B cell maturation antigen-binding proteins

专利摘要:
B cell maturation antigen-binding proteins with enhanced binding affinities and improved ability to mediate the killing of cancer cells expressing B cell maturation antigen (BCMA) are disclosed in this document. Compositions are further provided which comprise the binding proteins disclosed herein and methods of treating a cancer or metastasis of it using such formulations.
公开号:BR112020007249A2
申请号:R112020007249-4
申请日:2018-10-12
公开日:2020-10-13
发明作者:Holger Wesche；Bryan D. LEMON；Richard J. Austin
申请人:Harpoon Therapeutics, Inc.；
IPC主号:

专利说明:

[0001] [0001] This application claims the benefit of Provisional Application No. US 62 / 572,375 filed on October 13, 2017, which is incorporated by reference in this document in its entirety.
[0002] [0002] Cancer is the second leading cause of human death after coronary heart disease. Worldwide, millions of people die from cancer each year. In the United States alone, cancer causes the death of more than half a million people each year, with some 1.4 million new cases diagnosed each year. Although deaths from heart disease have declined significantly, those resulting from cancer in general are on the rise. Early in the next century, cancer is predicted to become the leading cause of death.
[0003] [0003] Furthermore, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients suffer from strong anxieties triggered by the science of the potential for recurrence or treatment failure. Many cancer patients suffer from significant physical weakness after treatment.
[0004] [0004] In general terms, the fundamental problem in the management of the most deadly cancers is the lack of effective and non-toxic systemic therapies. Cancer is a complex disease characterized by genetic mutations that lead to uncontrolled cell growth. Cancer cells are present in all organisms and, under normal circumstances, their overgrowth is strictly regulated by several physiological factors. SUMMARY OF THE INVENTION
[0005] [0005] The present disclosure provides single domain B cell maturation antigen (BCMA) binding proteins that can be used to diagnose and treat indications correlated to BCMA expression.
[0006] [0006] A single domain B cell maturation agent (BCMA) binding protein is provided herein, comprising CDR1, CDR2 and CDR3 complementarity determining regions, where (a) the amino acid sequence of CDR1 is as shown in X1X2X3XaXsXeX7PXsG (SEQ ID NO: 1), where X1h is T or S; X2 is N, D, or S; X; 3 is I, D, Q, H, Vou E; Xa is F, S, E, A, T, M, V, I, D, OQ, P, Rou G; Xs is S, M, R or N; X; is LI, K, S, T, R, E, D, N, V, H, L, A, QouG; X7 is S, T, Y, RouN; and Xg is M, G or Y; (b) the amino acid sequence of CDR2 is as shown in AIXsGX: oX: 1: TX12YADSVK (SEQ ID NO: 2), where x; is H, N, or S; X1 is F, G, K, R, P, D, OQ, H, E, N, T, S, A, IT, L or V; Xu is S, Q, E, T, K or D; and X12 is L, V, 1, F, Y, or W; and (c) the amino acid sequence of CDR3 is as shown in VPWGX: 3YHPX14X15sVX16 (SEQ ID NO: 3), where X13 is D, I, T, K, R, A, E, S or Y; Xu is R, G, L, K, T, Q, S or N; X15 is N, K, E, V, R, M or D; and Xi; It is Y, A, V, K, H, L, M, T, R, Qi, C, S or N.
[0007] [0007] In one embodiment, CDR1 does not comprise an amino acid sequence of SEQ ID NO: 473. In one embodiment, CDR2Q does not comprise an amino acid sequence of SEQ ID NO: 474. In one embodiment, CDR3 does not comprise an amino acid sequence of SEQ ID NO:
[0008] [0008] A single domain BCMA-binding protein is provided herein in which said protein comprises the following formula: fl-rl-f2-r2-f3-r3-f4, where, rl is SEQ ID NO: 1; r2 is SEQ ID NO: 2; and r3 is SEQ ID NO: 3; and where fl, f2, f £ 3 and f4 are framework residues selected so that said protein is about eighty percent (80%) to about 99% identical to the amino acid sequence shown in SEQ ID NO: 346 or
[0009] [0009] In some non-limiting examples, rl comprises an amino acid sequence shown as any of SEQ ID NOS: 4-117.
[0010] [0010] In some non-limiting examples, r2 comprises an amino acid sequence shown as any of SEQ ID NOS: 118-231.
[0011] [0011] In some non-limiting examples, r3 comprises an amino acid sequence shown as any of SEQ ID NOS: 232-345.
[0012] [0012] In other non-limiting examples, the protein comprises an amino acid sequence shown as any of SEQ ID NOS: 346-460.
[0013] [0013] In a single domain BCMA binding protein, fl can comprise SEQ ID NO: 461 or 462.
[0014] [0014] In a single domain BCMA binding protein, f2 can comprise SEQ ID NO: 463.
[0015] [0015] In a single domain BCMA binding protein, £ 3 can comprise SEQ ID NO: 464 or 465.
[0016] [0016] In a single domain BCMA binding protein, where f4 can comprise SEQ ID NO: 466 or 467.
[0017] [0017] In a non-limiting example, rl comprises SEQ ID NO: 76, 114, 115, 116 or 117. In a non-limiting example, rl comprises SEQ ID NO: 76.
[0018] [0018] In a non-limiting example, rl comprises SEQ ID
[0019] [0019] In a non-limiting example, rl comprises SEQ ID NO: 114, r2 comprises SEQ ID NO: 228 and r3 comprises SEQ ID NO: 342.
[0020] [0020] In a non-limiting example, rl comprises SEQ ID NO: 115, r2 comprises SEQ ID NO: 229 and r3 comprises SEQ ID NO: 343.
[0021] [0021] In a non-limiting example, rl comprises SEQ ID NO: 117, r2 comprises SEQ ID NO: 231 and r3 comprises SEQ ID NO: 345.
[0022] [0022] In a non-limiting example, rl comprises SEQ ID NO: 116, r2 comprises SEQ ID NO: 230 and r3 comprises SEQ ID NO: 344.
[0023] [0023] A domain BCMA-binding protein can have an elimination half-life of at least 12 hours, at least 20 hours, at least 25 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 100 hours or more. In some embodiments, the single-domain BCMA-binding protein further comprises an Fc domain. In some embodiments, the single domain BCMA-binding protein further comprises an anti-cancer agent.
[0024] [0024] A single domain anti-BCMA BCMA binding protein 253BH10 SEQ ID NO: 472 or a humanized version of that llama sequence, BH2T, SEQ ID NO 346, in which one or more residues is provided, is provided herein amino acid selected from amino acid positions 26, 27, 28, 29, 30, 31, 32 and 34 of CDR1; positions 52, 54, 55 and 57 of CDR2; and positions 101, 105, 106 and 108 of CDR3 are substituted, where amino acid position 26, if substituted, is replaced by S; amino acid position 27, if substituted, is replaced by D or S; amino acid position 28, if substituted, is replaced by D, Q, H, V or E; amino acid position 29, if substituted, is replaced by S, E, A, T, M, V, I, D, Q, P, R or G; amino acid position 30, if substituted, is replaced by M, R or N; amino acid position 31, if substituted, is replaced by K, S, T, R, E, D, N, V, H, L, A, Q or G; amino acid position 32, if substituted, is replaced by T, Y, R or N; amino acid position 34, if substituted, is replaced by G or Y; amino acid position 52, if substituted, is replaced by N or S; amino acid position 54, if substituted, is replaced by G, K, R, P, D, Q, H, E, N, T, S, A, I, L or V; amino acid position 55, if substituted, is replaced by Q, E, T, K or D; amino acid position 57, if substituted, is replaced by V, IT, F, Y, or NWNW; amino acid position 101, if substituted, is replaced by I, T, K, R, A, E, S or Y; amino acid position 105, if substituted, is replaced by G, L, K, T, Q, S or N; amino acid position 106, if substituted, is replaced by K, E, V, R, M or D; and amino acid position 108, if substituted, is replaced by A, V, K, H, L, M, T, R, Q, C, S or N.
[0025] Such single domain BCMA binding protein can be human, humanized, affinity matured or a combination thereof.
[0026] [0026] A method for treating or ameliorating B cell lineage cancer in an individual in need thereof is provided herein, which comprises administering to the individual a single domain BCMA-binding protein described herein.
[0027] [0027] A multispecific binding protein comprising the single domain BCMA binding protein described herein is provided herein.
[0028] [0028] A method for treating or ameliorating B cell lineage cancer in an individual in need thereof is provided herein comprising administering to the individual a multispecific binding protein described herein.
[0029] [0029] B-cell lineage cancer can be a primary cancer or a metastatic cancer.
[0030] [0030] A cancer cell line B to be treated with the methods described in this document can be a multiple myeloma, a leukemia, a lymphoma. INCORPORATION BY REFERENCE
[0031] [0031] All publications, patents and patent applications mentioned in this specification are incorporated into this document, by reference, to the same extent as if each individual publication, patent or patent application had been specifically indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] [0032] The innovative features of the invention are presented with particularity in the attached claims. A better understanding of the features and advantages of the invention will be obtained with reference to the following detailed description that defines the illustrative modalities, in which the principles of the invention are used, and the accompanying drawings, in which:
[0033] [0033] Fig. 1 illustrates the effect of exemplary BCMA targeting molecules (01H08, O01FO0O7, 02F02 and BH253), containing an anti-BCMA binding protein according to the present disclosure in the extermination of BCMA-expressing EJM cells compared to a negative control.
[0034] [0034] Fig. 2 is a SDS-PAGE image of representative purified BCMA-specific molecules. Line 1: O1FO7-M34Y TriTAC not reduced; Line 2: 01FO7-M34G-TriTAC not reduced; Line 3: 02B05 TriTAC not reduced; Line 4: 02GO0O2-M34Y TriTAC not reduced; Line 5: 02G0O2 M34G TriTAC not reduced; Line 6: Wide Range SDS-PAGE Standard (Bio-Rad nº 1610317); Line 7: O1FOT-M34Y TriTAC not reduced; Line 8: 01F0O7-M34G- TriTAC not reduced; Line 9: 02B05 TriTAC not reduced; Line 10: 02G02-M34Y TriTAC not reduced; Line 11: 02GO02 M34G TriTAC not reduced; and Line 12: Wide Range SDS-PAGE Standard (Bio-Rad nº 1610317).
[0035] [0035] Figs. 3A-3I illustrate the effect of exemplary BCMA triespecific targeting molecules containing an anti-BCMA binding protein according to the present disclosure on the extermination of Jekol cells (Figs. 3A-3C), MOLP-8 (Figs. 3D-3F ) or OPM-2 (Figs. 3G-3I) that express BCMA compared to a negative control.
[0036] [0036] Figs. 4A-4D illustrates the binding of an exemplary BCMA-specific targeting protein containing a BCMA-binding protein of this disclosure (02B05) to purified T cells from four different human donors, donor 02 (Fig. 4A), donor 35 (Fig. 4B),
[0037] [0037] Figs. 5A-5F illustrates the binding of an exemplary BCMA triespecific targeting protein (02B05) to cells expressing BCMA, NCI-H929 (Fig. 5A), EJM (Fig. 5B), OPM2 (Fig. 5D), RPMI8226 (Fig . 5E); Or cell lines that lack expression of BCMA, NCI-H510A (Fig. 5C), and DMS-153 (Fig. 5F).
[0038] [0038] Fig. 6 illustrates the results of a TDCC assay using an exemplary BCMA-specific targeting protein (02B05) and BCMA-expressing EJM cells, in the presence or absence of human serum albumin (HSA).
[0039] [0039] Fig. 7 illustrates the results of a TDCC assay using an exemplary BCMA triespecific targeting protein (02B05) and BCMA-expressing EJM cells, using varying ratio of effector cells to target cells.
[0040] [0040] Fig. 8 illustrates the results of a TDCC assay using an exemplary BCMA triespecific targeting protein (02B05) and BCM expressing OPM2 cells, using varying effector-to-target cell ratios.
[0041] [0041] Fig. 9 illustrates the results of a TDCC assay using an exemplary BCMA triespecific targeting protein (02B05) and NCI-H929 cells expressing BCMA, using varying time points and an effector cell to cell ratio 1: 1 target.
[0042] [0042] Fig. 10 illustrates the results of a TDCC assay using an exemplary BCMA-specific targeting protein (02B05), EJM cells that express
[0043] [0043] Fig. 11 illustrates the results of a TDCC assay using an exemplary BCMA triespecific targeting protein (02B05), NCI-H92º cells expressing BCMA, and T cells from four different donors, in the presence of serum albumin (HSA).
[0044] [0044] Fig. 12 illustrates the results of a TDCC assay using an exemplary BCMA triespecific targeting protein (02B05), BCM expressing OPM2 cells, and T cells from four different donors in the presence of human serum albumin ( HSA).
[0045] [0045] Fig. 13 illustrates the results of a TDCC assay using an exemplary BCMA triespecific targeting protein (02B05), RPMI8226 cells expressing BCMA, and T cells from four different donors, in the presence of human serum albumin ( HSA).
[0046] [0046] Fig. 14 illustrates the results of a TDCC assay using an exemplary BCMA triespecific targeting protein (02B05), OVCAR8 cells that do not express BCMA, and T cells from four different donors in the presence of human serum albumin (HSA).
[0047] [0047] Fig. 15 illustrates the results of a TDCC assay using an exemplary BCMA triespecific targeting protein (02B05), NCI-HS510A cells that do not express BCMA, and T cells from four different donors, in the presence of albumin human serum (HSA).
[0048] [0048] Fig. 16 illustrates the results of a TDCC assay using an exemplary BCMA triespecific targeting protein (02B05), NCI-H929 cells that express BCMA, and peripheral blood mononuclear cells (PBMC) from two different cinomolgos, in the presence of human serum albumin (HSA).
[0049] [0049] Fig. 17 illustrates the results of a TDCC assay using an exemplary BCMA-specific targeting protein containing a BCMA-binding protein from this disclosure (02B05), RPMI8226 cells that express BCMA, and peripheral blood mononuclear cells ( PBMC) from two different cinomolgus donors in the presence of human serum albumin (HSA).
[0050] [0050] Fig. 18 illustrates the expression level of CD69 T cell activation biomarker, after a TDCC assay using an exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure (02B05) and cells from BCJ expressing FMA.
[0051] [0051] Fig. 19 illustrates the CD25 T cell activation biomarker expression level after a TDCC assay using an exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure (02B05) and cells from BCJ expressing FMA.
[0052] [0052] Fig. 20 illustrates the CD69 T cell activation biomarker expression level after a TDCC assay using an exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure (02B05) and cells from OPM2 that express BCMA.
[0053] [0053] Fig. 21 illustrates the expression level of CD25 T cell activation biomarker, after a
[0054] [0054] Fig. 22 illustrates the CD69 T cell activation biomarker expression level after a TDCC assay using an exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure (02B05) and cells from RPMI8226 that express BCMA.
[0055] [0055] Fig. 23 illustrates the CD25 T cell activation biomarker expression level after a TDCC assay using an exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure (02B05) and cells from RPMI8226 that express BCMA.
[0056] [0056] Fig. 24 illustrates the level of CD69 T cell activation biomarker expression after a TDCC assay using an exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure (02B05) and cells from OVCAR8 that do not express BCMA.
[0057] [0057] Fig. 25 illustrates the expression level of CD25 T cell activation biomarker after a TDCC assay using an exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure (02B05) and cells from OVCAR8 that do not express BCMA.
[0058] [0058] Fig. 26 illustrates the expression level of CD69 T cell activation biomarker, after a
[0059] [0059] Fig. 27 illustrates the level of CD25 T cell activation biomarker expression after a TDCC assay using an exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure (02B05) and cells from NCI-H510A that do not express BCMA.
[0060] [0060] Fig. 28 illustrates the level of expression of a cytokine, TNF-a, in co-cultures of T cells and target cells expressing BCMA (EJM cells) treated with increasing concentrations of an exemplary BCMA targeting specific protein containing a BCMA-binding protein from this disclosure (02B05) or with a negative control GFP-specific protein.
[0061] [0061] Fig. 29 illustrates reduction of tumor growth in an RPMI8226 xenograft model, treated with an exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure (02B05), in varying concentrations, or with a control.
[0062] [0062] Fig. 30 illustrates reduced tumor growth in a Jekol xenograft model, treated with an exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure (02B05), in varying concentrations, or with a control.
[0063] [0063] Fig. 31 illustrates concentration of BCMA targeting specific protein in serum samples from cynomolgus monkeys dosed with varying concentrations of an exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure (02B05).
[0064] [0064] Fig. 32 illustrates the results of a TDCC assay using BCMA triespecific targeting protein obtained from serum samples of dosed cynomolgus monkeys with varying concentrations of an exemplary BCMA triespecific targeting protein containing a protein binding to BCMA of this disclosure (02B05), BCMA-expressing EJM cells, and purified human T cells, in the presence of serum from cynomolgus monkeys that have not been exposed to a specific BCMA targeting protein. DETAILED DESCRIPTION OF THE INVENTION
[0065] [0065] Although the preferred embodiments of the present invention have been presented and described in this document, it will be obvious to those skilled in the art that such modalities are provided by way of example only. Various variations, changes and substitutions will now occur for those skilled in the art without distancing themselves from the invention. It is to be understood that various alternatives to the modalities of the invention described in this document can be employed in the practice of the invention. It is intended that the following claims define the scope of the invention and that the methods and structures within the scope of those claims and their equivalents are covered by them. Certain definitions
[0066] [0066] The terminology used in this document is for the purpose of describing particular cases only and is not intended to be limiting. As used in this document, the singular forms "one", "one", "o" and "a" are intended to also include plural forms, unless the context clearly indicates otherwise. In addition, to the extent that the terms "which includes", "includes", "which has", "has", "with", or variants thereof are used in the detailed description and / or in the claims, such terms are intended to be inclusive in a similar way to the term “who understands”.
[0067] [0067] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by an element of common knowledge in the art, which will depend in part on how the value is measured or determined, for example, the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, by practice at the given value. Where particular values are described in the application and in the claims, unless otherwise stated, the term "about" must be assumed to mean an acceptable error range for the particular value.
[0068] [0068] The terms "individual", "patient" or "subject" are used interchangeably. None of the terms require or are limited to the situation characterized by the supervision (for example, constant or intermittent) of a health care worker (for example, a doctor, a registered nurse, a clinical nurse, a medical assistant, a worker or a hospice).
[0069] [0069] An "antibody" typically refers to a Y-shaped tetrameric protein that comprises two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Human light chains comprise a varied domain (VL) and a constant domain (CL) in which the constant domain can be readily classified as kappa or lambda based on the amino acid sequence and gene locations. Each heavy chain comprises a varied domain (VH) and a constant region, which, in the case of IgG, IgA and IgD, comprises three domains called CH1, CH2 and CH3 (IgM and Ige have a fourth domain, CH4). In the IgG, IgA and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a segment rich in proline and cysteine of varying length (usually about 10 to about 60 amino acids in IgG). The varied domains in both the light and heavy chain are linked to the constant domains by a "J" region of about 12 or more amino acids and the heavy chain also has a "D" region of about 10 additional amino acids. Each class of antibody further comprises intrachain and interchain disulfide bonds formed by paired cysteine residues. There are two types of native disulfide bonds or bridges in immunoglobulin molecules: intrachain and interchain disulfide bonds. The location and number of interchain disulfide bonds would come according to the species and class of immunoglobulin. Interchain disulfide bonds are located on the surface of the immunoglobulin, are accessible to the solvent and are, in fact, relatively and easily reduced. In the human Igel isotype, there are four interchain disulfide bonds, one from each heavy chain to the light chain and two between the heavy chains. Interchain disulfide bonds are not necessary for chain association. As is well known, the heavy chain cysteine-rich IgG1 hinge region has generally been retained to consist of three parts: an upper hinge, a central hinge and a lower hinge. One skilled in the art will appreciate that the IgGl hinge region contains the cysteines in the heavy chain comprising the interchain disulfide bonds (two heavy / heavy, two heavy / light), which provide structural flexibility that facilitates Fab movements. of disulfide interchain between the IgGl light and heavy chain is formed C214 of the kappa or lambda light chain and C220 in the upper hinge region of the heavy chain. The interchain heavy chain disulfide bonds are at positions C226 and C229 (all numbered by the EU index according to Kabat, et al., Infra.)
[0070] [0070] As used herein, the term "antibody" includes polyclonal antibodies, multiclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, grafted CDR antibodies, human antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies, antibodies bispecific, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies,
[0071] [0071] The term "framework" or "FR" residues (or regions) refer to residues of varied domain other than residues from the hypervariate region or CDR as defined in this document. A “human consensus framework” is a framework that represents the most common amino acid residue in a selection of human immunoglobulin VH or VL framework sequences.
[0072] [0072] As used herein, the term "varied region" or "varied domain" refers to the fact that certain portions of the varied domains differ extensively in sequence between antibodies and are used in the binding and specificity of each particular antibody to your particular antigen. However, variability is not evenly distributed across all varied domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariate regions in the varied domains of heavy and light chains. The most highly conserved portions of varied domains are called framework (FR). The varied domains of native light and heavy chains each comprise four FR regions, largely adopting a B-leaf configuration, connected by three CDRs, which form loops that connect and, in some cases, form part of the leaf structure -bB. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs in the other chain, contribute to the formation of the antibody antigen binding site (see, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions, such as dividing the antibody into antibody-dependent cellular toxicity. "Variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof, refers to the numbering system used for heavy chain variable domains or antibody light chain variable domains in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md, USA. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or more amino acids that correspond to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a varied heavy chain domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (eg residues 82a, 82b and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by aligning regions of homology of the antibody sequence with a "standard" numbered Kabat sequence. The CDRs in the present disclosure are not intended to necessarily correspond to the Kabat numbering convention.
[0073] [0073] In some embodiments, BCMA binding proteins comprise a heavy chain only antibody, such as a VH domain or a VHH domain. In some cases, BCMA binding proteins comprise a heavy chain only antibody that is a engineered human VH domain. In some examples, the manipulated human VH domain is produced by moving phage display libraries. In some embodiments, BCMA binding proteins comprise a VHH. The term "VHH", as used herein, refers to the light chain devoid of single chain antibody binding domain. In some cases, a VHH is derived from an antibody of the type that can be found in Camelidae or cartilaginous fish that is naturally devoid of light chains or for an unimmunized, synthetic VHH that can be built accordingly. Each heavy chain comprises a variable region encoded by exons V, D and J. A VHH, in some cases, is a natural VHH, like a VHH derived from Camelids or a recombinant protein that comprises a varied heavy chain domain. In some modalities, the VHH is derived from a species selected from the group consisting of camels, llamas, vicuñas, guanacos and cartilaginous fish (such as, but without limitation, sharks). In another modality, the VHH is derived from an alpaca (such as, but not limited to, a Huacaya Alpaca or a Suri Alpaca).
[0074] [0074] As used herein, the term "percentage (%) of amino acid sequence identity" in relation to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the sequence specific, after aligning the sequences and inserting gaps if necessary, to achieve the maximum percentage of sequence identity and not considering any conservative substitutions as part of the sequence identity. The alignment for purposes of determining percentage of amino acid sequence identity can be achieved in a number of ways that are included in the skill of the element versed in the technique, for example, with the use of publicly available computer software programs, such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN software , BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). The elements skilled in the art can determine p aramers suitable for measuring alignment, which includes any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.
[0075] [0075] As used herein, the term “elimination half-life” is used in its common sense, as it is described in Goodman and Gilliman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman and Alfred Gilman, eds., 6th ed. 1980). Briefly, the term is intended to cover a quantitative measure of the course of drug elimination time. The elimination of most drugs is exponential (that is, it follows first-order kinetics), since drug concentrations generally do not address those required for saturation of the elimination process. The rate of an exponential process can be expressed by its rate constant, k, which expresses the fractional change per unit of time, or per half-life, ti / 2 the time required to complete 50% of the process. The units of these two constants are time ** and time, respectively. A first order rate constant and the reaction half-life are simply related (kxt1 / 2 = 0.693) and can be interchanged accordingly. Since first-order elimination kinetics indicate that a constant drug fraction is lost per unit time, a plot of the drug concentration log versus time is linear at all times following the initial distribution phase (that is,
[0076] [0076] As used herein, the term "binding affinity" refers to the affinity of the proteins described in the disclosure to their binding targets and is expressed numerically using "Kd" values. If two or more proteins are indicated to have binding affinities comparable to their binding targets, then the Kd values for binding the respective proteins to their binding targets are within +2 times each. If two or more proteins are indicated to have binding affinities comparable to the single binding target, then the Kd values for binding the respective proteins to said single binding target are within t + 2 times each. If a protein is indicated to bind two or more targets with comparable binding affinities, then the Kd values for binding said protein to the two or more targets are within +2 times each. In general, a higher Kd value corresponds to a weaker bond. In some embodiments, “Kd” is measured by a radiolabeled antigen binding assay (RIA) or surface plasmon resonance assays using a BIACOREGS-2000 or BIACOREGO-3000 (BIAcore, Inc., Piscataway, NJ, USA ). In certain embodiments, a term "in fee" or "association fee" or "association fee" or "kon" and a term "out of fee" or "dissociation fee" or "dissociation fee" or "koff" are also determined with the surface plasmon resonance technique using a BIACOREG-2000 or a BIACOREG-3000 (BIAcCOre, Inc.,
[0077] [0077] BCMA binding proteins, pharmaceutical compositions as well as nucleic acids, recombinant expression vectors and host T cells for producing such BCMA binding proteins are described in this document. Also provided are methods of using the BCMA binding proteins disclosed in the prevention and / or treatment of diseases, conditions and disorders. BCMA binding proteins are capable of binding specifically to BCMA. In some embodiments, BCMA binding proteins include additional domains, such as a CD3 binding domain and an albumin binding domain. B cell maturation antigen (BCMA)
[0078] [0078] B cell maturation antigen (BCMA, TNFRSF17, CD269) is a transmembrane protein that belongs to the tumor necrosis family receptor (TNFR) superfamily that is mainly expressed in terminally differentiated B cells. BCMA expression is limited to the B cell lineage and mainly present in plasma and plasmoblast cells and, to some extent, in memory B cells, but virtually absent from peripheral and virgin B cells. BCMA is also expressed in multiple myeloma (MM) cells, in leukemia cells and lymphoma cells.
[0079] [0079] BCMA was marked by the molecular analysis of a t (4; 16) translocation (q26; p13) found in a human intestinal T cell lymphoma and a frame sequence was mapped to the chromosome line 16p13.1.
[0080] [0080] Human BCMA ccDNA has a 552 bp open reading frame encoding a 184 amino acid polypeptide. The BCMA gene is organized into three exons that are separated by two introns, each flanked by the consensus splicing sites of GT donors and AG acceptors, and encoded for a 1.2 Kb transcript. The BCMA protein structure includes an integral protein transmembrane based on a central 24 amino acid hydrophobic region in an alpha helix structure.
[0081] [0081] The murine BCMA gene is located on the chromosome
[0082] [0082] BCMA is not expressed on the cell surface, but is preferably located on the Golgi apparatus. The amount of BCMA expression is proportional to the stage of cell differentiation (highest in plasma cells).
[0083] [0083] BCMA is involved in the development of B cell and homeostasis due to its interaction with its BAFF ligands (cell activation factor B, also known as TALL-1 or TNFSF13B) and APRIL (a proliferation-inducing ligand).
[0084] [0084] BCMA regulates different aspects of humoral immunity, B cell development and homeostasis together with its family members TACI (cyclophilin ligand interactor and transmembrane activator) and BAFF-R (B cell activation factor receptor, also known as a member of the tumor necrosis factor 13C receptor superfamily). BCMA expression later appears in B cell differentiation and contributes to long-term survival of plasmoblasts and plasma cells in bone marrow. BCMA also supports the growth and survival of multiple myeloma (MM) cells.
[0085] [0085] BCMA is known mainly for its functional activity in mediating the survival of plasma cells that maintain humoral immunity in the long term.
[0086] [0086] There is a need to have treatment options for solid tumor diseases related to BCMA overexpression, such as multiple cancer myeloma, leukemias and lymphomas. The present disclosure provides, in certain embodiments, single domain proteins that specifically bind to BCMA on the surface of tumor target cells. BCMA binding proteins
[0087] [0087] BCMA binding proteins are contemplated in this document. In this document, in certain embodiments, binding proteins, such as single-domain anti-BCMA antibodies or antibody variants, are provided that bind to an epitope on the BCMA protein. In some embodiments, the BCMA-binding protein binds to a human BCMA protein that comprises the sequence of SEQ ID NO: 468. In some embodiments, the BCMA-binding protein binds to a human BCMA protein that comprises a truncated sequence compared to SEQ ID NO: 468. In a non-limiting example, the BCMA-binding protein binds to a human BCMA protein that comprises amino acid residues 5-51 of SEQ ID NO: 468.
[0088] [0088] In some embodiments, the BCMA binding proteins of the present disclosure can be expressed in a multiple domain protein that includes additional immunoglobulin domains. Such multiple domain proteins can act through inhibition based on tumor growth immunotoxin and induction of antibody-dependent cell cytotoxicity (ADCC). In some embodiments, the multiple domain proteins containing the BCMA binding proteins of the present disclosure exhibit complement-dependent cytotoxicity (CDC) activity. In some embodiments, the multiple domain proteins containing the BCMA binding proteins of the present disclosure exhibit both ADCC and CDC activity against cancer cell expression BCMA. An amino acid sequence from an Fc domain can be added to the BCMA binding proteins described herein to induce ACDD or CDC. Fcs domain amino acid sequences are known in the art and are contemplated herein.
[0089] [0089] A BCMA-binding protein described herein binds to the extracellular domain of BCMA. In one case, a BCMA-binding protein described herein binds to amino acid residues 5-51 of human BCMA.
[0090] [0090] In some embodiments, the BCMA-binding protein is an anti-BCMA antibody or an antibody variant. As used herein, the term "antibody variant" refers to the variants and derivatives of an antibody described herein. In certain embodiments, the amino acid sequence variants of the anti-BCMA antibodies described herein are contemplated. For example, in certain embodiments, the amino acid sequence variants of anti-BCMA antibodies described herein are contemplated to improve the binding affinity and / or other biological properties of the antibodies. Exemplary methods for preparing amino acid variants include, but are not limited to, introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions of and / or insertions in and / or substitutions of residues within the amino acid sequences of the antibody.
[0091] [0091] Any combination of deletion, insertion and substitution can be done to achieve the final construct, since the final construct has the desired characteristics, for example, antigen binding. In certain embodiments, antibody variants that have one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include the framework regions and CDRs. Examples of such substitutions are described below. Amino acid substitutions can be introduced into an antibody of interest and the products displayed for a desired activity, for example, binding to retained / enhanced antigen, decreased immunogenicity or enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Both conservative and non-conservative amino acid substitutions are contemplated to prepare antibody variants.
[0092] [0092] In another example of a substitution to create a variant anti-BCMA antibody, one or more residues of the hypervariable region of a parental antibody are replaced. In general, variants are then selected based on improvements in desired properties compared to a parent antibody, for example, increased affinity, reduced affinity, reduced immunogenicity, increased pH dependence on binding. For example, an affinity matured variant antibody can be generated, for example, using phage display-based affinity maturation techniques such as those described herein and known in the field.
[0093] [0093] In some embodiments, the BCMA-binding protein described herein is a single domain antibody such as a heavy chain (VA) varied domain, a sdAb derived domain (llama, peptide, linker or a BCMA-specific small molecule entity. In some embodiments, the BCMA-binding domain of the BCMA-binding protein described herein is any domain that binds to BCMA that includes, but is not limited to, domains of a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In certain embodiments, the BCMA-binding protein is a single domain antibody. In other embodiments, the BCMA-binding protein is a peptide. In additional embodiments, the BCMA-binding protein is a small molecule.
[0094] [0094] In general, it should be noted that the term single domain antibody as used in this document in its broadest sense is not limited to a specific biological source or a specific preparation method. Single domain antibodies are antibodies whose complementary determination regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and different domain domains from those derived from antibodies. The single domain antibodies can be any of the art or any future single domain antibodies. single domain antibodies can be derived from any species that includes, but is not limited to, mouse, human, camel, llama, goat, rabbit and bovine. For example, in some embodiments, the single domain antibodies of the disclosure are obtained: (1) by isolating the VHH domain from a naturally occurring heavy chain antibody;
[0095] [0095] In one embodiment, a single domain antibody corresponds to the VHH domains of naturally occurring heavy chain antibodies directed against BCMA. As further described herein, such VHH sequences can generally be generated or obtained by adequately immunizing a Llama species with BCMA, (i.e., in order to enhance an immune response and / or heavy chain antibodies directed against BCMA), by obtaining an appropriate biological sample from said Llama (such as a blood sample, serum sample or B cell sample), and by generating VHH sequences directed against BCMA, starting from the example type, using any suitable technique known in the field.
[0096] [0096] In another embodiment, such naturally occurring VHH domains against BCMA are obtained from virgin libraries of Camelid VHH sequences, for example, by displaying such a library using BCMA, or at least one part, fragment , antigenic determinant or epitope thereof using one or more display techniques known in the field. Such libraries and techniques are, for example, described in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively, enhanced semisynthetic or synthetic libraries derived from virgin VHH libraries are used, as VHH libraries obtained from virgin VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as, for example, described in WO 00 / 43507.
[0097] [0097] In an additional embodiment, yet another technique for obtaining VHH sequences directed against BCMA, involves adequately immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e., in order to increase an immune response and / or heavy chain antibodies directed against BCMA), obtain a suitable biological sample from said transgenic mammal (such as a blood sample, serum sample or B cell sample) and then generate VHH sequences directed against BCMA, starting from said using any suitable technique known in the field. For example, for that purpose, heavy chain antibody expression mice or rats and the additional methods and techniques described in WO 02/085945 and WO 04/049794 can be used.
[0098] [0098] In some embodiments, an anti-BCMA antibody, as described herein, comprises single domain antibody with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but which has been "humanized", that is, by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and, in particular, in the framework sequences) by one or more of the amino acid residues that occur at the position (or positions ) corresponding in a VH domain of a conventional human 4-chain antibody (for example, as indicated above). This can be done in a manner known in the field, which will be clear to the conversant person, for example, based on the additional description in this document. Again, it should be noted that such humanized anti-BCMA single domain antibodies of the disclosure are obtained in any suitable manner known per se (i.e., as indicated under points (1) - (8) above) and are thus not strictly limited to the polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material. In some additional embodiments, a single domain BCMA antibody, as described herein, comprises a single domain antibody with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but which has been "camelized ", that is, by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody with one or more of the amino acid residues that occur at the position (or positions ) corresponding in a VHH domain of a heavy chain antibody.
[0099] [0099] Other methods and techniques suitable for obtaining the single domain anti-BCMA antibody of the disclosure and / or nucleic acids encoding it, starting from naturally occurring VH VH sequences or VHH sequences, for example, comprise combining one or more parts of one or more naturally occurring VH VH sequences (such as one or more framework sequences (FR) and / or complementarity determining region (CDR) sequences), one or more parts of one or more more naturally occurring VHH sequences (such as one or more RF sequences or CDR sequences) and / or one or more synthetic or semi-synthetic sequences, in a suitable manner, to provide an anti-BCMA single domain antibody of the disclosure or a sequence of nucleotides or nucleic acid that encodes it.
[0100] [0100] It is contemplated that in some modalities the BCMA-binding protein is very small and not greater than kD, not greater than 20 kD, not greater than 15 kD or not greater than 10 kD in some modalities. In certain cases, the BCMA-binding protein is 5 kD or less if it is a small molecule entity or peptide.
[0101] [0101] In some embodiments, the BCMA-binding protein is a specific anti-BCMA antibody comprising a heavy chain variable CDR1 complementarity determining region, a heavy chain variable CDR2, a heavy chain variable CDR3, a variable CDR1l light chain, a light chain variable CDR2 and a light chain variable CDR3. In some embodiments, the BCMA-binding protein comprises any domain that binds to BCMA which includes, but is not limited to, domains of a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody or binding fragments antigen, such as single domain antibodies (sdAb), Fab, Fab ', F (ab) 2 and Fv fragments, fragments comprised of one or more CDRs, single chain antibodies (for example, single chain Fv fragments (scFv) ), disulfide stabilized Fv fragments (dsFv), heteroconjugate antibodies (eg bispecific antibodies), pFv fragments, heavy chain dimers and monomers, light chain dimers and monomers and dimers consisting of a heavy chain and a light chain . In some embodiments, the BCMA-binding protein is a single domain antibody. In some embodiments, the anti-BCMA single domain antibody comprises complementarity determining regions (CDR), CDR1, CDR2 and CDR3 variable heavy chain.
[0102] [0102] In some embodiments, the BCMA-binding protein of the present disclosure is a polypeptide comprising an amino acid sequence that is comprised of four regions / framework sequences (f1-f4) interrupted by three complementarity / sequence determining regions, as represented by the formula: fl-rl-f2-r2-f3-r3-f4, where rl, r2 and r3 are complementarity determining regions CDRl1, CDR2 and CDR3, respectively, and fl, £ f2, f3 and f4 are residues of framework. The rl residues of the BCMA-binding protein of the present disclosure comprise, for example, amino acid residues 26, 27, 28, 29, 30, 31, 32, 33 and 34; the r2 residues of the BCMA-binding protein of the present disclosure comprise, for example, amino acid residues, for example, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59.60, 61, 62 and 63; and the r3 residues of the BCMA-binding protein of the present disclosure comprise, for example, amino acid residues, for example, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 and 108. In In some embodiments, the BCMA-binding protein comprises an amino acid sequence selected from SEQ ID NOs: 346-460.
[0103] [0103] In one embodiment, CDR1 does not comprise an amino acid sequence of SEQ ID NO: 473. In one embodiment, CDR2 does not comprise an amino acid sequence of SEQ ID NO: 474. In one embodiment, CDR3 does not comprise an amino acid sequence of SEQ ID NO:
[0104] [0104] In some embodiments, CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 1 or a variant thereof that has one, two, three, four, five, six, seven, eight, nine, or ten substitutions of amino acid. An exemplary CDR1l comprises the amino acid sequence as shown in SEQ ID NO: 4. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 5. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: : 6. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 7. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 8. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 9. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 10. Another exemplary CDRI1 comprises the amino acid sequence as shown in SEQ ID NO: 11. Another exemplary CDR1 comprises the sequence amino acid as shown in SEQ ID NO: 12. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 13. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 14. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 15. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 16. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 17. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 18. Another Exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 19. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 20. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 21. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 22. Another exemplary CDR1 with comprises the amino acid sequence as shown in SEQ ID NO: 23. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 24. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 25. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 26. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 27. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 28. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 29. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 30. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 31. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO : 32. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 33. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 34. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 35. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 36. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 37. Another exemplary CDR1 comprises the sequence amino acid as shown in SEQ ID NO: 38. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 39. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 40. Another CDR1 exemplary comprises the amino acid sequence as shown in SEQ ID NO: 41. Another exemplary CDR1 comprises the sequence of a mino acids as shown in SEQ ID NO: 42. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 43. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 44, Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 45. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 46. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 47. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 48. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 49. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 50. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 51. Another CDR1 exe The amplifying sequence comprises the amino acid sequence as shown in SEQ ID NO: 52. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 53. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 54. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 55. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 56. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 57. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 58. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 59. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 60. Another exemplary CDR1 comprises the amino acid sequence as shown SEQ ID NO: 61. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 62. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 63. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 4. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 65. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 66. Another Exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 67. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 68. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 69. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 70. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 71. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 72. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 73. Another Exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 74, Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 75. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 76. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 77. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 78. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 79. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 80. A another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 81. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 82. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: : 83. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 84. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 85. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 86. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 87. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 88. Another exemplary CDR1 comprises the sequence amino acid as shown in SEQ ID NO: 89. Another exemplary CDR1 comprises the amino acid sequence s as shown in SEQ ID NO: 90. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 91. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 92. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 93. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 94, Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 95. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 29%. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 97. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 98. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 99. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 100. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 101. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 102. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 103. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 104. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 105. Another exemplary CDR1 comprises the a mino acids as shown in SEQ ID NO: 106. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 107. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 108. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 109. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 110. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 111. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 112. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 113. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 114. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 115. Another the exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 116. Another exemplary CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 117.
[0105] [0105] In some embodiments, CDR2 comprises a sequence as shown in SEQ ID NO: 2 or a variant that has one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in SEQ ID NO: 2.
[0106] [0106] In some embodiments, CDR3 comprises a sequence as shown in SEQ ID NO: 3 or a variant that has one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in SEQ ID NO: 3. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 232. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 233. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 234. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 235. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 236. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 237. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 238. Another exemplary CDR3 comprises addresses the amino acid sequence as shown in SEQ ID NO: 239. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 240. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 241. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 242. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 243. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 244. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 245. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 246. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 247. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 248. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 249. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 250. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 251. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 252. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 253. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 254. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 255. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 256. One another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 257. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 258. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 259. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 260. One another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 261. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 262. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 262. : 263. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 264. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 265. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 266. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID N O: 267. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 268. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 269. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 270. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 271. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 272. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 273. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 274. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 275. Another Exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 276. Another exemplary CDR3 comprises the sequence amino acid sequence as shown in SEQ ID NO: 277. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 278. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 279. Another Exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 280. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 281. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 282. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 283. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 284. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 285. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 28 6. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 287. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 288. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 289. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 290. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 291. Another exemplary CDR3 comprises the sequence of amino acids as shown in SEQ ID NO: 292. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 293. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 294. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 295. Another exemplary CDR3 comprises the sequence that of amino acids as shown in SEQ ID NO: 296. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 297. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 298. Another Exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 299. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 300. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 301. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 302. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 303. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 304. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 305. One the other exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 306. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 307. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 308. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 309. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 310. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 311. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 312. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 313. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 314. Another exemplary CDR3 comprises the sequence of amino acids as shown in SEQ ID NO: 315. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 316. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 317. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 318. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 319. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 320. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 321. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 322. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 323. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 324. One out An exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 325. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 326. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: : 327. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 328. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 329. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 330. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 331. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 332. Another exemplary CDR3 comprises the sequence amino acid as shown in SEQ ID NO: 333. Another exemplary CDR3 comprises the amino sequence acids as shown in SEQ ID NO: 334. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 335. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 336. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 337. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 338. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 339. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 340. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 341. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 342. Another exemplary CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 343. Another CD Exemplary R3 comprises the amino acid sequence as shown in SEQ ID NO: 344. Another exemplary R3 comprises the amino acid sequence as shown in SEQ ID NO: 345.
[0107] [0107] In various embodiments, the BCMA-binding protein of the present disclosure has a CDR1 that has an amino acid sequence that is at least about 75%, about 76%, about 77%, about 78%, about 7%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87 &%, about 88 %, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95 &%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 4-117.
[0108] [0108] In various embodiments, the BCMA-binding protein of the present disclosure has a CDR2 that has an amino acid sequence that is at least about 75%, about 76%, about 77%, about 78%, about of 79º &%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87 &%, about 88% , about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95 &%, about 96%, about 97%, about 98 %, about 99%,
[0109] [0109] In various embodiments, a BCMA-binding protein complementarity determining region of the present disclosure has a CDR3 that has an amino acid sequence that is at least about 10%, about 20%, about 30%, about about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 232-345.
[0110] [0110] In various embodiments, a BCMA-binding protein of the present disclosure has an amino acid sequence that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 346-460.
[0111] [0111] In various embodiments, a BCMA-binding protein of the present disclosure has a 1 (fl) framework that has an amino acid sequence that is at least about%, about 20%, about 30%, about 40 %, about 50%, about 60%, about 70%, about 80%, about
[0112] [0112] In various embodiments, a BCMA-binding protein of the present disclosure has a framework 2 (f £ 2) that has an amino acid sequence that is at least about%, about 20%, about 30%, about about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98 8%, about 99 8%, or about 100% identical to the amino acid sequence shown in SEQ ID NO: 463.
[0113] [0113] In various embodiments, a BCMA-binding protein of the present disclosure has a 3 (f3) framework that has an amino acid sequence that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99 3%, or about 100% identical to the amino acid sequence shown in SEQ ID
[0114] [0114] In various embodiments, a BCMA-binding protein of the present disclosure has a 4 (f4) framework that has an amino acid sequence that is at least about%, about 20%, about 30%, about 40 %, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86 %, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96 %, about 97%, about 98%, about 99 8%, or about 100% identical to the amino acid sequence shown in SEQ ID NO: 466 or SEQ ID NO: 467.
[0115] [0115] In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 346. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 347. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 348. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 349. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 350. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 351. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 352. In some embodiments, the binding protein BCMA is a dominant antibody the unique one comprising the sequence of SEQ ID NO: 353. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 354. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 355. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 356. In some embodiments, the protein BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 357. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 358. In some embodiments embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 359.
[0116] [0116] In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 360. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 361. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 362. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 363. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 364. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 365.
[0117] [0117] In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 370. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 371. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 372. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 373. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 374. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 375. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 376. In some embodiments, the binding protein BCMA is a dominant antibody The unique sequence comprising SEQ ID NO: 377. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 378. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 379.
[0118] [0118] In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 380. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 381. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 382. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 383. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 384. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 385. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 386. In some embodiments, the binding protein BCMA is a dominant antibody The unique protein comprising the sequence of SEQ ID NO: 387. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 388. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 389.
[0119] [0119] In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 390. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 391. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 392. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 393. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 394. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 395. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 396. In some embodiments, the binding protein BCMA is a dominant antibody the unique one comprising the sequence of SEQ ID NO: 397. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 398. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 399.
[0120] [0120] In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 400. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 401. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 402. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 403. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 404. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 405. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 406. In some embodiments, the binding protein BCMA is a dominant antibody the unique one comprising the sequence of SEQ ID NO: 407. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 408. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 409.
[0121] [0121] In some embodiments, the BCMA-binding protein is a humanized single domain antibody comprising the sequence of SEQ ID NO: 410. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 411. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 412. In some embodiments, the BCMA-binding protein is a domain antibody unique which comprises the sequence of SEQ ID NO: 413. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 414. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of
[0122] [0122] In some embodiments, the BCMA binding protein is a humanized single domain antibody comprising the sequence of SEQ ID NO: 420. In some embodiments, the BCMA binding protein is a humanized single domain antibody comprising the sequence of SEQ ID NO: 421. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 422. In some embodiments, the BCMA-binding protein is an antibody of single domain comprising the sequence of SEQ ID NO: 423. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 424. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 425. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 426. In some embodiments, the protein BCMA binding is a single domain antibody comprising the sequence of SEQ ID NO: 427. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 428. In some embodiments, the BCMA binding is a single domain antibody comprising the sequence of SEQ ID NO: 429.
[0123] [0123] In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 430. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 431. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 432. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 433. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 434. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 435. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 436. In some embodiments, the binding protein BCMA is a dominant antibody the unique sequence comprising SEQ ID NO: 437. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 438. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 439.
[0124] [0124] In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 440. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 441. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 442. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 443. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 444. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 445. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 446. In some embodiments, the binding protein BCMA is a dominant antibody the unique one comprising the sequence of SEQ ID NO: 447. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 448. In some embodiments, the BCMA-binding protein it is a single domain antibody comprising the sequence of SEQ ID NO: 449. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 450.
[0125] [0125] In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 451. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 452. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 453. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 454. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 455. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 456. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 457. In some embodiments, the binding protein BCMA is a dominant antibody The unique protein comprising the sequence of SEQ ID NO: 458. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 459. In some embodiments, the BCMA-binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 460.
[0126] [0126] A BCMA-binding protein described herein can bind to human BCMA with a Kd in the range of about 0.1 nM to about 500 nM. In some embodiments, hKd is in the range of about 0.1 nM to about 450 nM. In some embodiments, hKd is in the range of about 0.1 nM to about 400 nM. In some embodiments, hKd is in the range of about 0.1 nM to about 350 nM. In some embodiments, hKd is in the range of about 0.1 nM to about 300 nM. In some embodiments, hKd is in the range of about 0.1 nM to about 250 nM. In some embodiments, hKd is in the range of about 0.1 nM to about 200 nM. In some embodiments, hKd in the range of about 0.1 nM to about
[0127] [0127] In some embodiments, any of the foregoing BCMA binding proteins are labeled affinity peptide to facilitate purification. In some embodiments, the affinity peptide tag consists of six consecutive histidine residues, also called a His tag or a 6X-His tag (for example, SEQ ID NO: 471).
[0128] [0128] In certain embodiments, the BCMA binding proteins according to the present disclosure can be incorporated into the specific BCMA targeting protein. In some examples, the triespecific binding protein comprises a CD3 binding domain, a human serum albumin binding domain (HSA) and an anti-BCMA binding domain according to the present disclosure. In some cases, the triespecific binding protein comprises the domains described above in the following orientation: BCMA-HSA-CD3.
[0129] [0129] In certain embodiments, the BCMA binding proteins of the present disclosure preferentially bind membrane-bound BCMA by soluble BCMA. Membrane-bound BCMA refers to the presence of BCMA on or on the cell membrane surface of a cell that expresses BCMA. Suitable BCMA refers to BCMA that is no longer on or on the cell membrane surface of a cell that expresses or expressed BCMA. In certain cases, soluble BCMA is present in the blood and / or lymphatic circulation in an individual. In one embodiment, BCMA binding proteins bind membrane bound BCMA at least 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times, 100 times, 500 times or 1000 times more than soluble BCMA. In one embodiment, the antigen-binding proteins of the present disclosure preferably bind membrane-bound BCMA 30 times more than soluble BCMA. The determination of the preferred binding of an antigen protein binding to membrane bound BCMA over soluble BCMA can be readily determined using assays well known in the art. Integration into chimeric antigen (CAR) receptors
[0130] [0130] The BCMA binding proteins of the present disclosure, for example, a single domain anti-BCMA antibody, can, in certain examples, be incorporated into a chimeric antigen (CAR) receptor. An engineered immune effector cell, for example, a T cell or NK cell, can be used to express a CAR that includes an anti-BCMA single domain antibody as described herein. In one embodiment, the CAR that includes an anti-BCMA single domain antibody as described herein is connected to a transmembrane domain through a hinge region and, in addition, a co-stimulatory domain, for example, a functional signaling domain obtained from OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) or 4-1BB. In some embodiments, the CAR further comprises a sequence that encodes an intracellular signaling domain, such as 4-1BB and / or CD3 zeta. BCMA multispecific protein targeting
[0131] [0131] One embodiment provides a multispecific protein comprising a BCMA binding domain, wherein the BCMA binding domain is in accordance with any of the above modalities. In some embodiments, the multispecific protein comprises the BCMA binding domain according to any of the above modalities (anti-BCMA domain), a CD3 binding domain (anti-CD3 domain) and an albumin binding domain ( anti-ALB domain). In some embodiments, the BCMA multispecific targeting protein is a triespecific protein, wherein the triespecific protein has an HN- (C) - (a) - (B) -COOH domain order. In some embodiments, the anti-BCMA domain (the anti-target domain, T), the anti-CD3 domain (C) and the anti-ALB domain (a) are in an anti-CD3 orientation: anti-ALB: anti-BCMA (CAT). In some embodiments, the anti-BCMA domain (the anti-target domain, T), the anti-CD3 domain (C) and the anti-ALB domain (a) are in an anti-BCMA orientation: anti-ALB: anti-CD3 (TAC).
[0132] [0132] In certain embodiments, the BCMA binding proteins of the disclosure reduce tumor cell growth in vivo when administered to an individual who has tumor cells that express BCMA. The measurement of tumor cell growth reduction can be determined by several different methodologies well known in the art. Non-limiting examples include direct measurement of tumor size, measurement of excised tumor mass and comparison to control subjects, measurement using imaging techniques (for example, CT or MRI) that may or may not use isotopes or luminescent molecules (for example, luciferase) for improved analysis and the like. In the specific embodiments, administration of the antigen-binding agents of the disclosure results in a reduction of in vivo growth of tumor cells as compared to a control antigen-binding agent by at least about 10%, 20%, 30% , 40%, 50 8%, 60%, 70 &, 80%, 90% or 100%, with a reduction of about 100% in tumor growth indicating a complete response and tumor disappearance. In additional embodiments, administration of the antigen-binding agents of the disclosure results in a reduction of in vivo growth of tumor cells as compared to a control antigen-binding agent by about 50-100%, about 75- 100% or about 90-100%. In additional embodiments, administration of the antigen-binding agents of the disclosure results in a reduction in tumor cell growth in vivo as compared to a control antigen-binding agent by about 50-60%,
[0133] [0133] The BCMA binding proteins described in this document cover derivatives or analogues in which (i) an amino acid is replaced by an amino acid residue that is not one encoded by the genetic code, (ii) the mature polypeptide is fused to a another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, as a leader or secretory sequence or a sequence to block an immunogenic domain and / or to purify the protein.
[0134] [0134] Typical modifications include, but are not limited to, acetylation, acylation, ADP ribosylation, amidation, covalent bond of flavin, covalent bond of a heme fraction, covalent bond of a nucleotide or nucleotide derivative, covalent bond of a lipid or lipid derivative, covalent phosphatidylinositol bond, crosslinking, cyclization, disulfide bond formation, demethylation, covalent bond formation, cystine formation, pyroglutamate formation, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodation , methylation, myristylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition to proteins, such as arginylation and ubiquitination.
[0135] [0135] Modifications are made anywhere in the BCMA binding proteins described in this document, including the main peptide chain, the amino acid side chains and the amino or carboxyl terminus. Certain common peptide modifications that are useful for modifying BCMA binding proteins include glycosylation, lipid binding, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation, blocking of the amino or carboxyl group on a polypeptide, or both, by a covalent modification and ADP ribosylation. Polynucleotide-encoding BCMA binding proteins
[0136] [0136] Polynucleotide molecules encoding a BCMA-binding protein are also provided in some embodiments as described herein. In some embodiments, polynucleotide molecules are provided as DNA constructs. In other embodiments, polynucleotide molecules are provided as transcripts of messenger RNA.
[0137] [0137] Polynucleotide molecules are constructed by methods known as combining the genes that encode the anti-BCMA binding protein, operably linked to a suitable promoter and, optionally, a suitable transcription terminator, and which express the same in bacteria or other appropriate expression system, for example, CHO cells.
[0138] [0138] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents an additional modality. This recombinant vector can be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, viruses
[0139] [0139] A variety of host / expression vector systems can be used to contain and express the polynucleotide encoding the described BCMA-binding protein polypeptide. Examples of expression vectors for expression in E.coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285 (1): 111-27), pcDNAS5 (Invitrogen) for expression in mammalian cells, Systems Expression of PICHIAPINK "(Invitrogen), Baculovirus Expression System of BACUVANCE" "(GenScript).
[0140] [0140] Thus, the BCMA binding proteins as described in this document, in some embodiments, are produced by introducing a vector that encodes the protein as described above into a host cell and culturing said host cell under conditions, through In addition, protein domains are expressed, can be isolated and, optionally, further purified. Pharmaceutical compositions
[0141] [0141] Pharmaceutical compositions comprising a BCMA-binding protein described herein, a vector comprising the polynucleotide encoding the polypeptide of BCMA-binding proteins or a host cell transformed by that vector are also provided in some embodiments and at least one pharmaceutically acceptable carrier. the term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Such carriers can be formulated by conventional methods and can be administered to the individual in an appropriate dose. Preferably, the compositions are sterile. Such compositions may also contain adjuvants such as a preservative, emulsifying agents and dispersing agents. The prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents. An additional embodiment provides one or more of the binding proteins described above, as anti-BCMA single domain antibodies or antigen binding fragments thereof packaged in lyophilized form, or packaged in an aqueous medium.
[0142] [0142] In some embodiments of the pharmaceutical compositions, the BCMA-binding protein described herein is encapsulated in nanoparticles. In some embodiments, nanoparticles are fullerenes, liquid crystals, liposomes, quantum dots, superparamagnetic nanoparticles, dendrimers or nanobonds. In other embodiments of the pharmaceutical compositions, the BCMA-binding protein is attached to liposomes. In some cases, the BCMA-binding protein is conjugated to the surface of liposomes. In some cases, the BCMA-binding protein is encapsulated in the housing of a liposome. In some cases, the liposome is a cationic liposome.
[0143] [0143] The BCMA-binding proteins described in this document are contemplated for use as a medicine. Administration is carried out in different ways, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the route of administration depends on the type of therapy and the type of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the doctor in charge and other clinical factors. Dosages for any patient depend on many factors, which include the size of the patient, body surface area, age, sex, the particular compound to be administered, time and route of administration, the type of therapy, general health and other drugs which are administered concomitantly. An "effective dose" refers to amounts of the active ingredient that are sufficient to affect the course and severity of the disease, leading to a reduction or remission of such a pathology and can be determined using known methods.
[0144] [0144] In some embodiments, the BCMA-binding proteins of this disclosure are administered at a dosage of up to mg / kg at a frequency of once a week. In some cases, the dosage is in the range of about 1 ng / kg to about 10 mg / kg. In some embodiments, the dose is from about 1 ng / kg to about 10 ng / kg, about 5 ng / kg to about 15 ng / kg, about 12 ng / kg to about 20 ng / kg, about 18 ng / kg to about 30 ng / kg, about 25 ng / kg to about 50 ng / kg, about 35 ng / kg to about 60 ng / kg, about 45 ng / kg to about from 70 ng / k9g, about 65 ng / kg to about 85 ng / kg, about 80 ng / kg to about 1 unug / kg, about 0.5 pug / kg to about 5 ug / kg, about 2 ug / kg to about 10 pg / kg, about 7 pg / kg to about 15 ug / kg, about 12 pug / kg to about 25 pg / kg, about 20 pg / kg to about 50 vg / kg, about 35 pg / kg to about 70 ug / kg, about 45 pg / kg to about 80 ug / kg, about 65 pog / kg to about 90 ug / kg, about 85 ug / kg to about 0.1 mg / kg, about 0.095 mg / kg to about 10 mg / kg. In some cases, the dosage is about 0.1 mg / kg to about 0.2 mg / kg; about 0.25 mg / kg to about 0.5 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.75 mg / kg to about 3 mg / kg, about 2.5 mg / kg to about 4 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 6 mg / kg, about 5.5 mg / kg to about 7 mg / kg, about 6.5 mg / kg to about 8 mg / kg, about 7.5 mg / kg to about 9 mg / kg, or about 8 , 5 mg / kg to about 10 mg / kg. The frequency of administration, in some modalities, is about less than daily, alternating days, less than once a day, twice a week, weekly, once in 7 days, once in two weeks, once two weeks, once in three weeks, once in four weeks or once a month. In some cases, the frequency of administration is weekly. In some cases, the frequency of administration is weekly and the dosage is up to 10 mg / kg. In some cases, the duration of administration is from about 1 day to about 4 weeks or more.
[0145] [0145] Also provided in this document are, in some embodiments, methods and uses to stimulate the immune system of an individual in need thereof which comprises administration of a BCMA-binding protein as described herein. In some cases, administration of a BCMA-binding protein described herein induces and / or sustains cytotoxicity to a cell that expresses a target antigen. In some cases, the cell that expresses a target antigen is a terminally differentiated B cell that is a cancerous or tumor cell, or a metastatic cancer or tumor cell.
[0146] [0146] Also provided herein are methods and uses for treating a BCMA-associated disease, disorder or condition comprising administering to an individual in need of it a BCMA-binding protein or a multispecific binding protein comprising BCMA-binding protein described herein.
[0147] [0147] Diseases, disorders or conditions associated with BCMA include, but are not limited to, cancer or metastasis that is from a B cell line.
[0148] [0148] Cancers that can be treated, prevented or managed by the BCMA-binding proteins of the present disclosure, and methods of using them, include, but are not limited to, a primary cancer or metastatic cancer.
[0149] [0149] Examples of such leukemias include, but are not limited to: acute lymphoblastic leukemia (ALL) acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML), as well as numerous less common types such as , for example, Hair cell leukemia (HCL), T cell prolymphocytic leukemia (T-PLL), Large granular lymphocytic leukemia and Adult T cell leukemia, etc. The subtypes of acute lymphoblastic leukemia (ALL) to be treated include, but are not limited to, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt's leukemia, and acute biphenotypic leukemia. The subtypes of chronic lymphocytic leukemia (CLL) to be treated include, but are not limited to, B-cell prolymphocytic leukemia. The subtypes of acute myelogenic leukemia (AML) to be treated include, but are not limited to, acute promyelocytic leukemia, leukemia — acute myeloblastic and acute megacarioblastic leukemia. The subtypes of chronic myelogenous leukemia (CML) to be treated include, but are not limited to, chronic myelomonocytic leukemia.
[0150] [0150] Examples of lymphoma to be treated with the methods in question include, but are not limited to, Hodgkin's disease, non-Hodgkin's disease or any lymphoma subtype.
[0151] [0151] Examples of such multiple myelomas include, but are not limited to, a multiple myeloma of bone or other tissues including, for example, an asymptomatic multiple myeloma, a non-secretory myeloma, an osteosclerotic myeloma, etc.
[0152] [0152] For an analysis of such disorders, see Fishman et al., 1985, Medicine, 2nd Ed., JB Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA., Inc., United States of America).
[0153] [0153] As used herein, in some modalities, "treatment" or "treating" or "treated" refers to therapeutic treatment in which the objective is to slow down (decrease) an unwanted physiological condition, disorder or disease, or to obtain results beneficial or desired clinical features. For the purposes described in this document, beneficial or desired clinical results include, but are not limited to, symptom relief; decrease in the extent of the condition, disorder or disease; stabilization (that is, it does not worsen) of the condition, disorder or disease; delay in starting or slowing the progression of the condition, disorder or disease; improvement in condition, disorder or disease; and remission (if partial or total), if detectable or undetectable, or improvement or improvement of the condition, disorder or disease. Treatment includes inducing a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to expected survival if you don't receive treatment. In other modalities, "treatment" or "treating" or "treated" refers to prophylactic measures, in which the objective is to delay the onset of or reduce the severity of an unwanted physiological condition, disorder or disease, such as, for example, a person who is predisposed to a disease (for example, an individual who carries a genetic marker for a disease such as breast cancer).
[0154] [0154] In some embodiments of the methods described herein, BCMA-binding proteins as described herein are administered in combination with an agent to treat the particular disease, disorder or condition. Agents include, but are not limited to, therapies that involve antibodies, small molecules (for example, chemotherapy), hormones (steroidal, peptide and the like)
[0155] [0155] In some embodiments, a BCMA-binding protein as described in this document is administered before, during or after surgery.
[0156] [0156] In some embodiments, the anticancer agent is conjugated through any suitable medium for the specific protein. Methods of detecting BCMA expression and diagnosing BCMA-associated cancer
[0157] [0157] According to another embodiment of the disclosure, kits are provided to detect BCMA expression in vitro or in vivo. The kits include the aforementioned BCMA-binding proteins (for example, an anti-BCMA-labeled single domain antibody or antigen-binding fragments thereof), and one or more compounds to detect the label. In some embodiments, the label is selected from the group consisting of a fluorescent label, an enzymatic label, a radioactive label, an active nuclear magnetic resonance label, a luminescent label and a chromophore label.
[0158] [0158] In some cases, BCMA expression is detected in a biological sample. The sample can be any sample, which includes, but is not limited to, tissue from biopsies, autopsies and pathology specimens. Biological samples also include sections of tissue, for example, frozen sections removed for histological purposes. Biological samples additionally include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine. A biological sample is typically obtained from a mammal, such as a human or non-human primate.
[0159] [0159] In one embodiment, a method is provided to determine whether an individual has cancer by contacting a sample of the individual with an anti-BCMA single domain antibody as disclosed herein; and detecting binding of the single domain antibody to the sample. An increase in antibody binding to the sample compared to antibody binding to a control sample identifies the individual as having cancer.
[0160] [0160] In another embodiment, a method is provided to confirm a cancer diagnosis in an individual by contacting a sample from an individual diagnosed with cancer with a single domain anti-BCMA antibody as disclosed herein; and detecting binding of the antibody to the sample. An increase in antibody binding to the sample compared to antibody binding to a control sample confirms the diagnosis of cancer in the individual.
[0161] [0161] In some examples of the disclosed methods, the single domain antibody is directly labeled.
[0162] [0162] In some examples, the methods additionally include contacting a second antibody that specifically binds the single domain antibody to the sample; and detecting the binding of the second antibody. An increase in binding of the second antibody to the sample compared to binding of the second antibody to a control sample detects cancer in the individual or confirms the diagnosis of cancer in the individual.
[0163] [0163] In some cases, the cancer is lymphoma, leukemia or multiple myeloma.
[0164] [0164] In some instances, the control sample is a sample of an individual without cancer. In particular examples, the sample is a blood or tissue sample.
[0165] [0165] In some cases, the antibody that binds (for example, it specifically binds) to BCMA is directly labeled with a detectable tag. In another embodiment, the antibody that binds (for example, specifically binds) to BCMA (the first antibody) is unlabeled and a second antibody or other molecule that can bind to the antibody that specifically binds to BCMA is labeled. A second antibody is chosen so that it is able to specifically bind to the specific species and class of the first antibody. For example, if the first antibody is a llama IgG, then the secondary antibody can be an anti-llama-IgG. Other molecules that can bind to antibodies include, without limitation, protein A and protein G, both of which are commercially available. Suitable identifications for the antibody or secondary antibody are described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbeliferone, fluorescein, fluorescein isothiocyanate, rhodamine, fluorescein dichlorotriazinylamine, dansyl chloride or phycoerythrin. An exemplary non-limiting luminescent material is luminol; a non-limiting example of a magnetic agent is gadolinium, and non-limiting exemplary radioactive identifications include 1251, 1311, 35S or 3H.
[0166] [0166] In an alternative embodiment, BCMA can be assayed in a biological sample by a competition immunoassay using BCMA standards labeled with a detectable substance and an unlabeled antibody that specifically binds to BCMA. In this assay, the biological sample, the labeled BCMA standards and the antibody that specifically binds to BCMA are combined and the amount of labeled BCMA standard bound to the unlabeled antibody is determined. The amount of BCMA in the biological sample is inversely proportional to the amount of labeled BCMA standard bound to the antibody that specifically binds to BCMA.
[0167] [0167] The immunoassays and method disclosed in this document can be used for a number of purposes. In one embodiment, the antibody that specifically binds to BCMA can be used to detect BCMA production in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of BCMA in a biological sample, such as a tissue sample, or a blood or serum sample. In some examples, BCMA is cell surface BCMA. In other examples, BCMA is soluble BCMA (for example, BCMA in a supernatant cell culture or BCMA soluble in a body fluid sample, such as a blood or serum sample).
[0168] [0168] In one embodiment, a kit is provided to detect BCMA in a biological sample, such as a blood sample or tissue sample. For example, to confirm a diagnosis of cancer in an individual, a biopsy can be performed to obtain a tissue sample for histological examination. Alternatively, a blood sample can be obtained to detect the presence of soluble BCMA protein or fragment. Kits for detecting a polypeptide will typically comprise a single domain antibody according to the present disclosure, which specifically binds to BCMA. In some embodiments, an antibody fragment, such as an scFv fragment, a VH domain, or a Fab is included in the kit. In an additional embodiment, the antibody is labeled (for example, with a fluorescent, radioactive or enzymatic label).
[0169] [0169] In one embodiment, a kit includes instructional materials that reveal ways to use an antibody that binds to BCMA. Instructional materials can be written, in an electronic form (such as a floppy disk or computer compact disc) or can be visual (such as video files), or provided over an electronic network, for example, over the internet, World Wide Web , an intranet or another network. Kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means for detecting a label (such as enzyme substrates for enzymatic labels, filter sets for detecting fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like). Kits can additionally include buffers and other reagents routinely used to practice a particular method. Such kits and appropriate contents are well known to those skilled in the art.
[0170] [0170] In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of immunoassays may vary with the particular format employed, the method of detecting BCMA in a biological sample generally includes the steps of bringing the biological sample into contact with an antibody that specifically reacts, under immunologically reactive conditions, to a polypeptide BCMA. The antibody is allowed to bind specifically under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (antibody bound) is detected directly or indirectly.
[0171] [0171] Methods of determining the presence or absence of a cell surface marker are well known in the art. For example, antibodies can be conjugated to other compounds that include yqá but without limitation, enzymes, magnetic microspheres, colloidal magnetic microspheres, haptens, fluorochromes, metallic compounds, radioactive compounds or drugs. The antibodies can also be used in immunoassays such as, but not limited to, radioimmunoassays (RIASs), ELISA or immunohistochemical assays. Antibodies can also be used for fluorescence-activated cell classification (FACS). FACS employs a plurality of color channels, obtuse and low-angle diffusion detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or classify cells (see US Patent No. 5,061,620 ). Any of the single domain antibodies that bind to BCMA, as disclosed herein, can be used in these assays. Thus, antibodies can be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation. EXAMPLES
[0172] [0172] The application can be better understood by reference to the following non-limiting examples, which are provided as exemplary modalities of the application. The following examples are presented in order to more fully illustrate the modalities and should in no way be interpreted, however, as limiting the broad scope of the application. Example 1 Ability of an exemplary anti-BCMA trypecific domain antibody containing a BCMA-binding protein of the present disclosure to mediate T cell extermination of BCMA-expressing cancer cells in TDCC (T cell dependent cell cytotoxicity) assays
[0173] [0173] Protein Production
[0174] [0174] The sequences of BCMA targeting specific molecules, containing a BCMA binding protein according to the present disclosure, were cloned into the mammalian expression vector pcDNA 3.4 (Invitrogen) preceded by a leader sequence and followed by a 6x Histidine tag (SEQ ID NO: 471). Expi293 cells (Life Technologies A1l4527) were kept in suspension in Optimum Growth Flasks (Thomson) between 0.2 to 8 x l and 6 cells / ml in Expi293 media. The purified plasmid DNA was transfected into Expi293 cells according to the Expi293 Expression System Kit protocols (Life Technologies, Al4635), and maintained for 4-6 days post-transfection. The amount of the exemplary triespecific proteins being tested, in conditioned media, from the transfected Expi293 cells was quantified using an Octet instrument with protein A tips and using a specific trying control protein for a standard curve.
[0175] [0175] T cell dependent cell cytotoxicity assays
[0176] [0176] Titrations of conditioned media have been added to TDCC assays (T Cell Dependent Cell Cytotoxicity assays) to assess whether anti-BCMA single domain antibody is capable of forming a synapse between T cells and a cell line that expresses BCMA and target T cells to exterminate the cell line that expresses BCMA. In this essay (Nazarian et al.,
[0177] [0177] In this example, EJM cells were used, which is a cell line that serves as an in vitro model for multiple myelin and plasma leukemia cells. The viability of the EJM cells is measured after 48 hours. It was seen that the specific proteins mediated T cell extermination. Fig. 1 shows an example of cell viability assay with test proteins 01H08, 01F07, 02F02 and BH253 compared to a negative control. The ECso for the TDCC activity of several other test-specific proteins is listed below in Table 1.
[0178] [0178] Connection affinity
[0179] [0179] In the present study, the human BCMA protein binding affinity of the BCMA targeting specific proteins containing a BCMA binding protein according to the present disclosure was determined. Affinity measurements are listed in Table 1.
[0180] [0180] Table 1: Binding affinity and TDCC activity of various BCMA targeting specific proteins. Kp ”name of human BCMA (M) | TDCC EC50 (M) 01FO07 4.18E-09 02B05 5.39E-09 02F02 6, 73E-09 O1EO8 5.56E-09 02E06 6.31E-09
[0181] [0181] ND: Not determined.
[0182] [0182] Molecules 01H08, O1FO07, O1H06, 02GO02, 02B05, 01CO01, 02F02, O2EO5, O1EO8, 0O02COl, 02EO06, 02B06, 02FO04, 01GO08, 02CO6, 01H09, O1FO4, O1OO, O1O1, O1O1, , 02H09, O1E03, 0O02FO5, O1B0O5, O1CO5, 0O2F12, 01H11, 02GO6, O1EO6, O1Gl11, 02A05, O1A08, O02GO5, 0O1B09, 01G01, O1B06, O1FO, O01, 02, O01, 02, 02 , 0O1DO3, O1AO5, O2Fl1, O1DO4, O1BO04, 02C0O5, 02E03, O1DO5, O1CO4, O1EO7, O1GO6, 02FO6, O1BO0O1, O01DO7, 0O2A08, O1A02, O2G11, O1A4, O2G11, O1GO4, O1G twice and also show increased affinity compared to a molecule with parental CDRs, 253BH10.
[0183] [0183] The 01H08, O1FO07, O1H06, 02GO02, 02B05 molecules,
[0184] [0184] An anti-GFP specific molecule, included in these assays as a negative control, had no detectable BCMA binding and no effect on cell viability in the TDCC assay (data not shown). Example 2 Methods for evaluating binding and cytotoxic activities of exemplified purified trypspecific antigen binding proteins containing a BCMA binding domain according to the present disclosure against Jekol, MOLP8 and OPM2 cells
[0185] [0185] Protein Production
[0186] [0186] The sequences of BCMA targeting specific molecules, containing a BCMA binding protein according to the present disclosure, preceded by a leader sequence and followed by a Histidine Tag 6x (SEQ ID NO: 471), were expressed using the vectors and methods previously described (Running Deer and Allison,
[0187] [0187] Cytotoxicity tests
[0188] [0188] A human T cell dependent cell cytotoxicity (TDCC) assay was used to measure the ability of T cell engagers, which include specific molecules, to target T cells to exterminate tumor cells (Nazarian et al., 2015. JS Biomol Screen, 20: 519-27). In that assay, T cells and target cells of cancer cell line are mixed at a 10: 1 ratio in a 384-well plate, and varying amounts of the specific tries being tested are added. Tumor cell lines are designed to express luciferase protein. After 48 hours, to quantify the remaining viable tumor cells, STEADY-GLOS Luminescent Assay (Promega) was used.
[0189] [0189] In the present study, purified protein titrations were added to TDCC assays (T Cell Dependent Cell Cytotoxicity assays) to assess whether the anti-BCMA single domain antibody was able to form a synapse between T cells and cancer cell lines Jekol, MOLP8 and OPM2 that express BCMA. Jekol is a B cell lymphoma cell line. MOLP-8 is a myeloma cell line. OPM-2 is a human myeloma cell line.
[0190] [0190] Cell viability was measured after 48 hours. It was seen that the specific proteins mediated T cell extermination. Fig. 3 shows an example of a cell viability assay with test proteins compared to a negative control. The ECs for the TDCC activity of several other triespecific test proteins are listed below in Table 2. An anti-GFP triespecific molecule, included in these assays as a negative control, had no effect on cell viability (data not shown).
[0191] [0191] Table 2: ECso TDCC values for 3 cell lines to select ligand sequences in TriTAC EC50 format from EC50 to EC50 from Construct name by 25 [25 | 8% |
[0192] [0192] Connection affinity
[0193] [0193] In the present study, the human BCMA protein binding affinity of the BCMA targeting specific proteins containing a BCMA binding protein according to the present disclosure was determined.
[0194] [0194] Table 3: Binding affinity of purified triespecific targeting proteins containing a BCMA binding protein according to the present disclosure. Example 3 ADCC activity of an anti-BCMA multi-domain antibody exemplifying the present disclosure
[0195] [0195] This study is aimed at determining the ability of a multi-domain anti-BCMA antibody exemplifying the present disclosure to mediate ADCC as compared to a parental anti-BCMA llama antibody that has no sequence modifications or substitutions like the exemplary antibody of revelation. Both antibodies are expressed as multi-domain proteins that include an additional immunoglobulin Fc domain.
[0196] [0196] Materials
[0197] [0197] Donors are subjected to leukophoresis, and NK cells are isolated from Leukopack by the Cell Purification Group using the Milteni AUTOMACSO Pro negative selection system. NK cells are kept overnight at 4 ºC on a shaker, then , washed, counted and resuspended at 4x10º cells / ml in complete RPMI for use in the ADCC assay.
[0198] [0198] Targets: tumor cell targets are selected based on BCMA expression. The targets are washed and counted. 6 * x10º targets are resuspended in complete RPMI and marked in a final concentration of 10 µM calcein (Sigma calcein nº C1359-00UL at 4 mm in anhydrous DMSO) for 40 minutes at 37 “ºC, 5% CO». The cells are washed twice in PBS, resuspended in complete RPMI and incubated at 37 ºC, 5% CO, for 2 h. After labeling, the target cells are washed, recounted and resuspended at 0.2x106º cells / ml in complete RPMI for use in the ADCC assay.
[0199] [0199] Methods
[0200] [0200] The ADCC assay is performed on a 96-well round-bottom tissue culture plate (Corning 3799). Test proteins are titrated from ug / ml to 0.0002 pg / ml by removing 10 µl in 1000 µl of complete RPMI containing 10% FCS (a 1:10 dilution). Marked calcein targets are added, 50 µl to contain 10,000 cells. Target cells and various concentrations of “multi-domain proteins containing — the exemplary anti-BCMA single domain antibody or comparator antibody are incubated for 40 minutes at 4 ° C, then added NK cell effectors, 50 μl to contain
[0201] [0201] Statistical Analysis
[0202] [0202] The percentage (%) of specific lysis is defined as (sample fluorescence) - (spontaneous lysis fluorescence) / (100% spontaneous lysis fluorescence). Spontaneous lysis is determined by wells containing only targets and 100% lysis is determined by wells where the targets are lysed with IGEPAL CA 630 detergent. The raw data are entered into an Excel spreadsheet with built-in formulas to calculate% specific lysis and resulting values transferred to a graphics program (GraphPad Prism) where the data are transformed into a curve fitting graph. Subsequent analyzes (linear regression calculations) are performed on GraphPad to generate ECrso values. Example 4 CDC activity of an anti-BCMA single domain antibody exemplifying the present disclosure
[0203] [0203] To evaluate the anti-tumor activity of exemplary anti-BCMA single domain antibody, according to the present disclosure, against cancer cells, the cytotoxic activity in cell models A431 / H9 and NCI-H226 in the presence of human serum as a complement source is tested. the exemplary anti-BCMA single domain antibody is expressed as a multi-domain protein containing an Fc domain.
[0204] [0204] A multi-domain protein containing the anti-BCMA single domain antibody exemplary of the present disclosure exerts potent CDC activity by exterminating cancer cell lines, and shows no activity on a BCMA negative cell line. Example 5 Xenograft tumor model
[0205] [0205] An exemplary BCMA targeting specific protein containing an exemplary BCMA binding protein of this disclosure, 02B05 (SEQ ID NO: 383), was evaluated in a xenograft model.
[0206] [0206] On day 0, NCG mice were subcutaneously inoculated with RPMI-8226 cells, and also intraperitoneally implanted with normal human peripheral blood mononuclear cells (PBMCs). Treatment with the exemplary BCMA bleaching specific protein was also started on day O (qgdx10) (once daily for 10 days). The dosage for administration was 5 vg / kg, 50 ug / kg, or 500 ug / kg of the BCMA 02B05 specific targeting protein, or a vehicle as a control. Tumor volumes were determined for days. As shown in Fig. 29, mean tumor volumes were significantly lower in mice treated with the exemplary BCMA targeting specific protein (02B05) (at 50 µg / kg, or 500 µg / kg), as compared to treated mice. with oThe vehicle or the lower dose of BCMA targeting specific protein (02B05) (at 5 µg / kg).
[0207] [0207] On day 0, NCG mice were subcutaneously inoculated with Jeko 1 cells, and also intraperitoneally implanted with normal human peripheral blood mononuclear cells (PBMCs). Treatment with the exemplary BCMA targeting specific protein was started on day 3 (qgdx10) (once daily for 10 days). The dosage of administration was 1 µg / kg, 50 µg / kg, or 500 µg / kg of the BCMA 02B05 targeting specific protein, or a vehicle as a control. Tumor volumes were determined for 25 days. As shown in Fig. 30, mean tumor volumes were significantly lower in mice treated with the exemplary BCMA targeting specific protein (02B05) (at 500 ug / kg), as compared to mice treated with the vehicle or doses BCMA (02B05) triespecific bleaching protein (at 5 pg / kg or 50 ug / kg). Example 6 Affinity measurements for human BCMA and cinomolgo, CD3e, and albumin, using a BCMA targeting specific containing a BCMA binding protein from this disclosure
[0208] [0208] The objective of this study was to evaluate the affinity of an exemplary BCMA targeting specific protein containing a BCMA binding protein of this disclosure (02B05) (SEQ ID NO: 383), in relation to human BCMA, BCMA cinomolgo, CcD3e human, CcD3e cinomolgo, human albumin, cinomolga albumin, and mouse albumin. Affinities were measured using an instrument - Octet. For these measurements, streptavidin tips were first loaded with 2.5 nM human BCMA-Fc, 2.5 nM BCMA cinomolgo-Fc, 2.5 nM human CD3e-Fec, 2.5 nM CD3e-Fec cinomolgo, 50 nM human serum albumin (HSA), 50 nM cinomolga serum albumin or 50 nM murine serum albumin. Subsequently, the exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure 02B05 was incubated with the tips, and after a period of association, the tips were moved to a buffer solution to allow the specific targeting protein of exemplary BCMA containing a BCMA-binding protein of this disclosure (02B05) disassociated. Affinities for binding to human BCMA and CD3e and cinomolgos were measured in the presence of mg / ml human serum albumin. The averaged Ko »values from these studies are given in Table 4 (n indicates the number of independent measurements, n / d indicates no link detected under the conditions tested). The binding was detected with human BCMA, human CD3e, CD3: and cinomolgo, human serum albumin, cinomolga serum albumin and murine serum albumin. Under the conditions tested, no binding was detected to BCMA cinomolgo.
[0209] [0209] Table 4. K values, measured for exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure, 02B05, for protein ligands.
[0210] [0210] The exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure, 02B05 (SEQ ID NO: 383) was tested for its ability to bind to purified T cells. In short, the BCMA-specific protein or phosphate-buffered saline (PBS) was incubated with purified T cells from 4 different anonymous human donors. After washing the unbound protein, T cells were then incubated with an Alexa Fluor 647 conjugated antibody that recognizes the antialbumin domain in the BCMA 02B05 triespecific antigen binding protein. The T cells were then analyzed by flow cytometry. It was observed that human T cells incubated with the BCMA 02B05 antigen-specific antigen-binding protein had notable deviations associated with staining with Alexa Fluor 647 compared to cells that were incubated with PBS. The results are shown in Figs. 4A, 4B, 4C and 4D. In conclusion, that study indicated that the exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure was able to bind to human T cells. Example 8 Ability of a try-specific BCMA targeting protein exemplary of this disclosure to bind to cells that express BCMA
[0211] [0211] The exemplary BCMA targeting specific protein containing a BCMA binding protein of this disclosure 02B05 (SEQ ID NO: 383) has been tested for its ability to bind to cells expressing BCMA. In short, the BCMA 02B05 antigen-specific antigen-binding protein was incubated with cell lines that express BCMA (NCI-H929; EJM; RPMI-8226; OPM2) or lack BCMA (NCI-H510A; DMS-153). BCMA RNA expression in these cells is indicated by FPKM values (fragments per million kilobase) listed in Figs. 5A- F: RNA FPKM values are from the Cancer Cell Line Encyclopedia (Broad Institute, Cambridge, MA, USA). After washing the unbound protein, the cells were then incubated with an Alexa Fluor 647 conjugated antibody that recognizes the anti-albumin domain in the BCMA 02B05 triespecific antigen binding protein. The cells were then analyzed by flow cytometry. As a negative control, the cells were incubated with a specific protein that targets GFP. Cells expressing BCMA RNA and incubated with the BCMA triespecific protein had notable deviations associated with Alexa Fluor 647 staining compared to cells that were incubated with GFP triespecific protein (as in Figs. 5A, 5B, 5D and 5E) . Meanwhile, cells lacking BCMA RNA produced staining of Alexa Fluor 647 equivalent with the BCMA triespecific protein and the GFP triespecific protein (as seen in Figs. 5C and 5F). Thus, this study indicated that binding to the exemplary BCMA triespecific antigen was able to selectively bind to cells that express BCMA. Example 9 Ability of an exemplary BCMA targeting specific protein containing a BCMA-binding protein of this disclosure to mediate T cell extermination of cancer cells expressing BCMA
[0212] [0212] The exemplary BCMA triespecific protein containing a BCMA binding protein of this disclosure 02B05 (SEQ ID NO: 383) has been tested for its ability to target T cells to exterminate cells that express BCMA in the presence and absence of albumin human serum (HSA) using a standard TDCC assay as described in Example 1. Due to the fact that the exemplary BCMA-specific protein contains an anti-albumin domain, this experiment was carried out to confirm that binding to albumin would not prevent the protein from binding to BCMA tries specific antigen would target T cells to exterminate cells that express BCMA. Five cell lines expressing BCMA were tested: EJM, Jeko, OPM2, MOLP8 and NCI-H929. Representative data for an experiment with the EJM cells are shown in Fig.
[0213] [0213] Table 5 ECso TDCC values for an exemplary BCMA targeting specific protein containing a BCMA-binding protein of this disclosure in the presence or absence of human serum albumin with five cell lines expressing different BCMA Cell Line ECso without HSA (pM) ECso with HSA (pM)
[0214] [0214] In the standard TDCC assay (as described in
[0215] [0215] Table 6 ECso TDCC values for an exemplary BCMA targeting specific protein containing a BCMA-binding protein of this disclosure (02B05) with varying ratios from target cell (EJM cells) to effector cell (T cells) ( tested in the presence of mg / ml HSA) | EJM ECso ECso Target cell: (PM) OPM2 (pM) T cell Example 11 Ability of an exemplary BCMA targeting specific protein containing a BCMA binding protein of this disclosure to mediate T cell extermination of cancer cells that express BCMA, in a time course study, using a lower target cell to effector cell ratio
[0216] [0216] In the standard TDCC assay (Example 1), a ratio of 1 target cell to 10 effector cells (T cells) is used in a 48 hour assay. In this experiment, a time course was performed using a 1: 1 ratio of target cells (EJM cells) to effector cells (T cells). The expectation was that with increased time, a 1 to 1 ratio would result in target cell extermination. The TDCC assay was performed using EJM and a 1: 1 ratio of target cell to effector cell, and the experiment was performed in the presence of 15 mg / ml HSA. A specific GFP targeting protein was used as a negative control. Target cell viability was measured on days 1, 2, 3, and 4 after incubation of target cells and effector cells, at a l1: 1 ratio, in the presence of the exemplary BCMA 02B05 antigen-specific antigen-binding protein and 15 mg / ml HSA, or GFP targeting specific protein and 15 mg / ml HSA. Although no target cell extermination was observed on day 1, the extermination was actually observed at other points in time with the binding protein BCB 02B05 tries specific antigen, with the amount of extermination "increasing over time (Fig. 9) .No target cell extermination was observed with the GFP targeting specific protein The ECso values calculated for cell extermination each day are provided in Table 7 (n / a indicates insufficient extermination to determine an ECso value) From this study, it is concluded that the exemplary BCMA 02B05 specific protein was able to target T cell extermination with lower numbers of effector cells, but more time was needed to achieve more complete extermination.
[0217] [0217] Table 7 ECso DTCC values for an exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure (02B05) with 1 to 1 ratios from target cell (EJM cells) to effector cell (T cells ) (tested in the presence of mg / ml HSA), at varying points in time ECso
[0218] [0218] The exemplary BCMA triespecific protein containing a BCMA-binding protein of this disclosure 02B05 (SEQ ID NO: 383) was tested for its ability to target T cells from four different anonymous human donors to exterminate four different cells that express BCMA in the presence of mg / ml of human serum albumin (HSA) using a standard TDCC assay as described in Example 1. The cell lines expressing BCMA were EJM, NCI-H929, OPM2 and RPMI8226. As negative controls, two cell lines that lack BCMA expression, OVCAR8 and NCI-H510A, were also tested in the TDCC assays. A specific GFP targeting targeting protein was also used as a negative control. With the four cell lines expressing BCMA and all four T cell donors, cell viability decreased with increasing amounts of the BCMA triespecific protein, but not with the GFP triespecific protein (Figs. 10, 11, 12, and 13) . ECso values for cell extermination are provided in Table 8. The exemplary BCMA 02B05 antigen-specific antigen-binding protein did not direct extermination of cell lines that lack BCMA expression (Figs. 14 and 15). Thus, it was inferred that the exemplary BCMA 02B05 antigen-specific antigen-binding protein was able to target T cells from multiple donors to exterminate a spectrum of cell lines expressing BCMA.
[0219] [0219] Table 8 ECso values of BCMA triespecific protein exemplary of TDCC assays with four cell lines expressing BCMA and four T cell donors in the presence of 15 mg / ml HSA EC50 (pM) Donor 02 169 Example 13 Ability to an exemplary BCMA targeting specific protein containing a BCMA binding protein from this disclosure of targeting cinomolgus T cells to exterminate cells that express BCMA
[0220] [0220] The exemplary BCMA targeting specific protein containing a BCMA-binding protein of this disclosure 02B05 (SEQ ID NO: 383) was tested for its ability to target T-cells from cynomolgus monkeys to exterminate cells that express BCMA in the presence 15 mg / ml human serum albumin (HSA). The experimental conditions were the same as described in Example 1 except that peripheral blood mononuclear cells (PBMC) from cynomolgus monkeys were used as a source of T cells. Two cell lines expressing BCMA were tested, RPMI8226 and NCI-H929. As shown in Figs. 16 and 17, the BCMA triespecific protein was able to target T cells present in cinomolga PBMCs to exterminate the two cell lines that express BCMA. ECso values for cell extermination are listed in Table 9. A GFP-specific protein did not affect the viability of cells expressing BCMA. Thus, the specific protein that expresses BCMA, which can bind to CD3e cinomolgo (as shown in Example 6), can target cinomolgus T cells to exterminate cells that express human BCMA.
[0221] [0221] Table 9 ECso values of BCMA triespecific protein from TDCC assays with two cell lines and two donors of PMBC cinomolgos in the presence of 15 mg / ml of
[0222] [0222] The exemplary BCMA targeting specific protein containing a BCMA-binding protein of this disclosure 02B05 (SEQ ID NO: 383) was tested for its ability to activate T cells in the presence of cells that express BCMA.
[0223] [0223] Cynomolgus monkeys were administered with single intravenous doses of an exemplary BCMA targeting specific protein containing a BCMA-binding protein of this disclosure (02B05) (SEQ ID NO: 383), at 0.01 mg / kg, 0, 1 mg / kg, or 1 mg / kg. Two animals were included per dose group. After administration, serum samples were collected and analyzed by two different electrochemiluminescent assays. One assay used biotinylated CD3re as a capture reagent and detected with sulfo labeled BCMA (called a functional assay). Another assay used as a capture reagent a biotinylated antibody that recognizes the anti-albumin domain in the exemplary BCMA targeting specific protein containing a BCMA-binding protein of this disclosure 02B05 (SEQ ID NO: 383) and used as a detection reagent a sulfo-labeled antibody that recognizes the anti-CD3 binding domain in the exemplary BCMA targeting specific protein containing a BCMA binding protein of this disclosure (i.e., an anti-idiotype antibody). The results of the electrochemiluminescent assays are plotted in Fig. 31. As seen in Fig. 31, the exemplary BCMA targeting specific protein was detected in the cinomolga serum samples, even after 504 hours after administration. The exemplary attempted BCMA targeting protein was labeled using both sulfo labeled BCMA (lines marked using the term "functional" in Fig. 31) and the anti-idiotype antibody (lines marked using the term "anti-idiotype" in Fig . 31).
[0224] [0224] To confirm that the exemplary BCMA targeting specific protein retained the ability to target T cells to exterminate BCMA-expressing EJM cells, after in vivo administration, the 168-hour time point serum samples were tested in a TDCC (as described in Example 1) in the presence of 16.7% serum from a cynomolgus monkey that was not exposed to a BCMA targeting specific protein, titrating the exemplary BCMA targeting specific protein using the protein concentrations determined using electrochemiluminescent assays (shown in Fig. 32). The exemplary fresh diluted BCMA targeting specific protein containing a BCMA binding protein from this disclosure 02B05 (SEQ ID NO: 383) was compared with the BCMA trying specific protein collected from the test cinomolgy monkeys at 168 h. A specific GFP protein was included as a negative control. This study demonstrated that the exemplary BCMA targeting specific protein collected from the serum of test cynomolgus monkeys had activity identical to that of fresh diluted protein, and that the protein in the serum samples retained the ability to target T cells to exterminate NDE cells that express BCMA.
[0225] [0225] Although the preferred embodiments of the present invention have been presented and described in the present document, it will be obvious to those skilled in the art that such modalities are provided by way of example only. Various variations, changes and substitutions will now occur for those skilled in the art without distancing themselves from the invention. It is to be understood that various alternatives to the modalities of the invention described in this document can be employed in the practice of the invention. It is intended that the following claims define the scope of the invention and that the methods and structures within the scope of those claims and their equivalents are covered by them. Sequence Table
SEQ ID Description Ms 1 Sequence CDR1 X1X2X3XaXsXeX7PXsG where X1 is T Exemplary | or S; Xo is N, D, or S; X; is I, D, Q, H, Vou E; Xa is F, S, E, A, T, M, V, I, D, Q, P, R or G; Xs is S, M, Rou N; X; is I, K, S, T, R, E, D, N, V, H, L, A, Q or G; X; 7 is S, T, Y, R or N; and XséM GouY 2 CDR2 AIXSGX10X11 TX12YADSVK where X9 is Exemplary | H, N, or S; Xi is F, G, K, R, P, D, Q, H, E, N, T, S, A, L, L or V; X1 is S, OQ, E, T, K or D; and X12 is L, V, 1, F, Y, or W 3 CDR3 VPWGX: 3YHPX14X15VX16 where X13 is Exemplary | D, 1, T, K, R, A, E, S or Y; X1a is R, G, L, K, T, Q, S or N; X15 is N, K, E, V, R, M or D; and Xi6ç IS Y, A, V, K, H, L, M, T, R, Q, C, S or N and Pl
SEQ ID Description String
SEQ ID Description String FP TEE JE
SEQ ID Description Sequence FE ss DE ss DO o
SEQ ID Description Ts TFT Sequence FE SS
SEQ ID Description String
PE FO SEQ ID Name CDR2 Es
SEQ ID Description String
SEQ ID Description String
SEQ ID Description String
SEQ ID Description String
EF EF SEQ CDR3 ID NO: AND
SEQ ID Description String
SEQ ID Description String
SEQ ID Description String
SEQ ID Description String SEQ ID | Sequence Name VHH EE ç = = EVOLVESGGGLVOPGRSLTLSCAASTNIFSISPMGWYRQO
APGKORELVAAIHGFSTLYADSVKGRETISRDNAKNSIY 346 BH2T
LOMNSLRPEDTALYYCNKVPWGDYHPRNVYWGOGTOVTV Jo aa evqlvesgggLVOPGRSLTLSCAASTDIFSISPMGWYRO 347 O01A01 APGKQORELVAAIHGGSTLYADSVKGRFTISRDNAKNSIY LOMNSLRPEDTALYYCNKVPWGDYHPRNVAWGOGTOVTV
SEQ ID | VHH Sequence Name NO Construct LI A EVOLVESGGGLVOPGRSLTLSCAASTNDFSISPMGWYRQ
APGKORELVAAIHGGSTLYADSVKGRFTISRDNAKNSIY 348 | 02EO09
LOMNSLRPEDTALYYCNKVPWGDYHPRNVANGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSKSPMGWYRO
APGKORELVAAIHGKSTLYADSVKGRFTISRDNAKNSIY 349 | 01B03
LOMNSLRPEDTALYYCNKVPWGDYHPRNVVWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNDFSISPMGWYRO
APGKORELVAAIHGKSTLYADSVKGRFTISRDNAKNSIY 350 | 01B04
LOMNSLRPEDTALYYCNKVPWGDYHPRNVKWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNQFSISPMGWYRO
APGKORELVAAIHGKSTLYADSVKGRFTISRDNAKNSIY 351 | 02H05
LOMNSLRPEDTALYYCNKVPWGDYHPRNVVWGOGTOVTV ss evqalvesgggLVOPGRSLTLSCAASTNIFSSSPMGWYRQ
APGKORELVAAINGFSTLYADSVKGRFTISRDNAKNSIY 352 | 01A02
LOMNSLRPEDTALYYCNKVPWGDYHPRNVHWGQOGTOVTV ss evqglvesgggLVOPGRSLTLSCAASTNIFSSSPMGWYRO 353 | [01A05 APGKORELVAAIHGFSTLYADSVKGRFTISRDNAKNSIY
LOMNSLRPEDTALYYCNKVPWGDYHPRNVYWGOGTOVTV ss O01B12 EVOLVESGGGLVOPGRSLTLSCAASTNIFSTSPMGWYRO
SEQ ID | VHH Sequence Name NO Construct APGKORELVAAIHGFOTLYADSVKGRFTISRDNAKNSIY
LQOMNSLRPEDTALYYCNKVPWGDYHPRNVVWGOGTOVTV ss EVOLVESGGGLVOQPGRSLTLSCAASTNIFSRSPMGWYRQO
APGKORELVAAIHGFETLYADSVKGRFTISRDNAKNSIY 355 01G06
LOMNSLRPEDTALYYCNKVPWGDYHPRNVLWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSESPMGWYRQO
APGKQORELVAAIHGFTTLYADSVKGRETISRDNAKNSIY 356 02C05
LOMNSLRPEDTALYYCNKVPWGDYHPRNVTWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSDSPMGWYRQO
APGKQORELVAAIHGFSTLYADSVKGRFTISRDNAKNSIY 357 02G09
LOMNSLRPEDTALYYCNKVPWGDYHPRNVAWGOGTOVTV ss EVOLVESGGGLVOQPGRSLTLSCAASTNIFSNSPMGWYRQ
APGKORELVAAIHGGSTLYADSVKGRFTISRDNAKNSIY 358 01C08
LQOMNSLRPEDTALYYCNKVPWGDYHPRNVHWGOGTQOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSNSPMGWYRQO
APGKORELVAAIHGRSTLYADSVKGRFTISRDNAKNSIY 359 02B01
LQOMNSLRPEDTALYYCNKVPWGDYHPRNVMWGOGTQOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSNSPMGWYRQO
APGKORELVAAIHGPSTLYADSVKGRFTISRDNAKNSIY 360 02E03
LOMNSLRPEDTALYYCNKVPWGDYHPRNVTWGOGTOVTV ss
SEQ ID | VHH Sequence Name NO Construct EVOLVESGGGLVOPGRSLTLSCAASTNIFSNSPMGWYRQO
APGKQRELVAAIHGDSTLYADSVKGREFTISRDNAKNSIY 361 01DO03
LOMNSLRPEDTALYYCNKVPWGDYHPRNVRWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSRSPMGWYRQO
APGKQORELVAAIHGDSTLYADSVKGRFTISRDNAKNSIY 362 01D06
LQOMNSLRPEDTALYYCNKVPWGDYHPRNVTWGOGTOVTV ss EVOLVESGGGLVOQPGRSLTLSCAASTNIFSKSPMGWYRQ
APGKORELVAAIHGOSTLYADSVKGRFTISRDNAKNSIY 363 01H04
LQOMNSLRPEDTALYYCNKVPWGDYHPRNVTWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSSSPMGWYRQO
APGKORELVAAIHGHSTLYADSVKGRFTISRDNAKNSIY 364 02B07
LOMNSLRPEDTALYYCNKVPWGDYHPRNVTWGOGTOVTV ss evqlvesgggLVOPGRSLTLSCAASTNIFSSSPMGWYRQO
APGKQRELVAAIHGESTLYADSVKGREFTISRDNAKNSIY 365 O01A08
LOMNSLRPEDTALYYCNKVPWGDYHPRKVYWGOGTQOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSRSPMGWYRO
APGKQRELVAAIHGNSTLYADSVKGREFTISRDNAKNSIY 366 O1B07
LOMNSLRPEDTALYYCNKVPWGIYHPRNVYWGOGTOVTV ss
EVOLVESGGGLVOPGRSLTLSCAASTNIFSESPMGWYRQO 367 01FO3 APGKQORELVAAIHGNSTLYADSVKGRFTISRDNAKNSIY LQOMNSLRPEDTALYYCNKVPWGTYHPRNVYWGOGTOVTV
SEQ ID | VHH Sequence Name NO Construct LI EVOLVESGGGLVOQPGRSLTLSCAASTNIFSVSPMGWYRQ
APGKORELVAAIHGNSTLYADSVKGRFTISRDNAKNSIY 368 02F05
LQOMNSLRPEDTALYYCNKVPWGKYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSVSPMGWYRQO
APGKORELVAAIHGNSTLYADSVKGRETISRDNAKNSIY 369 02H04
LOMNSLRPEDTALYYCNKVPWGDYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSKSPMGWYRQO
APGKQRELVAAIHGNSTLYADSVKGRFTISRDNAKNSIY 370 02A07
LOMNSLRPEDTALYYCNKVPWGDYHPREVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSDSPMGWYRQ
APGKQORELVAAIHGTSTLYADSVKGRFTISRDNAKNSIY 371 01DO5
LQOMNSLRPEDTALYYCNKVPWGDYHPRNVYWGOGTOVTV ss EVOLVESGGGLVQPGRSLTLSCAASTNIFSRSPMGWYRQ
APGKORELVAAIHGTSTLYADSVKGRFTISRDNAKNSIY 372 O1EOS5
LQOMNSLRPEDTALYYCNKVPWGKYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSHSPMGWYRQO
APGKORELVAAIHGTSTLYADSVKGRFTISRDNAKNSIY 373 01F02
LQOMNSLRPEDTALYYCNKVPWGRYHPRNVYWGOGTOVTV ss
EVOLVESGGGLVOPGRSLTLSCAASTNIFSTSPMGWYRQO 374 02C06 APGKORELVAAIHGTSTLYADSVKGREFTISRDNAKNSIY
SEQ ID | VHH Sequence Name NO Construct
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSTSPMGWYRO
APGKORELVAAIHGTSTLYADSVKGRFTISRDNAKNSIY 375 02F11
LOMNSLRPEDTALYYCNKVPWGDYHPRNVOWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSLSPMGWYRO
APGKORELVAAIHGDSTLYADSVKGRFTISRDNAKNSIY 376 | 01E06
LOMNSLRPEDTALYYCNKVPWGDYHPRNVYWGQOGTOVTV ss evqalvesgggLVOPGRSLTLSCAASTNIFSISPGGWYRQO
APGKORELVAAIHGSSTLYADSVKGRFTISRDNAKNSIY 377 01A03
LOMNSLRPEDTALYYCNKVPWGDYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNHFSISPMGWYRO
APGKORELVAAIHGSSTLYADSVKGRFTISRDNAKNSIY 378 02A11
LOMNSLRPEDTALYYCNKVPWGDYHPRVVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSASPMGWYRO
APGKORELVAAIHGSSTLYADSVKGRFTISRDNAKNSIY 379 | 01DO7
LOMNSLRPEDTALYYCNKVPWGDYHPRNVNWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSASPMGWYRO
APGKORELVAAIHGSSTLYADSVKGRFTISRDNAKNSIY 380 O1D10
LOMNSLRPEDTALYYCNKVPWGRYHPRNVYWGOGTOVTV ss 01A07 evalvesgggLVOPGRSLTLSCAASTNISSISPMGWYRQO
SEQ ID | VHH Sequence Name NO Construct APGKORELVAAIHGTSTLYADSVKGRFTISRDNAKNSIY
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGQOGTOVTV ss EVOLVESGGGLVOQPGRSLTLSCAASTNIESISPMGWYRQO
APGKORELVAAIHGTSTLYADSVKGRFTISRDNAKNSIY 382 02F12
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSISPYGWYRQO
APGKQORELVAAIHGTSTLYADSVKGRETISRDNAKNSIY 383 02B05
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIASISPMGWYRO
APGKQORELVAAIHGTSTLYADSVKGRFTISRDNAKNSIY 384 O1EO4
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOQPGRSLTLSCAASTNIASISPMGWYRQO
APGKORELVAAIHGKSTLYADSVKGRFTISRDNAKNSIY 385 02A05
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGQOGTQOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIASISPMGWYRQO
APGKORELVAAIHGSSTLYADSVKGRFTISRDNAKNSIY 386 02C03
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGQOGTQOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNITSISPMGWYRQO
APGKORELVAAIHGDSTLYADSVKGRFTISRDNAKNSIY 387 O01EO03
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss
SEQ ID | VHH Sequence Name NO Construct EVOLVESGGGLVOPGRSLTLSCAASTNIMSISPMGWYRQO
APGKQRELVAAIHGNSTLYADSVKGREFTISRDNAKNSIY 388 01H09
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNITSISPMGWYRO
APGKQORELVAAIHGNSTLYADSVKGRFTISRDNAKNSIY 389 02GO05
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOQPGRSLTLSCAASTNIVSISPMGWYRQ
APGKORELVAAIHGHSTLYADSVKGRFTISRDNAKNSIY 390 01C01
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIVSISPMGWYRQO
APGKORELVAAIHGKSTLYADSVKGRFTISRDNAKNSIY 391 01DO02
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNVVSISPMGWYRO
APGKQRELVAAIHGKSTLYADSVKGREFTISRDNAKNSIY 392 02D09
LOMNSLRPEDTALYYCNKVPWGDYHPNNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIISISPMGWYRO
APGKQRELVAAIHGASTLYADSVKGRFTISRDNAKNSIY 393 02C01
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss
EVOLVESGGGLVOPGRSLTLSCAASTNIFSITPMGWYRO 394 02G02 APGKQORELVAAIHGASTLYADSVKGRFTISRDNAKNSIY LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV
SEQ ID | VHH Sequence Name NO Construct LI EVOLVESGGGLVOQPGRSLTLSCAASTNITSISPMGWYRQ
APGKORELVAAIHGFETLYADSVKGRFTISRDNAKNSIY 395 O1B05
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIQSISPMGWYRQO
APGKORELVAAIHGFETLYADSVKGRETISRDNAKNSIY 396 01GO8
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTQOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTSDFSISPMGWYRQO
APGKQRELVAAIHGFETLYADSVKGRFTISRDNAKNSIY 397 01H06
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIDSISPMGWYRQO
APGKQORELVAAIHGFOTLYADSVKGRFTISRDNAKNSIY 398 01FO04
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVQPGRSLTLSCAASTNIMSISPMGWYRQ
APGKORELVAAIHGFSTVYADSVKGRFTISRDNAKNSIY 399 01H08
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIESISPMGWYRQO
APGKORELVAAIHGFSTLYADSVKGRFTISRDNAKNSIY 400 02F07
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTQOVTV ss
EVOLVESGGGLVOPGRSLTLSCAASTNIFSSSPMGWYRQ 401 01CO05 APGKORELVAAIHGFKTLYADSVKGREFTISRDNAKNSIY
SEQ ID | VHH Sequence Name NO Construct
LOMNSLRPEDTARYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSSSPMGWYRO
APGKORELVAAIHGFSTLYADSVKGRFTISRDNAKNSIY 402 | 02FO04
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSNSPMGWYRO
APGKORELVAAIHGFSTLYADSVKGRFTISRDNAKNSIY 403 | 02B06
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGQOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSTSPMGWYRQ
APGKORELVAAIHGFSTIYADSVKGRFTISRDNAKNSIY 404 | 01FO7
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGQOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSTSPMGWYRO
APGKORELVAAIHGFSTIYADSVKGRFTISRDNAKNSIY 405 | 02B04
LOMNSLRPEDTALYYCNKVPWGDYHPLNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCVASTNIFSTSPMGWYRQO
APGKORELVAAIHGFSTLYADSVKGRFTISRDNAKNSIY 406 | 01H11
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSDSPMGWYRQO
APGKORELVAAIHGFSTFYADSVKGRFTISRDNAKNSIY 407 | 02EO06
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGQOGTOVTV ss 01EO8 EVOLVESGGGLVOPGRSLTLSCAASTNIFSQSPMGWYRO
SEQ ID | VHH Sequence Name NO Construct APGKORELVAAIHGDSTLYADSVKGRFTISRDNAKNSIY
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVCWGQOGTOVTV ss EVOLVESGGGLVOQPGRSLTLSCAASTNIFSQSPMGWYRQO
APGKORELVAAIHGKSTLYADSVKGRFTISRDNAKNSIY 409 02A04
LOMNSLRPEDTALYYCNKVPWGDYHPSNVYWGKGTQOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSRSPMGWYRQO
APGKQORELVAAIHGESTLYADSVKGRETISRDNAKNSIY 410 02A08
LOMNSLRPEDTALYYCNKVPWGRYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSRSPMGWYRQO
APGKQORELVAAIHGISTLYADSVKGRFTISRDNAKNSIY 411 02EO5
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss EVOLVESGGGLVOQPGRSLTLSCAASTNIFSRSPMGWYRQ
APGKORELVAAIHGSSTLYADSVKGRFTISRDNAKNSIY 412 02H09
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGQOGTQOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSGSPMGWYRQO
APGKORELVAAIHGNSTLYADSVKGRFTISRDNAKNSIY 413 02GO06
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGQOGTQOVTV ss EVOLVESGGGLVOPGRSLTLSCAASSNIFSISPMGWYRQO
APGKORELVAAIHGSSTLYADSVKGRFTISRDNAKNSIY 414 01B09
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss
SEQ ID | String Name VHH NO Construct evqlvesgggLVOPGRSLTLSCAASTNIFSIYPMGWYRQO
APGKQRELVAAIHGSSTLYADSVKGREFTISRDNAKNSIY 415 02F03
LOMNSLRPEDTALYYCNKVPWGDYHPKNVYWGOGTOVTV ss evqlvesgggLVOPGRSLTLSCAASTNIFSKSPMGWYRO
APGKQORELVAAIHGSSTLYADSVKGRFTISRDNAKNSIY 416 02F02
LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss evqlvesgggLVOPGRSLTLSCAASTNIFSKSPMGWYRQ
APGKORELVAAIHGSSTLYADSVKGRFTISRDNAKNSIY 417 02H01
LQOMNSLRPEDTALYYCNKVPWGDYHPRNVYWGOGTQOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNEFSISPMGWYRQO
APGKORELVAAIHGLSTLYADSVKGRFTISRDNAKNSIY 418 o01G10
LOMNSLRPEDTALYYCNKVPWGAYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNEFSISPMGWYRO
APGKQRELVAAIHGESTLYADSVKGREFTISRDNAKNSIY 419 02D11
LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss evqlvesgggLVOPGRSLTLSCAASTNIPSISPMGWYRQO
APGKQRELVAAIHGESTLYADSVKGRFTISRDNAKNSIY 420 O1B01
LOMNSLRPEDTALYYCNKVPWGDYHPRNVAWGOGTOVTV ss
EVOLVESGGGLVOPGRSLTLSCAASTNIPSISPMGWYRO 421 01G11 APGKQORELVAAIHGASTLYADSVKGRFTISRDNAKNSIY LQOMNSLRPEDTALYYCNKVPWGDYHPRNVAWGOGTOVTV
SEQ ID | String Name VHH NO Log Construct evqlvesgggLVOPGRSLTLSCAASTNIPSISPMGWYRQ
APGKORELVAAIHGESTLYADSVKGRFTISRDNAKNSIY 422 O1H10 LOMNSLRPEDTALYYCNKVPWGDYHPRNVYWGQOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIPSISPMGWYRO
APGKORELVAAIHGDSTLYADSVKGRFTISRDNAKNSIY 423 01C04
LOMNSLRPEDTALYYCNKVPWGDYHPRNVYWGQOGTOVTV ss EVOLVESGGGLVQPGRSLTLSCAASTNITSISPMGWYRQ
APGKORELVAAIHGVSTLYADSVKGRFTISRDNAKNSIY 424 01D04
LOMNSLRPEDTALYYCNKVPWGDYHPRNVONGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIPSISPMGWYRQO
APGKORELVAAIHGQOSTLYADSVKGRFTISRDNAKNSIY 425 01EO7
LOMNSLRPEDTALYYCNKVPWGDYHPRNVOWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIVSISPMGWYRO
APGKORELVAAIHGDSTLYADSVKGRFTISRDNAKNSIY 426 | 02B11
LOMNSLRPEDTALYYCNKVPWGDYHPRNVSWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASSNIFSISPMGWYRO
APGKORELVAAIHGESTLYADSVKGRFTISRDNAKNSIY 427 01F10
LOMNSLRPEDTALYYCNKVPWGDYHPRNVTWGQOGTOVTV ss 02GO08 EVOLVESGGGLVOPGRSLTLSCAASTNIDSISPMGWYRO
SEQ ID | VHH Sequence Name NO Construct APGKORELVAAIHGESTLYADSVKGRFTISRDNAKNSIY
LQOMNSLRPEDTALYYCNKVPWGDYHPRNVTWGOGTOVTV ss EVOLVESGGGLVOQPGRSLTLSCAASTNIDSISPMGWYRQO
APGKORELVAAIHGSSTLYADSVKGRFTISRDNAKNSIY 429 02G11
LOMNSLRPEDTALYYCNKVPWGDYHPRNVTWGOGTOVTV ss evqlvesgggLVOPGRSLTLSCAASTNIRSISPMGWYRQ
APGKQORELVAAIHGSSTLYADSVKGREFTISRDNAKNSIY 430 02H06
LOMNSLRPEDTALYYCNKVPWGDYHPRNVVWGOGTOVTV ss evqlvesgggLVOPGRSLTLSCAASTNITSISPMGWYRO
APGKQORELVAAISGFSTLYADSVKGRFTISRDNAKNSIY 431 O1B02
LOMNSLRPEDTALYYCNEVPWGDYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOQPGRSLTLSCAASTNITSISPMGWYRQO
APGKORELVAAIHGESTLYADSVKGRFTISRDNAKNSIY 432 02H11
LQOMNSLRPEDTALYYCNKVPWGDYHPRNVYWGQOGTQOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNITSVSPMGWYRQO
APGKORELVAAIHGPSTLYADSVKGRFTISRDNAKNSIY 433 01FO8
LQOMNSLRPEDTALYYCNKVPWGDYHPTNVYWGOGTQOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIGSISPMGWYRQO
APGKORELVAAIHGOSTLYADSVKGRFTISRDNAKNSIY 434 01H01
LOMNSLRPEDTALYYCNKVPWGDYHPONVYWGOGTOVTV ss
SEQ ID | VHH Sequence Name NO Construct EVOLVESGGGLVOPGRSLTLSCAASTNIESISPMGWYRQO
APGKQRELVAAIHGKSTLYADSVKGREFTISRDNAKNSIY 435 O1E10
LOMNSLRPEDTALYYCNKVPWGDYHPRRVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIVSISPMGWYRQO
APGKQORELVAAIHGDSTLYADSVKGRFTISRDNAKNSIY 436 01GO01
LQOMNSLRPEDTALYYCNKVPWGDYHPRRVYWGOGTOVTV ss EVOLVESGGGLVOQPGRSLTLSCAASTNIDSISPMGWYRQ
APGKORELVAAIHGNSTLYADSVKGRFTISRDNAKNSIY 437 01GO04
LQOMNSLRPEDTALYYCNKVPWGDYHPRMVYWGQOGTQOVTV ss evqlvesgggLVOPGRSLTLSCAASTNIFMISPMGWYRQ
APGKORELVAAIHGDSTLYADSVKGRFTISRDNAKNSIY 438 O01A04
LOMNSLRPEDTALYYCNKVPWGRYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFRISPMGWYRO
APGKQRELVAAIHGDSTLYADSVKGREFTISRDNAKNSIY 439 O01F12
LOMNSLRPEDTALYYCNKVPWGRYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSISPMGWYRO
APGKQRELVAAIHGDSTLYADSVKGRFTISRDNAKNSIY 440 O1B06
LOMNSLRPEDTALYYCNKVPWGEYHPRNVYWGOGTOVTV ss
EVOLVESGGGLVOPGRSLTLSCAASTNIFSISPMGWYRQO 441 01C06 APGKQORELVAAIHGDSTLYADSVKGRFTISRDNAKNSIY LQOMNSLRPEDTALYYCNKVPWGKYHPRNVYWGOGTOVTV
SEQ ID | VHH Sequence Name NO Construct LI EVOLVESGGGLVOQPGRSLTLSCAASTNIESISPMGWYRQ
APGKORELVAAIHGSSTLYADSVKGRFTISRDNAKNSIY 442 O1B08
LQOMNSLRPEDTALYYCNKVPWGRYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIESISPMGWYRQO
APGKORELVAAIHGNSTLYADSVKGRETISRDNAKNSIY 443 01C02
LOMNSLRPEDTALYYCNKVPWGRYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNISSISPMGWYRO
APGKQRELVAAIHGFSTLYADSVKGRFTISRDNAKNSIY 444 oO1C10
LOMNSLRPEDTALYYCNKVPWGYYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNISSISPMGWYRQO
APGKQORELVAAIHGHSTLYADSVKGRFTISRDNAKNSIY 445 01FO09
LQOMNSLRPEDTALYYCNKVPWGRYHPRNVYWGOGTOVTV ss EVOLVESGGGLVQPGRSLTLSCAASTNISSISPMGWYRQ
APGKORELVAAIHGFSTVYADSVKGRFTISRDNAKNSIY 446 02D06
LQOMNSLRPEDTALYYCNKVPWGRYHPRNVYWGOGTOVTV ss EvqlvesgggLVOPGRSLTLSCAASTNIFSIRPMGWYRQ
APGKORELVAAIHGFSTVYADSVKGRFTISRDNAKNSIY 447 01A06
LQOMNSLRPEDTALYYCNKVPWGDYHPRNVYWGOGTOVTV ss
EVOLVESGGGLVOPGRSLTLSCAASTNIFSIYPMGWYRQO 448 01C07 APGKORELVAAIHGFSTYYADSVKGREFTISRDNAKNSIY
SEQ ID | VHH Sequence Name NO Construct
LOMNSLRPEDTALYYCNKVPWGSYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFNISPMGWYRO
APGKORELVAAIHGFSTYYADSVKGRFTISRDNAKNSIY 449 | 01G09
LOMNSLRPEDTALYYCNKVPWGRYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSSSPMGWYRO
APGKORELVAAIHGFSTWYADSVKGRFTISRDNAKNSIY 450 | 01FO5
LOMNSLRPEDTALYYCNKVPWGRYHPRNVYWGQOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNISSISPMGWYRQO
APGKORELVAAIHGFDTLYADSVKGRFTISRDNAKNSIY 451 | 02B12
LOMNSLRPEDTALYYCNKVPWGDYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSINPMGWYRO
APGKORELVAAIHGFDTLYADSVKGRFTISRDNAKNSIY 452 | 02GO01
LOMNSLRPEDTALYYCNKVPWGDYHPRNVSWGOGTOVTV ss evglvesgggLVOPGRSLTLSCAASTNIFSITPMGWYRO
APGKORELVAAIHGRSTLYADSVKGRFTISRDNAKNSIY 453 | 01A09
LOMNSLRPEDTALYYCNKVPWGSYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOPGRSLTLSCAASTNIFSITPMGWYRO
APGKORELVAAIHGTSTLYADSVKGRFTISRDNAKNSIY 454 | 01H05
LOMNSLRPEDTALYYCNKVPWGRYHPRNVYWGOGTOVTV ss 02F06 EVOLVESGGGLVOPGRSLTLSCAASTNIFSITPMGWYRO
SEQ ID | String Name VHH EE —— APGKORELVAAIHGESTLYADSVKGRFTISRDNAKNSIY
LQOMNSLRPEDTALYYCNKVPWGRYHPRNVYWGOGTOVTV ss EVOLVESGGGLVOQPGRSLTLSCAASTNIFSITPMGWYRO
APGKORELVAAIHGESTLYADSVKGRFTISRDNAKNSIY 456 02G07
LOMNSLRPEDTALYYCNKVPWGDYHPRDVYWGOGTOVTV wow SO
EVOLVESGGGLVOPGRSLTLSCAASTNIFSTSPYGWYRO 01F07T- APGKORELVAAIHGFSTIYADSVKGRETISRDNAKNSIY o M34Y LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss
EVOLVESGGGLVOPGRSLTLSCAASTNIFSTSPGGWYRO 01F01- APGKQORELVAAIHGFSTIYADSVKGRFTISRDNAKNSIY aê M34G LOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss
EVOLVESGGGLVOPGRSLTLSCAASTNIFSITPYGWYRQO 02G02- APGKORELVAAIHGASTLYADSVKGRFTISRDNAKNSIY to M34Y LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss
EVOLVESGGGLVOQPGRSLTLSCAASTNIFSITPGGWYRO 02G02- APGKORELVAAIHGASTLYADSVKGRFTISRDNAKNSIY 2o M34G LQOMNSLRPEDTALYYCNKVPWGDYHPGNVYWGOGTOVTV ss
SEQ ID | VHH EEE ps String Name ps MLQOMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCOR
YCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLR 468 BOMA KINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPR GLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATIL VTTKTNDYCKSLPAALSATEIEKSISAR MAQQCFHSEYFDSLLHACKPCHLRCSNPPATCQPYCDPS
VTSSVKGTYTVLWIFLGLTLVLSLALFTISFLLRKMNPE 469 Pear ALKDEPQOSPGQLDGSAQLDKADTELTRIRAGDDRIFPRS Murano LEYTVEECTCEDCVKSKPKGDSDHFFPLPAMEEGATILV TTKTGDYGKSSVPTALQSVMGMEKPTHTR MLQOMARQCSQNEYFDSLLHDCKPCOLRCSSTPPLTCORY
CNASMTNSVKGMNAILWTCLGLSLIISLAVEVLTFLLRK 470 ONA MSSEPLKDEFKNTGSGLLGMANIDLEKGRTGDEIVLPRG cinomolgo LEYTVEECTCEDCIKNKPKVDSDHCFPLPAMEEGATILV
Label TTKTNDYCNSLSAALSVTEIEKSISAR His-His-His-His-His-His [= [o [Ames 253BH10 QVOLVESGGGLVOPGESLRLSCAASTNIFSISPMGWYRQO (anticorp | APGKORELVAAIHGFSTLYADSVKGRETISRDNAKNTIY 472 LQOMNSLKPEDTAVYYCNKVPWGDYHPRNVYWGOGTOVTV the anti-BCMA ss llama) [e 473 CDR1
SEQ ID | Sequence Name VHH NO Construct 253BH10 AIHGFSTLYADSVK 474 CDR2 253BH10 VPWGDYHPRNVY 475 CDR3

权利要求:
Claims (29)
[1]
1. Single domain B cell maturation agent (BCMA) binding protein characterized by comprising complementarity determining regions CDR1, CDR2 and CDR3, where (a) the amino acid sequence of CDR1 is as shown in X1X2X3XaXsXeX7PXgG (SEQ ID NO: 1), where X1 is Tou S; Xo is N, D, or S; X3 is I, D, Q, H, V or E; Xa is F, S, E, A, T, M, V, I, D, Q, P, R or G; Xs is S, M, RouN; X; is I, K, S, T, R, E, D, Nf, V, H, L, A, Q or G; X; is S, T, Y, Rou N; and Xgs is M, G or Y; (b) the amino acid sequence of CDR2 is as shown in AIXsGX: oX11TX12YADSVK (SEQ ID NO: 2), where X; is H, N, or S; X1w is F, G, K, R, P, D, Q, H, E, N, T, S, A, 1, Lou V; Xu is S, OQ, E, T, K or D; and X112 is L, V, II, F, Y, Or W; Ee (c) the amino acid sequence of CDR3 is as shown in VPWGX: 3YHPX: 4aX1sVX16 (SEQ ID NO: 3), where X13 is D, I, T, K, R, A, E, S or Y; Xu is R, G, L K, T, Q, S or N; X15s is N, K, E, V, R, M or D; and Xe IS Y, A, V, K, H, L, M, T, R, Q, C, S or N; and where CDR1I is not SEQ ID NO: 473, where CDR2 is not SEQ ID NO: 474, and where CDR3 is not SEQ ID NO: 475.
[2]
2. Single-domain BCMA-binding protein according to claim 1, the protein being characterized by the following formula: fl-rl-f2-r2-f3-r3-f4 where, rl is SEQ ID NO: 1; r2 is SEQ ID NO: 2; and r3 is SEQ ID NO: 3; and where fi, fa, f3 and fa are framework residues selected so that the protein is about eighty percent (80 8%) to about 99% identical to the amino acid sequence shown in SEQ ID NO: 346 or
472.
[3]
3. Single domain BCMA-binding protein according to claim 2, characterized in that rl comprises an amino acid sequence presented as any of SEQ ID NOS: 4-117.
[4]
4, Single domain BCMA-binding protein according to claim 2, characterized in that r2 comprises an amino acid sequence shown as any of SEQ ID NOS: 118-231.
[5]
5. Single domain BCMA-binding protein according to claim 2, characterized in that r3 comprises an amino acid sequence presented as any of SEQ ID NOS: 232-345.
[6]
A single domain BCMA-binding protein according to claim 2, the protein being characterized by comprising an amino acid sequence shown as any of SEQ ID NOS: 346-460.
[7]
7. Single domain BCMA-binding protein according to claim 2, characterized in that fl comprises SEQ ID NO: 461 or 462.
[8]
8. Single domain BCMA-binding protein according to claim 2, characterized by the fact that f2 comprises SEQ ID NO: 463.
[9]
9. Single domain BCMA-binding protein according to claim 2, characterized in that f3 comprises SEQ ID NO: 464 or 465.
[10]
10. Single domain BCMA-binding protein according to claim 2, characterized by the fact that f4 comprises SEQ ID NO: 466 or 467.
[11]
11. Single domain BCMA binding protein according to claim 2, characterized by the fact that rl comprises SEQ ID NO: 76, 114, 115, 116 or 117.
[12]
12. Single domain BCMA-binding protein according to claim 2, characterized by the fact that rl comprises SEQ ID NO: 76, r2 is SEQ ID NO: 190, and r3 is SEQ ID NO: 304.
[13]
13. Single domain BCMA-binding protein according to claim 2, characterized by the fact that rl comprises SEQ ID NO: 114, r2 comprises SEQ ID NO: 228 and r3 comprises SEQ ID NO: 342.
[14]
14. Single domain BCMA binding protein according to claim 2, characterized in that rl comprises SEQ ID NO: 115, r2 comprises SEQ ID NO: 229 and r3ê comprises SEQ ID NO: 343.
[15]
15. Single domain BCMA-binding protein according to claim 2, characterized in that rl comprises SEQ ID NO: 117, r2 comprises SEQ ID NO: 231 and r3 comprises SEQ ID NO: 345.
[16]
16. Single domain BCMA-binding protein according to claim 2, characterized in that rl comprises SEQ ID NO: 116, r2 comprises SEQ ID NO: 230 and r3 comprises SEQ ID NO: 344.
[17]
17. Single domain BCMA binding protein characterized by comprising CDR1, CDR2 and CDR3, comprising a sequence presented as SEQ ID NO: 346 or 472, in which one or more amino acid residues selected from amino acid positions 26, 27, 28, 29, 30, 31,
32 and 34 of CDR1; positions 52, 54, 55 and 57 of CDR2; and positions 101, 105, 106 and 108 of CDR3 are substituted, where amino acid position 26, if substituted, is replaced by S; amino acid position 27, if substituted, is replaced by D or S; amino acid position 28, if substituted, is replaced by D, Q, H, V or E; amino acid position 29, if substituted, is replaced by S, E, A, T, M, V, I, D, Q, D, P, R or G; amino acid position 30, if substituted, is replaced by M, R or N; amino acid position 31, if substituted, is replaced by K, S, T, R, E, D, N, V, H, L, A, Q or GS; amino acid position 32, if substituted, is replaced by T, Y, R or N; amino acid position 34, if substituted, is replaced by G or Y; amino acid position 52, if substituted, is replaced by N or S; amino acid position 54, if substituted, is replaced by G, K, R, P, D, OQ, H, E, N, T, S, A, LT, L or V; amino acid position 55, if substituted, is replaced by Q, E, T, K or D; amino acid position 57, if substituted, is replaced by V, IL, F, Y, or W;
amino acid position 101, if substituted, is replaced by I, T, K, R, A, E, S or Y; amino acid position 105, if substituted, is replaced by G, L, K, T, Q, S or N; amino acid position 106, if substituted, is replaced by K, E, V, R, M or D; and amino acid position 108, if substituted, is replaced by A, V, K, H, L, M, T, R, Q, C, S or N.
[18]
Domain BCMA-binding protein according to any one of claims 1-17, the protein being characterized by having an elimination half-life of at least 12 hours, at least 20 hours, at least 25 hours, at least at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, or at least 100 hours.
[19]
19. Single domain BCMA binding protein according to any one of claims 1-18, characterized by being human, humanized, affinity matured, or a combination thereof.
[20]
20. Single domain BCMA-binding protein according to any one of claims 1-19, characterized in that it further comprises an Fc domain.
[21]
21. Single domain BCMA binding protein according to any one of claims 1-19, characterized in that it further comprises an anticancer agent.
[22]
22. Single domain BCMA-binding protein according to claim 21, characterized in that the anti-cancer agent is conjugated to the single-domain BCMA-binding protein.
[23]
23. Method for treating or ameliorating B-cell lineage cancer in an individual in need of it characterized by comprising administering to the individual a single domain BCMA-binding protein as defined in any of claims 20-22.
[24]
24. Method according to claim 23, characterized by the fact that B cell lineage cancer is a primary cancer or a metastatic cancer.
[25]
25. Method according to claim 23 or 24, characterized by the fact that cancer of the B cell lineage is a multiple myeloma, a leukemia, a lymphoma.
[26]
26. Multispecific binding protein characterized by comprising the single domain BCMA binding protein, as defined in any one of claims 1-22.
[27]
27. Method for treating or ameliorating B-cell lineage cancer in an individual in need of it characterized by comprising administering to the individual a multispecific binding protein, as defined in claim 26.
[28]
28. Method according to claim 27, characterized by the fact that cancer of the B cell lineage is a primary cancer or a metastatic cancer.
[29]
29. Method according to claim 27 or 28, characterized by the fact that cancer of the B cell lineage is a multiple myeloma, a leukemia, a lymphoma.

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US10543271B2|2017-05-12|2020-01-28|Harpoon Therapeutics, Inc.|Mesothelin binding proteins|

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CN111465612A|2017-10-13|2020-07-28|哈普恩治疗公司|B cell maturation antigen binding proteins|

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JP2020537644A|2017-10-13|2020-12-24|ハープーン セラピューティクス，インク．|Trispecific protein and usage|

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CA3114038A1|2018-09-25|2020-04-02|Harpoon Therapeutics, Inc.|Dll3 binding proteins and methods of use|

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US11180563B2|2020-02-21|2021-11-23|Harpoon Therapeutics, Inc.|FLT3 binding proteins and methods of use|

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WO2021226543A2|2020-05-08|2021-11-11|Seattle Children's Hospital D/B/A Seattle Children's Research Institute|Chimeric antigen receptorstargeting natural killer cells|

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CN111909271B|2020-08-12|2021-03-23|深圳市茵冠生物科技有限公司|BCMA chimeric antigen receptor based on single domain antibody and application thereof|

法律状态:
2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762572375P| true| 2017-10-13|2017-10-13|

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US62/572,375|2017-10-13|

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PCT/US2018/055682|WO2019075378A1|2017-10-13|2018-10-12|B cell maturation antigen binding proteins|

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