was shown that the antibody bound to and induced killing of all patient samples in a dose-dependent manner after 2 h. The cell activation data correlate well with the killing data, as expected. The mean E of T cell activation C 50 These data indicate that BCMB72 has a potent in vitro It has been demonstrated that it can kill bone marrow cells in patients with advanced multiple myeloma.

[0308] Example 21: PBMC-humanized NSG mice with H929 human multiple myeloma xenografts Antitumor effects of BCMB72 in preventing tumor formation This study was performed using PBMC (peripheral blood mononuclear cells) humanized NSG (NOD SCID Gamma )BC in preventing tumor formation in H929 human multiple myeloma (MM) xenografts in mice The effect of MB72 on the proliferation and proliferation of mature functional T cells, B cells, and natural The strain is immunocompromised and lacks natural killer (NK) cells. Seven days before the test, 1×10 7 Each mouse was intravenously injected with 100 human PBMCs. Incubate the culture with H929 human MM cells (5 × 10 in 200 μL of PBS). 6 1 cell) on the right posterior side The mice were then subcutaneously implanted in the abdomen and then injected with PBS or 0.1 μg (0.005 mg / kg ), 0.5 μg (0.025 mg / kg), and 1 μg (0.05 mg / kg) / animal The treatments were administered intravenously (IV). 2 was administered every other day or every third day. H929 sc tumors showed rapid cell proliferation after tumor cell transplantation. For example, on day 8, the PBS and 0.1 μg BCMB72 treatment groups were detectable. Tumors in the group had a mean tumor volume of >500 mm on day 22. 3 It continued to grow until it reached 24. By day 1, the mean tumor volume in the PBS control group was 1000 mm 3 It was more than interesting. Importantly, sc H929 tumors were significantly higher in mice treated with 0.5 and 1 μg of BCMB72 than in mice treated with 0.5 and 1 μg of BCMB72. Thus, BCMB72 did not grow in mice at 0.5 and 1 μg / mL. It inhibited tumor formation in H929 human MM xenografts in all treated animals.

[0309] Example 22: H929 (human) in PBMC-humanized NSG mice treated with BCMB72 Quantification of soluble BCMA in mouse serum from multiple myeloma xenografts This study quantifies soluble BCMA concentrations in serum from H929 xenograft mice and to correlate soluble BCMA concentrations with tumor burden in these animals. Designed.

[0310] Briefly, serum from xenograft test samples was purchased from R&D Systems. The resulting BCMA was analyzed by enzyme-linked immunosorbent assay (ELISA). , diluted 1:50 in diluent and incubated overnight at 4°C. A was performed according to the manufacturer's protocol. The ELISA plate was set at 450 nm. The plate was read using the MD SpectraMax M5 (Molecular Device Each well in the ELISA was analyzed using a ELISA kit (Fabrics, Sunnyvale CA). Corresponding to serum from one mouse in the original xenograft study.

[0311] Soluble BC in mouse serum from mice treated with 1 μg and 0.5 μg of BCMB72 MA concentrations showed no significant effect when compared to PBS alone or 0.1 μg / mouse of BCMB72. These data were consistent with those from BCMB72 at 1 μg and 0.5 μg. There was no or minimal tumor growth in treated mice, supporting xenograft studies. These data suggest that soluble BCMA in serum samples may be an indicator of multiple myeloma. This provides insight into viable soluble BCMA as a potential biomarker for monitoring disease burden. This suggests that it may be useful to

[0312] [Table 14-1]

[0313] [Table 14-2]

[0314] [Table 14-3]

[0315] [Table 14-4]

[0316]

Table 14-5

[0317]

Table 14-6

[0318]

Table 14-7

[0319]

Table 14-8

[0320]

Table 14-9

[0321]

Table 14-10

[0322]

Table 14-11

Claims

1. A recombinant antibody, or antigen-binding fragment thereof, that immunospecifically binds to BCMA. wherein the antibody has a heavy chain and a light chain, the heavy chain comprising a. Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:4, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:6 D.R.3, b. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:4, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:6; c. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:7, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:6; d. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:4, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5 and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 19; e. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:4, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:8 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:6; f. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5 and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 19; g. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

19. Recombinant antibodies, or antigen-binding fragments thereof.

2. The antibody has a light chain CDR1 having the amino acid sequence of SEQ ID NO:24, an amino acid sequence of SEQ ID NO:25, and a light chain CDR2 having the amino acid sequence of SEQ ID NO:

26. The antibody, or antigen-binding fragment thereof, of claim 1 , further comprising:

3. The heavy chain of the antibody of (a) comprises the amino acid sequence of SEQ ID NO: 27, and the heavy chain of the antibody of (b) comprises (f) comprises the amino acid sequence of SEQ ID NO: 57, and the heavy chain of the antibody of (f) comprises the amino acid sequence of SEQ ID NO:

34. The heavy chain of the antibody of (k) comprises the amino acid sequence of SEQ ID NO: 39, and the heavy chain of the antibody of (l) comprises the amino acid sequence of SEQ ID NO:

39. The heavy chain comprises the amino acid sequence of SEQ ID NO: 40, and the heavy chain of the antibody of (m) comprises the amino acid sequence of SEQ ID NO:

58. or the heavy chain of the antibody (n) comprises the amino acid sequence of SEQ ID NO:

43.

2. The antibody or antigen-binding fragment of claim 1.

4. The antibody of claim 2 or 3, wherein the light chain of the antibody comprises the amino acid sequence of SEQ ID NO:

28. or an antigen-binding fragment.

5. The antibody or antigen-binding fragment thereof binds to the extracellular domain of human BCMA. The antibody or antigen-binding fragment according to any one of claims 1 to 4.

6. the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment; An antibody or antigen-binding fragment according to any one of claims 1 to 5.

7. The antigen-binding fragment may be a Fab fragment, a Fab2 fragment, or a single The antigen-binding fragment of any one of claims 1 to 6, which is a chain antibody.

8. The antibody or antigen-binding fragment thereof inhibits the interaction of BCMA and APRIL. The antibody or antigen-binding fragment of any one of claims 1 to 7.

9. The antibody or antigen-binding fragment has a BCMA and IC of approximately 5.9 nM for APRIL interaction ５０ The antibody or antibody according to claim 8, is the antigen-binding fragment.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof is an IgG.

2. The antibody or antigen-binding fragment according to claim 1 .

11. The antibody or antigen-binding domain according to any one of claims 1 to 10, which is of the IgG4 isotype. Combined fragment.

12. The IgG4 has an S228P substitution, an L234A substitution, and an L235A substitution in its Fc region. The antibody of claim 11 having a substitution.

13. The antibody or antigen-binding fragment thereof immunospecifically binds to human BCMA and The antibody or antigen according to any one of claims 1 to 12, which cross-reacts with macaque BCMA. Combined fragments.

14. The antibody or antigen-binding fragment thereof binds to BCMA on the surface of human myeloma cells. The antibody or antigen-binding fragment of any one of claims 1 to 13.

15. The antibody or antigen-binding fragment thereof binds to BCMA on the surface of human multiple myeloma cells. The antibody or antigen-binding fragment of any one of claims 1 to 14, which binds to

16. Expressing the antibody or antigen-binding fragment according to any one of claims 1 to 15. , recombinant cells.

17. The cell of claim 16 , wherein the cell is a hybridoma.

18. The cell of claim 16 , wherein the antibody is recombinantly produced.

19. Recombinant BCMAxCD3 bispecific antibody or its BCMAxCD3 bispecific binding fragment A segment, a) a first heavy chain (HC1); and b) a second heavy chain (HC2); and c) the first light chain (LC1); and d) a second light chain (LC2), HC1 is bound to LC1, and HC2 is bound to LC2, and they immunospecifically bind to CD3. To form a first antigen-binding site, HC1 is selected from SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:

70. LC1 includes sequence number 61, and LC2 includes sequence numbers 62, 63, and 64; To form a second antigen-binding site that immunospecifically binds to BCMA, HC2 has the sequence LC1 includes SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and LC2 includes SEQ ID NO: 24, SEQ ID NO: 25, and and SEQ ID NO:

26.

3. Bispecific Binding Fragments.

20. HC1 comprising SEQ ID NO:55, LC1 comprising SEQ ID NO:56, and HC comprising SEQ ID NO:65 2 and LC2 comprising SEQ ID NO:

76.

3. Bispecific antibodies or fragments thereof.

21. 21. The BCM of claim 20, wherein the antibody or bispecific binding fragment is an IgG. AxCD3 bispecific antibodies or bispecific binding fragments.

22. 19. The antibody or bispecific binding fragment is of the IgG4 isotype.

22. The BCMAxCD3 bispecific antibody or bispecific antibody according to any one of claims 20 to 21. Sex-binding fragment.

23. The antibody or bispecific binding fragment has 20. The antibody of claim 19, which immunospecifically binds to human BCMA with an affinity of at least 0.22 nM.

23. A BCMAxCD3 bispecific antibody or bispecific binding fragment according to claim 22.

24. The antibody or bispecific binding fragment thereof binds to BCMA on the surface of human myeloma cells.

24. The BCMAxCD3 bispecific antibody or bispecific Combined fragments.

25. The antibody or bispecific binding fragment thereof binds to B 25. The BCMAxCD3 bispecific antibody or bispecific antibody according to claims 19 to 24, which binds to CMA. Specific binding fragments.

26. The antibody or bispecific binding fragment has an EC ５０ Invitation BCMAxCD3 bispecific antibodies according to claims 19 to 25, which induce human T cell activation in vitro. Heteromeric antibodies or bispecific binding fragments.

27. The antibody or bispecific binding fragment has an EC ５０ Invitation The method according to any one of claims 19 to 26, which induces T cell-dependent cytotoxicity of BCMA-expressing cells in vitro. BCMAxCD3 bispecific antibodies or bispecific binding fragments.

28. 12. The antibody or bispecific binding fragment is not a BCMA agonist.

9. A BCMAxCD3 bispecific antibody or bispecific binding fragment according to claim 9-27.

29. The antibody or bispecific binding fragment exhibits NF-κB activation at a concentration of less than 10 nM. The BCMA×CD3 bispecific antibody or bispecific antibody according to claims 19 to 28 does not change Heteromer bond fragments.

30. Expressing the antibody or bispecific binding fragment according to any one of claims 19 to 29. Recombinant cells.

31. The cell of claim 30 , wherein the cell is a hybridoma.

32. A method for treating a subject having cancer, comprising administering to said subject a compound according to any one of claims 19 to 29. Therapeutic efficacy of the described BCMAxCD3 bispecific antibodies or bispecific binding fragments and administering to a subject in need thereof an amount sufficient to treat the cancer. method.

33. A method for inhibiting the growth or proliferation of cancer cells, comprising the step of: Therapeutic use of the BCMAxCD3 bispecific antibody or bispecific binding fragment according to one aspect of the present invention to inhibit the growth or proliferation of cancer cells.

34. A method for redirecting T cells to BCMA-expressing cancer cells, comprising the steps of: A BCMAxCD3 bispecific antibody or bispecific binding fragment according to any one of claims 1 to 4. to redirect T cells to the cancer.

35. 35. The method of claim 32, 33, or 34, wherein the cancer is a hematological cancer.

36. 36. The method of claim 35, wherein the hematological cancer is a BCMA-expressing B cell cancer.

37. 37. The method of claim 36, wherein the BCMA-expressing B cell cancer is multiple myeloma.

38. 33. The method of claim 32, further comprising administering a second therapeutic agent.

39. 39. The method of claim 38, wherein the second therapeutic agent is a chemotherapeutic agent or a targeted anti-cancer therapeutic agent. method.

40. The chemotherapeutic agent may be cytarabine, anthracycline, histamine dihydrochloride, or indomethacin.

40. The method of claim 39, wherein the antibody is talukin 2.

41. The BCMAxCD3 bispecific antibody or the bispecific antibody according to any one of claims 19 to 29. A pharmaceutical composition comprising an isomeric binding fragment and a pharma- ceutically acceptable carrier.

42. The cell according to any one of claims 30 to 31 is cultured to obtain a cell according to claims 19 to 2.

10. The BCMA×CD3 bispecific antibody or bispecific binding fragment according to any one of claims 9 to 10. A method for generating a metric.

43. The BCMAxCD3 bispecific antibody or the bispecific antibody according to any one of claims 19 to 29. An isolated polypeptide encoding the isomeric fragment HC1, HC2, LC1, or LC2. Synthetic polynucleotides.

44. A BCMAxCD3 bispecific antibody as defined in any one of claims 19 to 29. or a bispecific binding fragment, and / or a polynucleotide as defined in claim 44. A kit comprising:

Images