Anti-b-cell maturation antigen (BCMA) binding domains and uses thereof

Abstract

Anti-B-cell maturation antigen (BCMA) binding domains and uses thereof are provided. The binding domains can be used to target such cells expressing BCMA ex vivo or in vivo for research, diagnostic, or therapeutic purposes, for example in the treatment of multiple myeloma.

Description

F053-0202PCT / 25-116-WO-PCTANTI-B-CELL MATURATION ANTIGEN (BCMA) BINDING DOMAINS AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 777,455 filed on March 25, 2025, the entire contents of which are incorporated by reference herein.REFERENCE TO SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is 3LL9672.XML. The file is 196,543 bytes, was created on March 25, 2026, and is being submitted electronically via Patent Center.FIELD OF THE DISCLOSURE

[0003] The present disclosure provides anti-B-cell maturation antigen (BCMA) binding domains and uses thereof. The binding domains can be used to target cells expressing BCMA ex vivo or in vivo for research, diagnostic, or therapeutic purposes.BACKGROUND OF THE DISCLOSURE

[0004] Multiple myeloma (MM) is the second most common hematological malignancy characterized by clonal proliferation of plasma cells in the bone marrow or less frequently at extramedullary sites producing monoclonal immunoglobulins. Although the past decade has been marked by significant therapeutic advances, increasing patient survival, MM is still considered incurable and almost all MM patients eventually relapse. Effective and tolerable treatment options for relapsed and refractory (R / R) MM patients remain unsatisfactory with a median overall survival (OS) of 9.3 months in tri pie- refractory patients and 5.6 months in penta-refractory patients.

[0005] In recent years, more targeted therapies to treat cancer have been developed. Targeted therapies specifically target cancer cells by identifying and exploiting specific molecular and / or immunophenotypic changes seen primarily in those cells. For example, many cancer cells preferentially express markers on their cellular surfaces which may be used as targets for antibody-based therapeutics.

[0006] Significant progress has been made in genetically engineering cells of the immune system to target and kill unwanted cell types, such as cancer cells. Many of these immune cells are T cells that have been genetically engineered to express a recombinant receptor, for example, a chimeric antigen receptor (CAR). CAR are proteins including several distinct subcomponents that allow the genetically modified immune cells to recognize and kill targeted cell types. TheF053-0202PCT / 25-116-WO-PCTsubcomponents include at least an extracellular component and an intracellular component expressed as a single protein or in a form that assembles into a functional unit. The extracellular component includes a binding domain that specifically binds a marker (e.g., an antigen) that is preferentially present on the surface of unwanted cells, such as cancer cells. When the binding domain binds such markers, the intracellular component signals the immune cell to destroy the bound cell. CAR can additionally include a transmembrane domain that can link the extracellular component to the intracellular component.

[0007] For antibody-based therapeutics to be effective, an appropriate target antigen must be selected that preferentially targets and kills malignant cells. B cell maturation antigen (BCMA) is a transmembrane glycoprotein in the tumor necrosis factor receptor superfamily 17 (TNFRSF17). It is preferentially expressed by mature B lymphocytes, and its overexpression and activation are associated with MM.SUMMARY OF THE DISCLOSURE

[0008] The present disclosure provides anti-B-cell maturation antigen (BCMA) binding domains and uses thereof. The binding domains can be used to target cells expressing BCMA ex vivo or in vivo for research, diagnostic, or therapeutic purposes.

[0009] In particular embodiments, the disclosed anti-BCMA binding domains include an anti-BCMA antibody. In particular embodiments, the anti-BCMA antibody includes an anti-BCMA scFv. In particular embodiments, the anti-BCMA binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 24 and a variable light chain including the sequence of SEQ ID NO: 25. In particular embodiments, the anti-BCMA binding domain is included in a multidomain binding molecule, an antibody conjugate, and / or a recombinant receptor. In particular embodiments, a recombinant receptor includes a chimeric antigen receptor including the sequence of SEQ ID NO: 82 or SEQ ID NO: 83.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0010] Some of the drawings submitted herewith may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0011] FIGs. 1A,1B. The chimeric antigen receptor (CAR) T cells were tested for their cytotoxicity using a real-time killing assay. Anti-BCMA CAR T cells (effector) were cocultured with fluorescent target cells coexpressing green fluorescent protein (GFP) and either (1 A) mouse B cell maturation antigen (BCMA) antigen or (1B) human BCMA antigen (1x104cells / well) at the indicated effector: target (E:T) ratios in 96-well plates. Four images of each culture well were recorded in anF053-0202PCT / 25-116-WO-PCTIncucyte® every three hours for three days and analyzed using the Incucyte® image analysis software. Killing potency of the anti-BCMA CAR T cells was measured as the relative decrease in the number of fluorescent targets (normalized to time=0). T cells expressing an anti-CD19 CAR were used as negative controls. The anti-BCMA CAR T cells specifically target cells expressing the mouse BCMA antigen but did not target cells expressing the human BCMA antigen, demonstrating antigen-specific cytotoxicity relevant to treatment of BCMA-related disorders.

[0012] FIG. 2. Sequences supporting the disclosure.DETAILED DESCRIPTION

[0013] B cell maturation antigen (BCMA) is a transmembrane glycoprotein in the tumor necrosis factor receptor superfamily 17 (TNFRSF17). It is preferentially expressed by mature B lymphocytes, and its overexpression and activation are associated with multiple myeloma (MM).

[0014] The present disclosure provides anti-BCMA binding domains and uses thereof. The binding domains can be used to target cells expressing BCMA ex vivo or in vivo for research, diagnostic, or therapeutic purposes.

[0015] In particular embodiments, the disclosed anti-BCMA binding domains include an anti-BCMA antibody. In particular embodiments, the anti-BCMA antibody includes an anti-BCMA scFv. In particular embodiments, the anti-BCMA binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 24 and a variable light chain including the sequence of SEQ ID NO: 25. In particular embodiments, the anti-BCMA binding domain is included in a multidomain binding molecule, an antibody conjugate, and / or a recombinant receptor. In particular embodiments, a recombinant receptor includes a chimeric antigen receptor including the sequence of SEQ ID NO: 82 or SEQ ID NO: 83.

[0016] Aspects of the current disclosure are now described with additional details and options as follows: (i) Anti-BCMA Binding Domains; (ii) Antibody Variants; (iii) Multi-Domain Binding Molecules; (iv) Expression of Recombinant Proteins; (v) Antibody Conjugates; (vii) Compositions and Formulations; (viii) Kits; (ix) Methods of Use; (x) Exemplary Embodiments; and (xi) Closing Paragraphs. These headings are provided for organization purposes only and should not be construed to limit the teachings or interpretation of the current disclosure.

[0017] (i) Anti-BCMA Binding Domains. The present disclosure provides binding domains (e.g., antibodies) that bind BCMA. In particular embodiments, BCMA [Homo sapiens] (GenBank: BAB60895.1) includes the sequence:MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLS LIISLAVFVLMFLLRKISSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDF053-0202PCT / 25-116-WO-PCTCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR (SEQ ID NO: 1).

[0018] In particular embodiments, BCMA [Mus musculus] (GenBank: AAC23799.1) includes the sequence:MAQQCFHSEYFDSLLHACKPCHLRCSNPPATCQPYCDPSVTSSVKGTYTVLWIFLGLTLVLSL ALFTISFLLRKMNPEALKDEPQSPGQLDGSAQLDKADTELTRIRAGDDRIFPRSLEYTVEECTCE DCVKSKPKGDSDHFFPLPAMEEGATILVTTKTGDYGKSSVPTALQSVMGMEKPTHTR (SEQ ID NO: 2).

[0019] Naturally occurring antibody structural units include a tetramer. Each tetramer includes two pairs of polypeptide chains, each pair having one light chain and one heavy chain. The aminoterminal portion of each chain includes a variable region that is responsible for antigen recognition and epitope binding. The variable regions exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions (CDRs). The CDRs from the two chains of each pair are aligned by the framework regions, which enables binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions include the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4.

[0020] The assignment of amino acids to each domain can be in accordance with Kabat numbering (Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme)); Chothia (Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme)), Martin (Abinandan etal., Mol Immunol. 45:3832-3839 (2008), “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains”), Gelfand, Contact (MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (Contact numbering scheme)), IMGT (Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme)), AHo (Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme)), North (North et al., J Mol Biol. 406(2):228-256 (2011), “A new clustering of antibody CDR loop conformations”), or other numbering schemes.

[0021] Definitive delineation of a CDR and identification of residues including the binding site of an antibody can be accomplished by solving the structure of the antibody and / or solving the structure of the antibody-epitope complex. In particular embodiments, this can be accomplished by methods such as X-ray crystallography and cryoelectron microscopy. Alternatively, CDRs areF053-0202PCT / 25-116-WO-PCTdetermined by comparison to known antibodies (linear sequence) and without resorting to solving a crystal structure. To determine residues involved in binding, a co-crystal structure of the Fab (antibody fragment) bound to the target can optionally be determined. Software programs and bioinformatical tools, such as ABodyBuilder and Paratome can also be used to determine CDR sequences. Additionally, delineation of a CDR can be according to X-ray crystallography.

[0022] In particular embodiments, a binding domain that binds BCMA includes a variable heavy chain including a CDRH1, CDRH2, and CDRH3, and a variable light chain including a CDRL1, CDRL2, and CDRL3 defined in Table 1.

[0023] Table 1. CDR sequences as defined by IMGT, Kabat, Chothia, North, and Contact CDR definitions.CDR Definition CDR Sequence SEQ ID NO:CDRH1 GYAFTNYL 3CDRH2 INPGSGGT 4CDRH3 ARRHDTYYRNYFDY 5IMGT CDRL1 QSLLYSSNQKNY 6CDRL2 WAS N / ACDRL3 QQYYSYPFT 7CDRH1 NYLIE 8CDRH2 VINPGSGGTNYNEKFKG 9CDRH3 RHDTYYRNYFDY 10KabatCDRL1 KSSQSLLYSSNQKNYLA 11CDRL2 WASTRES 12CDRL3 QQYYSYPFT 7CDRH1 GYAFTNY 13CDRH2 NPGSGG 14CDRH3 RHDTYYRNYFDY 10ChothiaCDRL1 KSSQSLLYSSNQKNYLA 11CDRL2 WASTRES 12CDRL3 QQYYSYPFT 7CDRH1 KASGYAFTNYLIE 15CDRH2 VINPGSGGTN 16NorthCDRH3 ARRHDTYYRNYFDY 5CDRL1 KSSQSLLYSSNQKNYLA 11F053-0202PCT / 25-116-WO-PCTCDRL2 YWASTRES 17CDRL3 QQYYSYPFT 7CDRH1 TNYLIE 18CDRH2 WIGVINPGSGGTN 19CDRH3 ARRHDTYYRNYFD 20ContactCDRL1 KNYLAWY 21CDRL2 LLIYWASTRE 22CDRL3 QQYYSYPF 23

[0024] In particular embodiments, the anti-BCMA binding domain includes a variable heavy chain including the sequence:QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTNYNE KFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARRHDTYYRNYFDYWGQGTTLTVSS (SEQ ID NO: 24)and a variable light chain sequence including the sequence:DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRES GVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPFTFGSGTKLEIK (SEQ ID NO: 25).

[0025] In particular embodiments, the anti-BCMA binding domain includes a variable heavy chain that can be encoded by the sequence:CAGGTGCAGCTGCAGCAGAGCGGCGCGGAACTGGTGCGCCCGGGCACCAGCGTGAAAG TGAGCTGCAAAGCGAGCGGCTATGCGTTTACCAACTATCTGATTGAATGGGTGAAACAGC GCCCGGGCCAGGGCCTGGAATGGATTGGCGTGATTAACCCGGGCAGCGGCGGCACCAAC TATAACGAAAAATTTAAAGGCAAAGCGACCCTGACCGCGGATAAAAGCAGCAGCACCGCG TATATGCAGCTGAGCAGCCTGACCAGCGAAGATAGCGCGGTGTATTTTTGCGCGCGCCGC CATGATACCTATTATCGCAACTATTTTGATTATTGGGGCCAGGGCACCACCCTGACCGTGA GCAGC (SEQ ID NO: 102)and a variable light chain sequence that can be encoded by the sequence:GATATTGTGATGAGCCAGAGCCCGAGCAGCCTGGCGGTGAGCGTGGGCGAAAAAGTGAC CATGAGCTGCAAAAGCAGCCAGAGCCTGCTGTATAGCAGCAACCAGAAAAACTATCTGGC GTGGTATCAGCAGAAACCGGGCCAGAGCCCGAAACTGCTGATTTATTGGGCGAGCACCCG CGAAAGCGGCGTGCCGGATCGCTTTACCGGCAGCGGCAGCGGCACCGATTTTACCCTGA CCATTAGCAGCGTGAAAGCGGAAGATCTGGCGGTGTATTATTGCCAGCAGTATTATAGCTA TCCGTTTACCTTTGGCAGCGGCACCAAACTGGAAATTAAA (SEQ ID NO: 103).

[0026] The carboxy- terminal portion of each chain of a naturally occurring antibody defines aF053-0202PCT / 25-116-WO-PCTconstant region, which can be responsible for effector function particularly in the heavy chain (the Fc). Examples of effector functions include: C1q binding and complement dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B-cell receptors); and B-cell activation. A portion of an Fc region is a fragment of an Fc region. The fragment can include 10% of an Fc region, 20% of an Fc region, 30% of an Fc region, 40% of an Fc region, 50% of an Fc region, 60% of an Fc region, 70% of an Fc region, 80% of an Fc region, 90% of an Fc region, or 95% of an Fc region. A portion of an Fc region can also include a characterized segment of an Fc region, such as a CH2 region or a CH3 region.

[0027] Human light chains are classified as kappa (IgK) and lambda (IgA) light chains. In particular embodiments, a human IgK Fc region includes the sequence: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 26). In particular embodiments, a human IgA Fc region includes the sequence: GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 27).

[0028] Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, lgG1, lgG2, lgG3, and lgG4. IgM has subclasses including lgM1 and lgM2. IgA is similarly subdivided into subclasses including lgA1 and lgA2. IgG causes opsonization and cellular cytotoxicity and crosses the placenta, IgA functions on the mucosal surface, IgM is most effective in complement fixation, and IgE mediates degranulation of mast cells and basophils. The function of IgD is still not well understood. Resting B cells, which are immunocompetent but not yet activated, express IgM and IgD. Once activated and committed to secrete antibodies these B cells can express any of the five isotypes. The heavy chain isotypes of IgG, IgA, IgM, IgD and IgE are respectively designated the y, a, p, 5, and £ chains.

[0029] The constant region of the antibody with multiple binding domains may be of any suitable immunoglobulin subtype. In particular embodiments the subtype of the antibody may be of the class IgG, IgD, IgE, IgA, or IgM. Such an antibody may further belong to any subclass, e.g., lgG1, lgG2a, lgG2b, lgG3 and lgG4. In particular embodiments, a constant region includes a light chain constant region and a heavy chain constant region. A “functional constant heavy chain” or “functional CH” activates an aspect of the immune response.

[0030] In particular embodiments, a human lgG1 Fc region includes the sequence: THTCPPCPAPEFFGGPSVFFFPPKPKDTFMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHF053-0202PCT / 25-116-WO-PCTNAKTKPREEQYNSTYRVVSVETVFHQDWENGKEYKCKVSNKAFPVPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28).

[0031] In particular embodiments, a human lgG2 Fc region includes the amino acid sequence: PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 29)

[0032] In particular embodiments, a human lgG3 Fc region includes the amino acid sequence: PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 30).

[0033] In particular embodiments, a human lgG4 Fc region includes the amino acid sequence: PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 31).

[0034] The human IgD constant region typically includes the amino acid sequence: APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYY MTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKAT TAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFT CFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTL NHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQRE VNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVSY VTDHGPMK (SEQ ID NO: 32).

[0035] The human IgE constant region typically includes the amino acid sequence: ASTQSPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTWDTGSLNGTTMTLPATTLTLSG HYATISLLTVSGAWAKQMFTCRVAHTPSSTDWVDNKTFSVCSRDFTPPTVKILQSSCDGGGHF PPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTY TCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLWDLAPSKGTVNLTW SRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTS GPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTK GSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK (SEQ ID NO: 33).F053-0202PCT / 25-116-WO-PCT

[0036] The human lgA1 constant region typically includes the amino acid sequence: ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDL YTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR LSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVL PGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLAR GFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMV GHEALPLAFTQKTIDRUkGKPTHVNVSVVMAEVDGTCY (SEQ ID NO: 34).

[0037] The human lgA2 constant region typically includes the amino acid sequence ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGD LYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLL LGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHG ETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRW LQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQK TIDRLAGKPTHVNVSVVMAEVDGTCY (SEQ ID NO: 35).

[0038] The human IgM constant region typically includes the amino acid sequence GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 36).

[0039] Within full-length light and heavy chains, the variable and constant regions are joined by a “J” region of amino acids, with the heavy chain also including a “D” region of amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).

[0040] As indicated, antibodies bind epitopes on antigens. The term antigen refers to a molecule or a portion of a molecule capable of being bound by an antibody when in the non-blocked presence of the antibody. An epitope is a region of an antigen that is bound by the variable region of an antibody. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and / or specific charge characteristics. When the antigen is a protein or peptide, the epitope includes specific amino acids within that protein or peptide that contact the variable region of an antibody.F053-0202PCT / 25-116-WO-PCT

[0041] An epitope denotes the binding site on a cancer protein, viral peptide, bacterial peptide, or other antigen bound by a corresponding variable region of an antibody. The variable region either binds to a linear epitope, (e.g., an epitope including a stretch of 5 to 12 consecutive amino acids), or the variable region binds to a three-dimensional structure formed by the spatial arrangement of several short stretches of the protein target. Three-dimensional epitopes recognized by a variable region, e.g., by the epitope recognition site or paratope of an antibody or antibody fragment, can be thought of as three-dimensional surface features of an epitope molecule. These features fit precisely (in)to the corresponding binding site of the variable region and thereby binding between the variable region and its target protein (more generally, antigen) is facilitated. In particular embodiments, an epitope can be considered to have two levels: (i) the “covered patch” which can be thought of as the shadow an antibody variable region would cast on the antigen to which it binds; and (ii) the individual participating side chains and backbone residues that facilitate binding. Binding is then due to the aggregate of ionic interactions, hydrogen bonds, and hydrophobic interactions.

[0042] Unless otherwise indicated, the term “antibody” includes (in addition to antibodies having two full-length heavy chains and two full-length light chains as described above) variants, derivatives, and fragments thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies can include monoclonal antibodies (mAbs), human or humanized antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, multi-specific antibodies, polyclonal antibodies, linear antibodies, minibodies, domain antibodies, synthetic antibodies, chimeric antibodies, antibody fusions, single chain variable fragments (scFvs), polyclonal antibodies, and fragments thereof, respectively. In particular embodiments, antibodies can include oligomers or multiplexed versions of the antibodies disclosed herein.

[0043] A monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies including the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including the hybridoma method, recombinant DNA methods,F053-0202PCT / 25-116-WO-PCTphage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0044] A “human antibody” is one which includes an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-coding sequences.

[0045] A “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VLor VHsequences is from a subgroup of variable domain sequences. The subgroup of sequences can be a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In particular embodiments, for the VL, the subgroup is subgroup kappa I as in Kabat etal. (supra). In particular embodiments, for the VH, the subgroup is subgroup III as in Kabat et al. (supra).

[0046] In particular embodiments, the binding domains disclosed herein are part of an scFv. In particular embodiments, an scFv includes a variable light chain and variable heavy chain as described herein linked by a linker described elsewhere herein. In particular embodiments, the linker includes the sequence of SEQ ID NO: 99.

[0047] In particular embodiments, the binding domains disclosed herein are part of a full antibody. In particular embodiments, the antibody is an IgG antibody, an IgA antibody, an IgM antibody, an IgE antibody, or an IgD antibody. In particular embodiments, the IgG antibody is an I gG 1 antibody, an lgG2 antibody, an lgG3 antibody, or an lgG4 antibody. In particular embodiments, the IgG antibody is an lgG1 antibody. In particular embodiments, the IgA antibody is an lgA1 antibody or an lgA2 antibody. In particular embodiments, the IgA antibody is an lgA1 antibody. In particular embodiments, the IgA antibody is monomeric, dimeric, or polymeric.

[0048] (ii) Recombinant Receptors. Anti-BCMA binding domains disclosed herein can be utilized within recombinant receptors. In particular embodiments, a recombinant receptor is or includes a binding domain that binds a target antigen, wherein the recombinant receptor is expressed by a cell following the artificial introduction of a nucleic acid encoding the recombinant receptor into the cell. The recombinant receptor can be, e.g., a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a hybrid thereof.

[0049] As described previously, CAR include several distinct subcomponents that allow genetically modified cells to recognize and kill unwanted cells, such as cancer cells. The subcomponents include at least an extracellular component and an intracellular component. The extracellular component includes a binding domain that specifically binds a target antigen that isF053-0202PCT / 25-116-WO-PCTpreferentially present on the surface of diseased cells or within the immediate area thereof. When the binding domain binds such antigens, the intracellular component activates the genetically modified cell to destroy the bound diseased cell. CAR additionally include a transmembrane domain that directly or indirectly links the extracellular component to the intracellular component, and other subcomponents that can increase the CAR’s function. For example, the inclusion of a spacer region and / or one or more linker sequences can allow the CAR to have additional conformational flexibility, often increasing the binding domain’s ability to bind the target antigen. Binding domains for use in recombinant receptors are described in the (i) Anti-BCMA Binding Domains section and each of the other subcomponents are described in more detail in the following section (ii) subheadings.

[0050] Many considerations associated with CAR apply to eTCR as well. eTCR disclosed herein include a binding domain that binds a target antigen (e.g., an scFv) linked to the Ca and / or C chains of a TCR. A TCR is a heterodimeric fusion protein that typically includes an a and 3 chain. Each chain includes a variable region (Va and VfB) and a constant region (Ca and C|3) . In particular embodiments, an eTCR does not include the native TCR variable region but does include the native TCR constant region. In particular embodiments, the eTCR includes an scFv as the variable region of either the a or p chain. In particular embodiments, the eTCR includes an scFv as the variable region of both the a and p chain.

[0051] (ii-a) Intracellular Effector Domains. The intracellular effector domains of a recombinant receptor are responsible for activation of the cell in which the recombinant receptor is expressed. The term “effector domain” is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal. An effector domain can directly or indirectly promote a biological or physiological response in a cell when receiving the appropriate signal. In certain embodiments, an effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule, which triggers a signal from the effector domain. An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM). In other embodiments, an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response, such as costimulatory domains.

[0052] Effector domains can provide for activation of at least one function of a modified cell upon binding to the cellular marker expressed by a cancer cell. Activation of the modified cell can include one or more of differentiation, proliferation and / or activation or other effector functions. In particular embodiments, an effector domain can include an intracellular signaling componentF053-0202PCT / 25-116-WO-PCTincluding a T cell receptor and a co-stimulatory domain which can include the cytoplasmic sequence from co-receptor or co-stimulatory molecule.

[0053] An effector domain can include one, two, three or more intracellular signaling components (e.g., receptor signaling domains, cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof. Exemplary effector domains include signaling and stimulatory domains selected from: 4-1 BB (CD137), CARD11, CD3y, CD30, CD3c, CD3 , CD27, CD28, CD79A, CD79B, DAP10, FcRa, FcRp (FceRIb), FcRy, Fyn, HVEM (LIGHTR), ICOS, LAG3, LAT, Lek, LRP, NKG2D, NOTCH1, pTa, PTCH2, 0X40, ROR2, Ryk, SLAMF1 , Slp76, TCRa, TCRp, TRIM, Wnt, Zap70, or any combination thereof. In particular embodiments, exemplary effector domains include signaling and co-stimulatory domains selected from: CD86, FcyRlla, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8a, CD8 , 1 L2R , IL2Ry, IL7Ra, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, PAG / Cbp, NKp44, NKp30, NKp46, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, orTLR9. In particular embodiments, the effector domain includes a CD3 signaling domain.

[0054] Intracellular signaling component sequences that act in a stimulatory manner may include iTAMs. Examples of iTAMs including primary cytoplasmic signaling sequences include those derived from CD3y, CD35, CD3s, CD3 , CD5, CD22, CD66d, CD79a, CD79b, and common FcRy (FCER1G), FcyRlla, FcR (Fee Rib), DAP10, and DAP12. In particular embodiments, variants of CD3 retain at least one, two, three, or all ITAM regions.

[0055] In particular embodiments, an effector domain includes a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein including a plurality of ITAMs, a co-stimulatory domain, or any combination thereof.

[0056] Additional examples of intracellular signaling components include the cytoplasmic sequences of the CD3 chain, and / or co- receptors that act in concert to initiate signal transduction following binding domain engagement.

[0057] A co-stimulatory domain is a domain whose activation can be required for an efficient lymphocyte response to cellular marker binding. Some molecules are interchangeable asF053-0202PCT / 25-116-WO-PCTintracellular signaling components or co-stimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1 BB (CD 137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83. For example, CD27 co-stimulation has been demonstrated to enhance expansion, effector function, and survival of human CAR T cells in vitro and augments human T cell persistence and anti-cancer activity in vivo (Song et al. Blood. 2012; 119(3): 696-706). Further examples of such co-stimulatory domain molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8a, CD8[3, IL2R0, I L2Ry, IL7Ra, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD103, ITGAL, CDIIa, ITGAM, CDI lb, ITGAX, CDIIc, ITGBI, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD19a.

[0058] In particular embodiments, the intracellular signaling components include CD3^ (SEQ ID NOs: 64, 65, or 66), 4-1 BB (SEQ ID NOs: 69, 70, or 71), and / or CD28. In particular embodiments, the intracellular signaling components include a CD3 signaling domain(encoded by, for example, SEQ ID NOs: 67 or 68), a 4-1 BB signaling domain (encoded by, for example, SEQ ID NOs: 72, 73, or 74), and / or a CD28 signaling domain. In particular embodiments, the intracellular signaling component includes all or a portion of the signaling domain of CD3 and all or a portion of the signaling domain of 4-1 BB. In particular embodiments, the intracellular signaling components include a CD28-CD3 signaling domain (SEQ ID NO: 63).

[0059] Intracellular components may also include one or more of a protein of a Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), NOTCH signaling pathway (e.g., NOTCH1, NOTCH2, NOTCH3, or NOTCH4), Hedgehog signaling pathway (e.g., PTCH or SMO), receptor tyrosine kinases (RTKs) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomycin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE) receptor family, receptor tyrosine kinase-like orphan (ROR) receptor family, discoidin domain (DDR) receptor family, rearranged during transfection (RET) receptor family, tyrosine-protein kinase-like (PTK7) receptor family, related to receptor tyrosine kinase (RYK) receptor family, ormuscle specific kinase (MuSK) receptor family);F053-0202PCT / 25-116-WO-PCTG-protein-coupled receptors, GPCRs (Frizzled or Smoothened); serine / threonine kinase receptors (BMPR orTGFR); or cytokine receptors (IL1R, IL2R, IL7R, or IL15R).

[0060] (ii-b) Transmembrane Domain. As indicated, a transmembrane domain within a recombinant receptor serves to functionally and / or physically connect the extracellular component and intracellular component through the cell membrane. The transmembrane domain can anchor the recombinant receptor in the modified cell’s membrane.

[0061] The transmembrane domain can be derived either from a natural and / or a synthetic source. When the source is natural, the transmembrane domain can be derived from any membrane-bound or transmembrane protein. Transmembrane domains can include at least the transmembrane region(s) of the a, p or chain of a T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In particular embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rp, IL2Ry, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, GDI Id, ITGAE, CD103, ITGAL, CDI la, ITGAM, CDI lb, ITGAX, CDI Ic, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9(CD229), , PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C. In particular embodiments, a variety of human hinges can be employed as well including the human Ig (immunoglobulin) hinge (e.g., an lgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8a hinge. In particular embodiments, the recombinant receptor includes a CD28 transmembrane domain. It has been shown that a CD28 transmembrane domain reduces the antigen-threshold for second-generation 4-1 BB CAR T cell activation.

[0062] In particular embodiments, a transmembrane domain has a three-dimensional structure that is thermodynamically stable in a cell membrane, and generally ranges in length from 15 to 30 amino acids. The structure of a transmembrane domain can include an a helix, a p barrel, a p sheet, a p helix, or any combination thereof.

[0063] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid within the extracellular region of the recombinant receptor (e.g., up to 15 amino acids of the extracellular region) and / or one or more additional amino acids within the intracellular region of the recombinant receptor (e.g., up to 15F053-0202PCT / 25-116-WO-PCTamino acids of the intracellular components). In one aspect, the transmembrane domain is from the same protein that the signaling domain, co-stimulatory domain or the hinge domain is derived from. In another aspect, the transmembrane domain is not derived from the same protein that any other domain of the recombinant receptor is derived from. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other unintended members of the receptor complex. In particular embodiments, the transmembrane domain is encoded by the nucleic acid sequence encoding the CD28 transmembrane domain (SEQ ID NOs: 59, 60, 61, or 62). In particular embodiments, the transmembrane domain includes the amino acid sequence of the CD28 transmembrane domain (SEQ ID NOs: 55, 56, 57, or 58).

[0064] (ii-c) Spacers. Spacers are used to create appropriate distances and / or flexibility from other recombinant receptor sub-components. As indicated, in particular embodiments, the length of a spacer is customized for binding targeted antigen-expressing cells and mediating destruction. In particular embodiments, a spacer length can be selected based upon the location of a cellular marker antigen epitope, affinity of a binding domain for the antigen epitope, and / or the ability of the recombinant receptor to mediate cell destruction following target antigen binding.

[0065] Spacers typically include those having 10 to 250 amino acids, 10 to 200 amino acids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids.

[0066] In particular embodiments, a spacer is 5 amino acids, 8 amino acids, 10 amino acids, 12 amino acids, 14 amino acids, 20 amino acids, 21 amino acids, 26 amino acids, 27 amino acids, 45 amino acids, 50 amino acids, or 75 amino acids. These lengths qualify as short spacers.

[0067] In particular embodiments, a spacer is 76 amino acids, 90 amino acids, 100 amino acids, 110 amino acids, 120 amino acids, 125 amino acids, 128 amino acids, 131 amino acids, 135 amino acids, 140 amino acids, 150 amino acids, 160 amino acids, 170 amino acids, or 179 amino acids. These lengths qualify as intermediate spacers.

[0068] In particular embodiments, a spacer is 180 amino acids, 190 amino acids, 200 amino acids, 210 amino acids, 212 amino acids, 214 amino acids, 216 amino acids, 218 amino acids, 220 amino acids, 228 amino acids, 230 amino acids, 240 amino acids, 250 amino acids, 260 amino acids, or 270 amino acids. These lengths qualify as long spacers.

[0069] Exemplary spacers include all or a portion of an immunoglobulin hinge region. An immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wildtype immunoglobulin hinge region. In certain embodiments, an immunoglobulin hinge region is a human immunoglobulin hinge region. As used herein, a “wild type immunoglobulin hinge region”F053-0202PCT / 25-116-WO-PCTrefers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody.

[0070] An immunoglobulin hinge region may be an IgG, IgA, IgD, IgE, or IgM hinge region. An IgG hinge region may be an lgG1, lgG2, lgG3, or lgG4 hinge region. Sequences from lgG1, lgG2, lgG3, lgG4 or IgD can be used alone or in combination with all or a portion of a CH2 region; all or a portion of a CH3 region; or all or a portion of a CH2 region and all or a portion of a CH3 region.

[0071] In particular embodiments, the spacer is a short spacer including an lgG4 hinge region. In particular embodiments, the spacer is an intermediate (or medium) spacer including an lgG4 hinge region and an lgG4 CH3 region. In particular embodiments, the spacer is a long spacer including an lgG4 hinge region, an lgG4 CH2 region, and an lgG4 CH3 region. In particular embodiments, the spacer includes the long hinge (SEQ ID NO: 42).

[0072] Other examples of hinge regions that can be used in a recombinant receptor described herein include the hinge region present in the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7.

[0073] In particular embodiments, a spacer includes a hinge region that includes a type II C-lectin interdomain (stalk) region or a cluster of differentiation (CD) molecule stalk region. A “stalk region” of a type II C-lectin or CD molecule refers to the portion of the extracellular domain (ECD) of the type II C-lectin or CD molecule that is located between the C-type lectin-like domain (CTLD; e.g., similar to CTLD of natural killer cell receptors) and the hydrophobic portion (transmembrane domain). For example, the ECD of human CD94 (GenBank Accession No. AAC50291.1) corresponds to amino acid residues 34-179, but the CTLD corresponds to amino acid residues 61-176, so the stalk region of the human CD94 molecule includes amino acid residues 34-60, which are located between the hydrophobic portion (transmembrane domain) and CTLD (see Boyington et al., Immunity 10:15, 1999; for descriptions of other stalk regions, see also Beavil et al., Proc. Nat'l. Acad. Sci. USA 89:153, 1992; and Figdor et al., Nat. Rev. Immunol. 2:11, 2002). These type II C-lectin or CD molecules may also have junction amino acids (described below) between the stalk region and the transmembrane region or the CTLD. In another example, the 233 amino acid human NKG2A protein (GenBank Accession No. P26715.1) has a hydrophobic portion (transmembrane domain) ranging from amino acids 71-93 and an ECD ranging from amino acids 94-233. The CTLD includes amino acids 119-231 and the stalk region includes amino acids 99-116, which may be flanked by additional junction amino acids. Other type II C-lectin or CD molecules, as well as their extracellular ligand-binding domains, stalk regions, and CTLDs are known in the art (see, e.g., GenBank Accession Nos. NP 001993.2; AAH07037.1; NPF053-0202PCT / 25-116-WO-PCT001773.1; AAL65234.1; CAA04925.1 ; for the sequences of human CD23, CD69, CD72, NKG2A, and NKG2D and their descriptions, respectively).

[0074] (ii-d) Linkers. As used herein, a linker can include a chemical moiety that serves to connect two other subcomponents of a molecule. Examples of linkers are described elsewhere herein.

[0075] Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase- induced cleavage, and disulfide bond cleavage. Alternatively, linkers can be substantially resistant to cleavage (e.g., stable linker or noncleavable linker). In some aspects, the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid-based linker.

[0076] Junction amino acids can be a linker which can be used to connect sequences when the distance provided by a spacer is not needed and / or wanted. For example, junction amino acids can be short amino acid sequences that can be used to connect co-stimulatory intracellular signaling components. In particular embodiments, junction amino acids are 9 amino acids or less (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 amino acids). In particular embodiments, a glycine-serine doublet can be used as a suitable junction amino acid linker. In particular embodiments, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable junction amino acid.

[0077] (ii-e) Control Features Including Tag Cassettes, Transduction Markers, Selection Cassettes, and / or Suicide Switches. In particular embodiments, artificial expression constructs coding for recombinant receptors can further encode one or more tag cassettes and / or transduction markers. Tag cassettes and transduction markers can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and / or eliminate genetically modified cells in vitro, in vivo and / or ex vivo. "Tag cassette" refers to a unique synthetic peptide sequence affixed to, fused to, or that is part of an expressed molecule (e.g., recombinant receptor or chemokine receptor), to which a cognate binding molecule (e.g., ligand, antibody, or other binding partner) is capable of specifically binding where the binding property can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and / or eliminate the tagged protein and / or cells expressing the tagged protein. Transduction markers can serve the same purposes but are derived from naturally occurring molecules and are often expressed using a skipping element that separates the transduction marker from the rest of the expressed molecule.

[0078] Tag cassettes that bind cognate binding molecules include, for example, affinity tags described elsewhere herein.

[0079] Conjugate binding molecules that specifically bind tag cassette sequences disclosed herein are commercially available. For example, His tag antibodies are commercially availableF053-0202PCT / 25-116-WO-PCTfrom suppliers including Life Technologies, Pierce Antibodies, and GenScript. Flag tag antibodies are commercially available from suppliers including Pierce Antibodies, GenScript, and Sigma-Aldrich. Xpress tag antibodies are commercially available from suppliers including Pierce Antibodies, Life Technologies and GenScript. Avi tag antibodies are commercially available from suppliers including Pierce Antibodies, IsBio, and Genecopoeia. Calmodulin tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Pierce Antibodies. HA tag antibodies are commercially available from suppliers including Pierce Antibodies, Cell Signal and Abeam. Myctag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Cell Signal. Strep tag antibodies are commercially available from suppliers including Abeam, Iba, and Qiagen.

[0080] Transduction markers may be selected from at least one of a truncated CD19 (tCD19; see Budde et al., Blood 122: 1660, 2013); a truncated human EGFR (tEGFR or EGFRt; see Wang et al., Blood 118: 1255, 2011); an ECD of human CD34; and / or RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol. Therapy 1(5 Pt 1); 448-456, 2000) and CD20 antigens (see Philip et al, Blood 124: 1277-1278). In particular embodiments, cells are genetically modified to express EGFRt.

[0081] In particular embodiments, a selection cassette provides for positive selection or negative selection of a desired cell population. Negative selection is when several cell types are removed, leaving the cell type of interest. Positive selection involves targeting the desired cell population to retain desired cells.

[0082] A selection cassette can encode proteins that (a) confer resistance to antibiotics or other toxins, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. Any number of selection systems may be used to recover transformed cells. In particular embodiments, a positive selection cassette includes resistance genes to neomycin, hygromycin, ampicillin, puromycin, phleomycin, zeomycin, blasticidin, or viomycin. In particular embodiments, a selection cassette includes the DHFR (di hydrofol ate reductase) gene or DHFR double mutant (DHFRdm) gene providing resistance to methotrexate (MTX), the MGMT P140K gene responsible for the resistance to O6BG / BCNU, the HPRT (Hypoxanthine phosphoribosyl transferase) gene responsible for the transformation of specific bases present in the HAT selection medium (aminopterin, hypoxanthine, thymidine) or other genes for detoxification with respect to some drugs. In particular embodiments, the selection agent includes neomycin, hygromycin, puromycin, phleomycin, zeomycin, blasticidin, viomycin, ampicillin, O6BG / BCNU, MTX, tetracycline, aminopterin, hypoxanthine, thymidine kinase, DHFR, Gin synthetase, or ADA.F053-0202PCT / 25-116-WO-PCT

[0083] In particular embodiments, the selection cassette includes DHFRdm. In particular embodiments, the method does not require a selection cassette to acquire highly purified cell populations.

[0084] In particular embodiments, negative selection cassettes include a gene for transformation of a substrate present in the culture medium into a toxic substance for the cell that expresses the gene. These molecules include detoxification genes of diptheria toxin (DTA) (Yagi et al., Anal Biochem. 214(1):77-86, 1993; Yanagawa etal., Transgenic Res. 8(3):215-221, 1999), the kinase thymidine gene of the Herpes virus (HSV TK) sensitive to the presence of ganciclovir or FIAU. The HPRT gene may also be used as a negative selection by addition of 6-thioguanine (6TG) into the medium, and for all positive and negative selections, a poly A transcription termination sequence from different origins, the most classical being derived from SV40 poly A, or a eukaryotic gene poly A (bovine growth hormone, rabbit p-globin, etc.).

[0085] In particular embodiments, artificial expression constructs can include a polynucleotide that encodes a self-cleaving polypeptide, wherein the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the recombinant receptor and a polynucleotide encoding a transduction marker (e.g., EGFRt). In particular embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding a transduction marker and a polynucleotide encoding a CXCR3. In particular embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding a transduction marker and a polynucleotide encoding a selection cassette (e.g., DHFRdm).

[0086] Exemplary self-cleaving polypeptides include 2A peptide from porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A). Further exemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example, Kim et al. (PLOS One 6:e18556 (2011). In particular embodiments, cells are genetically modified to include a self-cleaving polypeptide. In particular embodiments, the selfcleaving polypeptide includes T2A. In particular embodiments, the sequence encoding the selfcleaving polypeptide is between the sequence encoding the recombinant receptor and the sequence encoding the transduction marker. In particular embodiments, the sequence encoding the self-cleaving polypeptide is between the sequence encoding the recombinant receptor and the sequence encoding the chemokine receptor. In particular embodiments, the sequence encoding the self-cleaving polypeptide is between the sequence encoding the transduction marker and the sequence encoding the chemokine receptor.

[0087] Control features may be present in multiple copies in an artificial expression construct orF053-0202PCT / 25-116-WO-PCTcan be expressed as distinct molecules with the use of a skipping element For example, an artificial expression construct can have one, two, three, four or five tag cassettes and / or one, two, three, four, or five transduction markers could also be expressed and / or one, two, three, four or five selection cassettes. For example, embodiments can include an artificial expression construct having two Myc tag cassettes, or a His tag and an HA tag cassette, or a HA tag and a Softag 1 tag cassette, or a Myc tag and a SBP tag cassette. Exemplary transduction markers and cognate pairs are described in US 13 / 463,247.

[0088] One advantage of including at least one sequence encoding a control feature in an artificial expression construct is that cells expressing the artificial expression construct administered to a subject can be increased or depleted using the cognate binding molecule to a tag cassette. In certain embodiments, the present disclosure provides a method for depleting a modified cell expressing an artificial expression construct by using an antibody specific for the tag cassette, using a cognate binding molecule specific for the control feature, or by using a second modified cell expressing a recombinant receptor (e.g., CAR) and having specificity for the control feature. Elimination of modified cells may be accomplished using depletion agents specific for a control feature. For example, if EGFRt is used, then an anti-EGFRt binding domain (e.g., antibody, scFv) fused to or conjugated to a cell-toxic reagent (such as a toxin, radiometal) may be used, or an anti-EGFRt / anti-CD3 bispecific scFv, or an anti-EGFRt CAR T cell may be used.

[0089] In particular embodiments, a polynucleotide encoding an iCaspase9 construct (iCasp9) may be inserted into an artificial expression construct as a suicide switch.

[0090] In certain embodiments, modified cells expressing an artificial expression construct may be detected or tracked in vivo by using antibodies that bind with specificity to a control feature (e.g., anti-Tag antibodies), or by other cognate binding molecules that specifically bind the control feature, which binding partners for the control feature are conjugated to a fluorescent dye, radiotracer, iron-oxide nanoparticle or other imaging agent known in the art for detection by X-ray, CT-scan, MRI-scan, PET-scan, ultrasound, flow-cytometry, near infrared imaging systems, or other imaging modalities (see, e.g., Yu, etal., Theranostics 2:3, 2012).

[0091] Thus, modified cells expressing at least one control feature can be, e.g., more readily identified, isolated, sorted, induced to proliferate, tracked, and / or eliminated as compared to a modified cell without a tag cassette.

[0092] Methods to genetically modify cells to express recombinant receptors are well-known in the art. Some example methods include vector delivery (e.g., lentiviral vector delivery) and targeted genetic engineering approaches (e.g., using the CRISPR / Cas system, zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), MegaTALs,F053-0202PCT / 25-116-WO-PCTtransposon-based systems). In particular embodiments, cells that are genetical modified to express a recombinant receptor include immune cells. In particular embodiments, cells that are genetical modified to express a recombinant receptor include T-cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, lymphocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPC), and / or a mixture of HSC and HPC (i.e. , HSPC). In particular embodiments, genetically modified cells include T-cells. In particular embodiments, T cells include CD8+ and / or CD4+ T cells.

[0093] (iii) Antibody Variants. Binding domains disclosed herein can be utilized to prepare various forms of relevant binding domain molecules. For example, particular embodiments can include binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, and single chain Fv fragments (scFvs) or any biologically effective fragments of an immunoglobulin that bind specifically to an epitope described herein.

[0094] In particular embodiments, an antibody fragment is used. An “antibody fragment” denotes a portion of a full-length antibody that retains the ability to bind to an epitope. Antibody fragments can be made by various techniques, including proteolytic digestion of an intact antibody as well as production by recombinant host-cells (e.g., mammalian suspension cell lines, E. coli or phage), as described herein. Antibody fragments can be screened for their binding properties in the same manner as intact antibodies. Examples of antibody fragments include Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2i diabodies; and linear antibodies.

[0095] A single chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. Fv fragments include the VL and VH domains of a single arm of an antibody but lack the constant regions. Although the two domains of the Fv fragment, VL and VH, are coded by separate genes, they can be joined, using, for example, recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLand VHregions pair to form monovalent molecules (single chain Fv (scFv)). For additional information regarding Fv and scFv, see e.g., Bird, et al., Science 242:423-426, 1988; Huston, et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York), (1994) 269-315; WO 1993 / 16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458.

[0096] Linker sequences that are used to connect the VL and VH of an scFv are generally five to 35 amino acids in length. In particular embodiments, a VL-VH linker includes from five to 35, ten to 30 amino acids or from 15 to 25 amino acids. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. Linker sequences of scFv areF053-0202PCT / 25-116-WO-PCTcommonly Gly-Ser linkers, described in more detail elsewhere herein.

[0097] Additional examples of antibody-based binding domain formats include scFv-based grababodies and soluble VH domain antibodies. These antibodies form binding regions using only heavy chain variable regions. See, for example, Jespers et al., Nat. Biotechnol. 22:1161, 2004; Cortez- Retamozo etal., Cancer Res. 64:2853, 2004; Baral etal., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.

[0098] A Fab fragment is a monovalent antibody fragment including VL, VH, CL and CH1 domains. A F(ab')2fragment is a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region. For discussion of Fab and F(ab')2fragments having increased in vivo half-life, see U.S. Patent 5,869,046. Diabodies include two epitope-binding sites that may be bivalent. See, for example, EP 0404097; WO1993 / 01161 ; and Holliger, et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993. Dual affinity retargeting antibodies (DART™; based on the diabody format but featuring a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117:4542-51, 2011)) can also be used. Antibody fragments can also include isolated CDRs. For a review of antibody fragments, see Hudson, et al., Nat. Med. 9:129-134, 2003.

[0099] In particular embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human lgG1, lgG2, lgG3or lgG4 Fc region) including an amino acid modification (e.g., a substitution) at one or more amino acid positions. Numerous Fc modifications are known in the art, and a representative sampling of such possible modifications are described herein.

[0100] In particular embodiments, variants (including Fc variants) have been modified from a reference sequence to produce an administration benefit. Exemplary administration benefits can include (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for forming protein complexes, (4) altered binding affinities, (5) reduced immunogenicity; and / or (6) extended half-life. While the disclosure below describes these modifications in terms of their application to antibodies, when applicable to another particular anti-BCMA binding domain format (e.g., bispecific antibodies), the modifications can also be applied to these other formats.

[0101] In particular embodiments the antibodies can be mutated to increase their affinity for Fc receptors. Exemplary mutations that increase the affinity for Fc receptors include: G236A / S239D / A330L / I332E (GASDALIE). Smith et al., Proceedings of the National Academy of Sciences of the United States of America, 109(16), 6181-6186, 2012. In particular embodiments, an antibody variant includes an Fc region with one or more amino acid substitutions which improveF053-0202PCT / 25-116-WO-PCTADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues). In particular embodiments, alterations are made in the Fc region that result in altered C1q binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184, 2000.

[0102] In particular embodiments, it may be desirable to create cysteine engineered antibodies, e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further below. In particular embodiments, residue 5400 (EU numbering) of the heavy chain Fc region is selected. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No.7,521,541.

[0103] Antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1 % to 80%, from 1 % to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., W02000 / 61739; WO 2001 / 29246; W02002 / 031140; US2002 / 0164328; W02003 / 085119; W02003 / 084570; US2003 / 0115614; US2003 / 0157108; US2004 / 0093621; US2004 / 0110704; US2004 / 0132140; US2004 / 0110282; US2004 / 0109865; W02005 / 035586; W02005 / 035778; W02005 / 053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka etal. Arch. Biochem. Biophys. 249:533-545, 1986, and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614, 2004; Kanda et al., Biotechnol. Bioeng., 94(4): 680-688, 2006; and W02003 / 085107).

[0104] In particular embodiments, the Fc moiety of an antibody includes a substitution at positionsF053-0202PCT / 25-116-WO-PCTCH2 4, CH2 5, or both. In general, the amino acid at positions 4 and 5 of CH2 of the wild-type IgG 1 and lgG3 is a leucine ("L"). In particular embodiments, the antibody includes an amino acid at position CH24, CH25, or both, that is not an L. In particular embodiments, an antibody includes an alanine ("A") at position CH2 4, or CH2 5, or both. In particular embodiments, the antibody includes both, a CH2 L4A and a CH2 L5A substitution. Such antibodies are referred to herein as a "l_AI_A" variant. Interestingly, a "LALA" mutation in the Fc moiety does not only result in a lack of contribution of the respective antibody in antibody-dependent enhancement (ADE), but also blocks ADE.

[0105] In particular embodiments, an lgG4 Fc region is mutated to form the lgG4_S228P Fc region. lgG4 antibodies can undergo a process called Fab arm exchange which results in functionally monovalent, bispecific antibodies with unknown specificity and thus potentially reduced therapeutic efficacy. Mutating the wildtype lgG4 serine at position 228 within the corehinge region to a proline creates the lgG4_S228P mutant. In particular embodiments, the lgG4_S228P mutant prevents Fab arm exchange.

[0106] In particular embodiments, modified antibodies include those wherein one or more amino acids have been replaced with a non-amino acid component, or where the amino acid has been conjugated to a functional group or a functional group has been otherwise associated with an amino acid. The modified amino acid may be, e.g., a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, or an amino acid conjugated to an organic derivatizing agent. Amino acid(s) can be modified, for example, co-translationally or post-translationally during recombinant production (e.g., N-linked glycosylation at N-X-S / T motifs during expression in mammalian cells) or modified by synthetic means. The modified amino acid can be within the sequence or at the terminal end of a sequence. Modifications also include nitrited constructs.

[0107] In particular embodiments, variants include glycosylation variants wherein the number and / or type of glycosylation site has been altered compared to the amino acid sequences of a reference sequence. In particular embodiments, glycosylation variants include a greater or a lesser number of N-linked glycosylation sites than the reference sequence. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (e.g., those that areF053-0202PCT / 25-116-WO-PCTnaturally occurring) are eliminated and one or more new N-linked sites are created. Additional antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the reference sequence. These cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. These cysteine variants generally have fewer cysteine residues than the reference sequence, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0108] PEGylation particularly is a process by which polyethylene glycol (PEG) polymer chains are covalently conjugated to other molecules such as proteins. Several methods of PEGylating proteins have been reported in the literature. For example, N-hydroxy succinimide (NHS)-PEG was used to PEGylate the free amine groups of lysine residues and N-terminus of proteins; PEGs bearing aldehyde groups have been used to PEGylate the amino-termini of proteins in the presence of a reducing reagent; PEGs with maleimide functional groups have been used for selectively PEGylating the free thiol groups of cysteine residues in proteins; and site-specific PEGylation of acetyl-phenylalanine residues can be performed.

[0109] Covalent attachment of proteins to PEG has proven to be a useful method to increase the half-lives of proteins in the body (Abuchowski, A. et al., Cancer Biochem. Biophys., 1984, 7:175-186; Hershfield, M. S. et al., N. Engl. J. Medicine, 1987, 316:589-596; and Meyers, F. J. et al., Clin. Pharmacol. Then, 49:307-313, 1991). The attachment of PEG to proteins not only protects the molecules against enzymatic degradation, but also reduces their clearance rate from the body. The size of PEG attached to a protein has significant impact on the half-life of the protein. The ability of PEGylation to decrease clearance is generally not a function of how many PEG groups are attached to the protein, but the overall molecular weight of the altered protein. Usually the larger the PEG is, the longer the in vivo half-life of the attached protein. In addition, PEGylation can also decrease protein aggregation (Suzuki etal., Biochem. Bioph. Acta 788:248, 1984), alter protein immunogenicity (Abuchowski etal., J. Biol. Chem. 252: 3582, 1977), and increase protein solubility as described, for example, in PCT Publication No. WO 92 / 16221).

[0110] Several sizes of PEGs are commercially available (Nektar Advanced PEGylation Catalog 2005-2006; and NOF DDS Catalogue Ver 7.1), which are suitable for producing proteins with targeted circulating half-lives. A variety of active PEGs have been used including mPEG succinimidyl succinate, mPEG succinimidyl carbonate, and PEG aldehydes, such as mPEG-propionaldehyde.

[0111] In particular embodiments, the antibody can be fused or coupled to an Fc polypeptide that includes amino acid alterations that extend the in vivo half-life of an antibody that contains theF053-0202PCT / 25-116-WO-PCTaltered Fc polypeptide as compared to the half-life of a similar antibody containing the same Fc polypeptide without the amino acid alterations. In particular embodiments, Fc polypeptide amino acid alterations can include M252Y, S254T, T256E, M428L, and / or N434S and can be used together, separately or in any combination. For example, M428L / N434S is a pair of mutations that increase the half-life of antibodies in serum, as described in Zalevsky etal., Nature Biotechnology 28, 157-159, 2010. Other alterations that can be helpful are described in US Patent No. 7,083,784, US Patent No. 7,670,600, US Publication No. 2010 / 0234575, PCT / US2012 / 070146, and Zwolak, Scientific Reports 7: 15521, 2017. In particular embodiments, any substitution at one of the following amino acid positions in an Fc polypeptide can be considered an Fc alteration that extends half-life: 250, 251, 252, 259, 307, 308, 332, 378, 380, 428, 430, 434, 436. Each of these alterations or combinations of these alterations can be used to extend the half-life of an antibody as described herein.

[0112] In particular embodiments, Fc modifications include hulgG4 ProAlaAla, hulgG2m4, and / or hulgG2sigma mutations. In particular embodiments, one or several amino acids at the amino or carboxy terminus of the light and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in a proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., US Patent No. 5,624,821; Tso etal., US Patent No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). For additional information regarding Fc mutations that create administration benefits, see Saunders, Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life, Frontiers in Immunology (2019) Vol. 10, Article 1296.

[0113] (iv) Multi-Domain Binding Molecules. Multi-domain binding molecules include at least two binding domains, wherein at least one binding domain includes an anti-BCMA binding domain disclosed herein. In particular embodiments, a multi-domain binding molecule includes at least one, at least two, at least, three, at least four binding domains that bind an epitope on BCM A. In particular embodiments, all of the binding domains of a multi-domain binding molecule bind BCMA. In particular embodiments, multi-domain binding molecules include bispecific antibodies, trispecific antibodies, and so on.

[0114] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (for example, F(ab')2bispecific antibodies). For example, WO 1996 / 016673 describes a bispecific anti-ErbB2 / anti-Fc gamma Rill antibody; US Pat. No. 5,837,234 describes a bispecific anti-ErbB2 / anti-Fc gamma Rl antibody; WO 1998 / 002463 describes a bispecific anti-ErbB2 / Fc alphaF053-0202PCT / 25-116-WO-PCTantibody; and US 5,821,337 describes a bispecific anti-ErbB2 / anti-CD3 antibody. In particular embodiments, a bispecific antibody can be in the form of a Bispecific T-cell Engaging (BiTE®) antibody.

[0115] Some additional exemplary bispecific antibodies have two heavy chains (each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain), and two immunoglobulin light chains that confer antigen-binding specificity through association with each heavy chain. However, as indicated, additional architectures are envisioned, including bi-specific antibodies in which the light chain(s) associate with each heavy chain but do not (or minimally) contribute to antigen-binding specificity, or that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding of one or both of the heavy chains to one or both epitopes.

[0116] scFv dimers ordiabodies may be used, rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains (usually including the variable domain components from both light and heavy chains of the source antibody), potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain “Janusins” described in Traunecker etal. (Embo Journal, 10, 3655-3659, 1991).

[0117] Exemplary bispecific antibodies with extended half-lives are described in, for example, US Patent No. 8,921,528 and US Patent Publication No. 2014 / 0308285.

[0118] Methods for making antibodies with two binding domains are known in the art. For example, traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, for example, Millstein et al. Nature 305:37-39, 1983). Similar procedures are disclosed in, for example, WO 1993 / 008829, Traunecker etal., EMBO J. 10:3655-3659, 1991 and Holliger & Winter, Current Opinion Biotechnol. 4, 446-449 (1993).

[0119] In particular embodiments, multi-domain binding molecules with two binding domains can be prepared using chemical linkage. For example, Brennan et al. (Science 229: 81, 1985) describes a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated then are converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives then is reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the antibody having two binding domains.F053-0202PCT / 25-116-WO-PCT

[0120] In particular embodiments, bispecific antibodies (or antibodies with two binding domains) can be prepared using knobs-into holes techniques. Knobs-into-holes refers to forcing the pairing of two different antibody heavy chains by introducing mutations into the CH3 domains to modify the contact interface. On one chain bulky amino acids are replaced by amino acids with short side chains to create a ‘hole’. Conversely, amino acids with large side chains were introduced into the other CH3 domain, to create a ‘knob’. By coexpressing these two heavy chains (and two identical light chains, which have to be appropriate for both heavy chains), high yields of heterodimer formation (‘knob-hole’) versus homodimer formation (‘hole-hole’ or ‘knob-knob’) is observed (Ridgway, J. B., Protein Eng. 9 (1996) 617-621; and WO 96 / 027011).

[0121] In particular embodiments, the ‘knob’ and / or the ‘hole’ may exist in the original polypeptide or may be introduced synthetically (e.g., by altering nucleic acid encoding the polypeptide). To synthetically introduce a knob and / or hole, the nucleic acid encoding the original amino acid residue (or other non-amino acid groups such as, for example carbohydrate groups) in the interface of the polypeptide is replaced with DNA encoding at least one import amino acid residue, wherein the interface refers to amino acid residues in contact between a first heavy chain constant region and one or more amino acid residues (or other non-amino acid groups) in a second heavy chain constant region.. The preferred import residues for the formation of a hole are amino acids with smaller side chain volumes than the original amino acid residue such as alanine (A), serine (S), threonine (T), valine (V), or glycine (G). The preferred import residues for the formation of a knob are amino acids with larger side chain volumes than the original amino acid residue such as tyrosine (Y), arginine (R), phenylalanine (F), or tryptophan (W). The percentage of heterodimer can be increased by remodeling the interaction surfaces of the two CH3 domains using a phage display approach and the introduction of a disulfide bridge to stabilize the heterodimers (Merchant A. M, etal., Nature Biotech 16 (1998) 677-681; Atwell, S.,J. Mol. Biol. 270 (1997) 26-35).

[0122] Two or more binding domains can be linked through a linker to form a multi-domain binding molecule. Examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0123] Commonly used flexible linkers include a linker sequence with the amino acids glycine and serine (Gly-Ser linkers). In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). Particular examples include (Gly4Ser)n(SEQ ID NO: 84), (Gly3Ser)n(Gly4Ser)n(SEQ ID NO: 85), (Gly3Ser)n(Gly2Ser)n (SEQ ID NO: 86), andF053-0202PCT / 25-116-WO-PCT(Gly3Ser)n(Gly4Ser)i (SEQ ID NO: 87). In particular embodiments, the linker is (Gly4Ser)4 (SEQ ID NO: 88), (Gly4Ser)3(SEQ ID NO: 89), (Gly4Ser)2(SEQ ID NO: 90), (Gly4Ser)i (SEQ ID NO: 91), (Gly3Ser)2(SEQ ID NO: 92), (Gly3Ser)i (SEQ ID NO: 93), (Gly2Ser)2(SEQ ID NO: 94) or (Gly2Ser)i, GGSGGGSGGSG (SEQ ID NO: 95), GGSGGGSGSG (SEQ ID NO: 96), or GGSGGGSG (SEQ ID NO: 97). In particular embodiments, the linker includes a Whitlow linker (GSTGSGSKPGSGEGSTKG; SEQ ID NO: 98). In particular embodiments, the linker includes GSTSGSGKPGSGEGSTKG (SEQ ID NO: 99).

[0124] Linkers that include one or more antibody hinge regions and / or immunoglobulin heavy chain constant regions, such as CH3 alone or a CH2CH3 sequence can also be used. Additional examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0125] In some situations, flexible linkers may be incapable of maintaining a distance or positioning of binding domains needed for a particular use. In these instances, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In particular embodiments, a proline-rich linker is a peptide sequence having more proline residues than would be expected based on chance alone. In particular embodiments, a proline-rich linker is one having at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51% proline residues. Particular examples of proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).

[0126] T-cell activation can be mediated by two distinct signals: those that initiate antigendependent primary activation and provide a T-cell receptor like signal (primary cytoplasmic signaling sequences) and those that act in an antigen independent manner to provide a secondary or co- stimulatory signal (secondary cytoplasmic signaling sequences). Immune cell activating multispecific (l-AMS) disclosed herein can target any T-cell activating epitope that upon binding induces T-cell activation. Examples of such T-cell activating epitopes are on T-cell markers including CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1 BB (CD 137), 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, and B7-H3. Binding domains that bind T-cell markers are known in the art. B cell activation can be initiated by binding of an antigen to the B cell receptor (BCR) (e.g., IgM or IgD). Exemplary immune cell activating factors for NK cells include IL-15 and CD137.

[0127] In particular embodiments, the CD3 binding domain (e.g., scFv) is derived from the OKT3 antibody (the same as the one utilized in blinatumomab). The OKT3 antibody is described in detail in US 5,929,212. In particular embodiments, a CD3 binding domain includes a variable heavyF053-0202PCT / 25-116-WO-PCTchain including a CDRH1 sequence including KASGYTFTRYTMH (SEQ ID NO: 101), a CDRH2 sequence including INPSRGYTNYNQKFKD (SEQ ID NO: 104), and a CDRH3 sequence including YYDDHYCLDY (SEQ ID NO: 105); and a variable light chain including a CDRL1 sequence including SASSSVSYMN (SEQ ID NO: 106), a CDRL2 sequence including RWIYDTSKLAS (SEQ ID NO: 107), and a CDRL3 sequence including QQWSSNPFT (SEQ ID NO: 108). In particular embodiments, the CD3 T-cell activating epitope binding domain is a human or humanized binding domain (e.g., scFv).

[0128] The following sequence is an scFv derived from OKT3 which retains the capacity to bind CD3:QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYN QKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGGG GSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYD TSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 109). It may also be used as a CD3 binding domain.

[0129] In particular embodiments, the CD3 T-cell activating epitope binding domain is a human or humanized binding domain (e.g., scFv) including a variable heavy chain including a CDRH1 sequence including GFTFTKAW (SEQ ID NO: 110), a CDRH2 sequence including IKDKSNSYAT (SEQ ID NO: 111), and a CDRH3 sequence including RGVYYALSPFDY (SEQ ID NQ:103); and a variable light chain including a CDRL1 sequence including QSLVHNNGNTY (SEQ ID NO: 112), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 113). These reflect CDR sequences of the 20G6-F3 antibody.

[0130] In particular embodiments, the CD3 T-cell activating epitope binding domain is a human or humanized binding domain (e.g., scFv) including a variable heavy chain including a CDRH1 sequence including GFTFSNAW(SEQ ID NO: 114), a CDRH2 sequence including IKARSNNYAT (SEQ ID NO: 115), and a CDRH3 sequence including RGTYYASKPFDY (SEQ ID NO: 116); and a variable light chain including a CDRL1 sequence including QSLVHDNGNTY (SEQ ID NO: 117), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 118). These reflect CDR sequences of the 4B4-D7 antibody.

[0131] In particular embodiments, the CD3 T-cell activating epitope binding domain is a human or humanized binding domain (e.g., scFv) including a variable heavy chain including a CDRH1 sequence including GFTFSNAW(SEQ ID NO: 119), a CDRH2 sequence including IKDKSNNYAT (SEQ ID NO: 120), and a CDRH3 sequence including RYVHYGIGYAMDA (SEQ ID NO: 121); and a variable light chain including a CDRL1 sequence including QSLEHNNGNTY (SEQ ID NO: 122), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQF053-0202PCT / 25-116-WO-PCTID NO: 123). These reflect CDR sequences of the 4E7-C9 antibody.

[0132] In particular embodiments, the CD3 T-cell activating epitope binding domain is a human or humanized binding domain (e.g., scFv) including a variable heavy chain including a CDRH1 sequence including GFTFTNAW(SEQ ID NO: 124), a CDRH2 sequence including KDKSNNYAT (SEQ ID NO: 125), and a CDRH3 sequence including RYVHYRFAYALDA (SEQ ID NO: 126); and a variable light chain including a CDRL1 sequence including QSLVHTNGNTY (SEQ ID NO: 127), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTHYPFT (SEQ ID NO: 128). These reflect CDR sequences of the 18F5-H10 antibody.

[0133] Additional examples of anti-CD3 antibodies, binding domains, and CDRs can be found in WO2016 / 116626. TR66 may also be used.

[0134] CD28 is a surface glycoprotein present on 80% of peripheral T-cells in humans and is present on both resting and activated T-cells. CD28 binds to B7-1 (CD80) and B7-2 (CD86) and is the most potent of the known co-stimulatory molecules (June et a!., Immunol. Today 15:321, 1994; Linsley et al., Ann. Rev. Immunol. 11:191, 1993). In particular embodiments, the CD28 binding domain (e.g., scFv) is derived from CD80, CD86 or the 9D7 antibody. Additional antibodies that bind CD28 include 9.3, KOLT-2, 15E8, 248.23.2, and EX5.3D10. Further, 1YJD provides a crystal structure of human CD28 in complex with the Fab fragment of a mitogenic antibody (5.11A1).

[0135] In particular embodiments, a CD28 binding domain is derived from TGN1412. In particular embodiments, a CD28 binding domain includes a variable heavy chain including the sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNE KFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSS (SEQ ID NO: 129) a variable light chain including the sequence:DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIK (SEQ ID NO: 130).

[0136] In particular embodiments, the CD28 binding domain includes a variable heavy chain including a CDRH1 sequence including GYTFTSYYIH (SEQ ID NO: 131), a CDRH2 sequence including CIYPGNVNTNYNEK (SEQ ID NO: 132), and a CDRH3 sequence including SHYGLDWNFDV (SEQ ID NO: 133); and a variable light chain including a CDRL1 sequence including HASQNIYVWLN (SEQ ID NO: 134), CDRL2 sequence including KASNLHT (SEQ ID NO: 135), and CDRL3 sequence including QQGQTYPYT (SEQ ID NO: 136).

[0137] In particular embodiments, the CD28 binding domain includes a variable heavy chain including a CDRH1 sequence including SYYIH (SEQ ID NO: 137), a CDRH2 sequence including CIYPGNVNTNYNEKFKD (SEQ ID NO: 138), and a CDRH3 sequence includingF053-0202PCT / 25-116-WO-PCTSHYGLDWNFDV (SEQ ID NO: 139); and a variable light chain including a CDRL1 sequence including HASQNIYVWLN (SEQ ID NO: 140), a CDRL2 sequence including KASNLHT (SEQ ID NO: 141), and a CDRL3 sequence including QQGQTYPYT (SEQ ID NO: 142).

[0138] Activated T-cells express 4-1 BB (CD137). In particular embodiments, the 4-1 BB binding domain includes a variable heavy chain including a CDRH1 sequence including YYWS (SEQ ID NO: 143), a CDRH2 sequence including INH, and a CDRH3 sequence including YGPGNYDWYFDL (SEQ ID NO: 144); and a variable light chain including a CDRL1 sequence including RASQSVS (SEQ ID NO: 145), a CDRL2 sequence including ASNRAT (SEQ ID NO: 146), and a CDRL3 sequence including QRSNWPPALT (SEQ ID NO: 147).

[0139] In particular embodiments, the 4-1 BB binding domain includes a variable heavy chain including a CDRH1 sequence including GYSFSTYWIS (SEQ ID NO: 148), a CDRH2 sequence including KIYPGDSYTNYSPS (SEQ ID NO: 149) and a CDRH3 sequence including GYGIFDY (SEQ ID NO: 150); and a variable light chain including a CDRL1 sequence including SGDNIGDQYAH (SEQ ID NO: 151), a CDRL2 sequence including QDKNRPS (SEQ ID NO: 152), and a CDRL3 sequence including ATYTGFGSI-AV (SEQ ID NO: 153).

[0140] Particular embodiments disclosed herein including binding domains that bind epitopes on CD8. In particular embodiments, the CD8 binding domain (e.g., scFv) is derived from the OKT8 antibody. For example, in particular embodiments, the CD8 T-cell activating epitope binding domain is a human or humanized binding domain (e.g., scFv) including a variable heavy chain including a CDRH1 sequence including GFNIKD (SEQ ID NO: 154), a CDRH2 sequence including RIDPANDNT (SEQ ID NO: 155), and a CDRH3 sequence including GYGYYVFDH (SEQ ID NO: 156); and a variable light chain including a CDRL1 sequence including RTSRSISQYLA (SEQ ID NO: 157), a CDRL2 sequence including SGSTLQS (SEQ ID NO: 158), and a CDRL3 sequence including QQHNENPLT (SEQ ID NO: 159). These reflect CDR sequences of the OKT8 antibody.

[0141] In particular embodiments natural killer cells (also known as NK-cells, K-cells, and killer cells) are targeted for localized activation by l-AMS. NK cells can induce apoptosis or cell lysis by releasing granules that disrupt cellular membranes and can secrete cytokines to recruit other immune cells.

[0142] Examples of activating proteins expressed on the surface of NK cells include NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, and several members of the natural cytotoxicity receptor (NCR) family. Examples of NCRs that activate NK cells upon ligand binding include NKp30, NKp44, NKp46, NKp80, and DNAM-1.

[0143] Examples of commercially available antibodies that bind to an NKcell receptor and induce and / or enhance activation of NK cells include: 5C6 and 1D11, which bind and activate NKG2DF053-0202PCT / 25-116-WO-PCT(available from BioLegend® San Diego, CA); mAb 33, which binds and activates KIR2DL4 (available from BioLegend®); P44-8, which binds and activates NKp44 (available from BioLegend®); SK1, which binds and activates CD8; and 3G8 which binds and activates CD16.

[0144] In particular embodiments, the l-AMS can bind to and block an NK cell inhibitory receptor to enhance NK cell activation. Examples of NK cell inhibitory receptors that can be bound and blocked include KIR2DL1, KIR2DL2 / 3, KIR3DL1, NKG2A, and KLRG1. In particular embodiments, a binding domain that binds and blocks the NK cell inhibitory receptors KIR2DL1 and KIR2DL2 / 3 includes a variable heavy chain region of the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAANYAQ KFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARIPSGSYYYDYDMDVWGQGTTVTVSS(SEQ ID NO: 160) and a variable light chain region of the sequence EIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG SGSGTDFTLTISSLEPEDFAVYYCQQRSNWMYTFGQGTKLEIKRT (SEQ ID NO: 161). Additional NK cell activating antibodies are described in WQ / 2005 / 0003172 and US Patent No.9,415,104.

[0145] In particular embodiments macrophages are targeted for localized activation by l-AMS. Macrophages are a type of leukocyte (or white blood cell) that can engulf and digest cells, cellular debris, and / or foreign substances in a process known as phagocytosis.

[0146] The l-AMS can be designed to bind to a protein expressed on the surface of macrophages. Examples of activating proteins expressed on the surface of macrophages (and their precursors, monocytes) include CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2 Toll-like receptors (TLRs) 1-9, IL-4Ra, and MARCO. Commercially available antibodies that bind to proteins expressed on the surface of macrophages include M1 / 70, which binds and activates CD11b (available from BioLegend®); KP1, which binds and activates CD68 (available from ABCAM®, Cambridge, United Kingdom); and ab87099, which binds and activates CD163 (available from ABCAM®).

[0147] In particular embodiments, l-AMS can target a pathogen recognition receptor (PRR). PRRs are proteins or protein complexes that recognize a danger signal and activate and / or enhance the innate immune response. Examples of PRRs include the TLR4 / M D-2 complex, which recognizes gram negative bacteria; Dectin-1 and Dectin-2, which recognize mannose moieties on fungus and other pathogens; TLR2 / TLR6 or TLR2 / TLR1 heterodimers, which recognize gram positive bacteria; TLR5, which recognizes flagellin; and TLR9 (CD289), which recognizes CpG motifs in DNA. In particular embodiments, l-AMS can bind and activate TLR4 / MD-2, Dectin-1, Dectin-2, TRL2 / TLR6, TLR2 / TLR1, TLR5, and / or TLR9.F053-0202PCT / 25-116-WO-PCT

[0148] In particular embodiments, l-AMS can target the complement system. The complement system refers to an immune pathway that is induced by antigen-bound antibodies and involves signaling of complement proteins, resulting in immune recognition and clearance of the antibody-coated antigens.

[0149] Binding domains of l-AMS and other engineered formats described herein may be joined through a linker. A linker is an amino acid sequence which can provide flexibility and room for conformational movement between the binding domains of a l-AM. Any appropriate linker may be used.

[0150] Examples of linkers can be found in Chen etal. (Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369) and described elsewhere herein. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0151] Cytolytic properties of l-AMS molecules can be confirmed in comparative in vitro assays. Briefly, for cell line experiments, target cells can be incubated in 96-well round bottom plates at 5-10,000 cells / well containing increasing concentrations of the various l-AMS antibodies with / without healthy donor T-cells (used at an E:T cell ratio of 1:1 and 3:1). After 48 hours, cell numbers and drug-induced cytotoxicity, using 4',6-diamidino-2-phenylindole (DAPI) to detect non-viable cells, can be determined by flow cytometry. In experiments where healthy donor T-cells are added, cells can be identified by forward / side scatter properties and negativity for CellVue Burgundy dye. Experiments can include technical duplicates.

[0152] In particular embodiments, T-cell activating epitope binding domains including l-AMS constructs include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the Va, Vp, Ca, or Cp of a known TCR. An insertion, deletion or substitution may be anywhere in a Va, Vp, Ca, or Cp region, including at the amino- or carboxy-terminus or both ends of these regions, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain including a modified Va, Vp, Ca, or Cp region can still specifically bind its target with an affinity similar to wild type.

[0153] T ri-specific antibodies are artificial proteins that simultaneously bind to three different types of antigens, wherein at least one of the antigens is BCMA. Tri-specific antibodies are described in, for example, WO2016 / 105450, WO 2010 / 028796; WO 2009 / 007124; WO 2002 / 083738; US 2002 / 0051780; and WO 2000 / 018806.

[0154] In some embodiments, a multi-domain binding molecule includes a basic immunoglobulinF053-0202PCT / 25-116-WO-PCTstructure such as an IgA domain or an IgM domain. Basic immunoglobulin structures in vertebrate systems are described above and are well understood. (See, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0155] In particular embodiments, multi-domain binding molecules are multimers of an antibody disclosed herein. Multimerization strategies include formation of a fusion protein using protein linkers or use of IgA or IgM constant regions as a multimerization scaffold. In certain aspects, multimerization is achieved by linking antibodies or binding domains of antibodies in a fusion protein with protein linkers. Fusion proteins include different protein domains linked to each other directly or through intervening linker segments such that the function of each included domain is retained.

[0156] Multimerized antibodies and antibody-like molecules such as IgA and IgM antibodies have emerged as promising drug candidates in the fields of, e.g., immuno-oncology and infectious diseases allowing for improved specificity, improved avidity, and the ability to bind to multiple binding targets. See, e.g., U.S. Patent Nos. 9,951,134, 10,400,038, and 9,938,347, U.S. Patent Application Publication Nos. US20190100597A1, US20180118814A1, US20180118816A1, US20190185570A1, and US20180265596A1, and PCT Publication Nos. WO 2018 / 017888, WO 2018 / 017763, WO 2018 / 017889, WO 2018 / 017761, and WO 2019 / 165340.

[0157] Particular embodiments include using IgA and IgM constant region domains to allow the binding portion of molecules provided herein to readily multimerize into dimers, pentamers or hexamers. Basic immunoglobulin structures in vertebrate systems are described above and are well understood. (See, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0158] Immunoglobulin A (IgA), as the major class of antibody present in the mucosal secretions of most mammals, represents a key first line of defense against invasion by inhaled and ingested pathogens. IgA is also found at significant concentrations in the serum of many species, where it functions as a second line of defense mediating elimination of pathogens that have breached the mucosal surface. Receptors specific for the Fc region of IgA, FcaR, are key mediators of IgA effector function. Native IgA is a tetrameric protein including two identical light chains (K or A) and two identical heavy chains. IgA, similarly to IgG, contains three constant domains (CA1-CA3), with a hinge region between the CA1 and CA2 domains. The main difference between lgA1 and lgA2 resides in the hinge region that lies between the two Fab arms and the Fc region. I gA1 has an extended hinge region due to the insertion of a duplicated stretch of amino acids, which is absent in lgA2. Both forms of IgA have the capacity to form dimers, in which two monomer units, are arranged in an end-to-end configuration stabilized by disulfide bridges and incorporation of aF053-0202PCT / 25-116-WO-PCTJ-chain. J-chains are also part of IgM pentamers and are discussed in more detail below.

[0159] Both IgA and IgM (discussed further below in relation to pentamers and hexamers) possess an 18-amino acid extension in the C terminus called the "tailpiece" (tp). The IgA and IgM tp is highly conserved among various animal species. The conserved penultimate cysteine residue in the IgA and IgM tp has been demonstrated to be involved in multimerization by forming a disulfide bond between heavy chains to permit formation of a multimer. Both tp contain an N-linked carbohydrate addition site, the presence of which is required for dimer formation in IgA and J-chain incorporation and pentamer formation in IgM. However, the structure and composition of the N-linked carbohydrates in the tp differ, suggesting differences in the accessibility of the glycans to processing by glycosyltransferases. Particularly, the IgA (atp) and IgM (ptp) tp differ at seven amino acid positions.

[0160] The human lgA1 constant region typically includes the amino acid sequence of SEQ ID NO: 34). Referring to this SEQ ID NO: 34, the human CA1 domain extends from amino acid 6 to amino acid 98; the human lgA1 hinge region extends from amino acid 102 to amino acid 124, the human CA2 domain extends from amino acid 125 to amino acid 219, the human CA3 domain extends from amino acid 228 to amino acid 330, and the tp extends from amino acid 331 to amino acid 352.

[0161] The human lgA2 constant region typically includes the amino acid sequence of SEQ ID NO: 35. Referring to this SEQ ID NO: 35, the human CA1 domain extends from amino acid 6 to amino acid 98, the human lgA2 hinge region extends from amino acid 102 to amino acid 111, the human CA2 domain extends from amino acid 113 to amino acid 206, the human CA3 domain extends from amino acid 215 to amino acid 317, and the tp extends from amino acid 318 to amino acid 340.

[0162] As indicated, two IgA binding units can form a complex with two additional polypeptide chains, the J chain (e.g., SEQ ID NO: 38, the mature human J chain) and the secretory component to form a bivalent secretory IgA (slgA)-derived binding molecule. An exemplary precursor secretory component includes the sequence MLLFVLTCLLAVFPAISTKSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGC ITLISSEGYVSSKYAGRANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVS QGPGLLNDTKVYTVDLGRTVTINCPFKTENAQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRL DIQGTGQLLFSVVINQLRLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRGSVTFH CALGPEVANVAKFLCRQSSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSFSVVITGLRKED AGRYLCGAHSDGQLQEGSPIQAWQLFVNEESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIK YWCLWEGAQNGRCPLLVDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNF053-0202PCT / 25-116-WO-PCTGDTLWRTTVEIKIIEGEPNLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALP SQDEGPSKAFVNCDENSRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAA GSRDVSLAKADAAPDEKVLDSGFREIENKAIQDPRLFAEEKAVADTRDQADGSRASVDSGSSE EQGGSSRALVSTLVPLGLVLAVGAVAVGVARARHRKNVDRVSIRSYRTDISMSDFENSREFGA NDNMGASSITQETSLGGKEEFVATTESTTETKEPKKAKRSSKEEAEMAYKDFLLQSSTVAAEA QDGPQEA (SEQ ID NO: 162). An exemplary mature secretory component includes KSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGCITLISSEGYVSSKYAGR ANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGLLNDTKVYTVDL GRTVTINCPFKTENAQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRLDIQGTGQLLFSVVINQL RLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRGSVTFHCALGPEVANVAKFLCR QSSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSFSVVITGLRKEDAGRYLCGAHSDGQLQE GSPIQAWQLFVNEESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLL VDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEP NLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDEN SRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAAGSRDVSLAKADAAPDEK VLDSGFREIENKAIQDPR (SEQ ID NO: 163). While not wishing to be bound by theory, and as indicated above, the assembly of two IgA binding units into a dimeric IgA-derived binding molecule is thought to involve the CA3 and tp domains. See, e.g., Braathen, R., el al., J. Biol. Chem.277:42755-42762 (2002). Accordingly, a multimerizing dimeric IgA-derived binding molecule provided in this disclosure typically includes IgA constant regions that include at least the CA3 and tp domains.

[0163] An engineered IgA heavy chain constant region can additionally include a CA2 domain or a fragment thereof, an IgA hinge region or fragment thereof, a CA1 domain or a fragment thereof, and / or other IgA (or other immunoglobulin, e.g., IgG) heavy chain domains, including, e.g., an IgG hinge region. In certain embodiments, a binding molecule as provided herein can include a complete IgA heavy chain constant region (e.g., SEQ ID NO: 34 or SEQ ID NO: 35), or a variant, derivative, or analog thereof.

[0164] In particular embodiments, the IgA heavy chain constant regions can include amino acids 125 to 353 of SEQ ID NO: 34 or amino acids 113 to 340 of SEQ ID NO: 35. In particular embodiments, the IgA heavy chain constant regions can each further include an IgA or IgG hinge region situated N-terminal to the IgA CA2 domains. For example, the IgA heavy chain constant regions can include amino acids 102 to 353 of SEQ ID NO: 34 or amino acids 102 to 340 of SEQ ID NO: 35. In particular embodiments, the IgA heavy chain constant regions can each further include an IgA CA1 domain situated N-terminal to the IgA hinge region.F053-0202PCT / 25-116-WO-PCT

[0165] Each of the strategies discussed above can be used to create IgA antibody-based dimers.

[0166] Particular embodiments include IgM immunoglobulin constant region domains that allow the binding portion of molecules provided herein to readily multimerize into pentamers or hexamers.

[0167] Particular embodiments include IgM constant regions (or variants thereof). These embodiments have the ability to form hexamers, or in association with a J-chain, form pentamers. Embodiments with an IgM constant region typically include at least the Cp4-tp domains of the IgM constant region but can include heavy chain constant region domains from other antibody isotypes, e.g., IgG, from the same species or from a different species. In particular embodiments, one or more constant region domains can be deleted so long as the IgM antibody is capable of forming hexamers and / or pentamers. Thus, an IgM antibody can be, e.g., a hybrid IgM / IgG antibody or can be a “multimerizing fragment” of an IgM-derived binding molecule.

[0168] The assembly of five or six IgM binding units into a pentameric or hexameric IgM antibody is thought to involve the Cp4 and tp domains. See, e.g., Braathen, R., et al., J Biol. Chem.277:42755-42762 (2002). Accordingly, a pentameric or hexameric IgM antibody described in this disclosure typically includes at least the Cp4 and / or tp domains (also referred to herein collectively as Cp4-tp). A “multimerizing fragment” of an IgM heavy chain constant region thus includes at least the Cp4-tp domains. An IgM heavy chain constant region can additionally include a Cp3 domain or a fragment thereof, a Cp2 domain or a fragment thereof, a Cp1 domain or a fragment thereof, and / or other IgM heavy chain domains.

[0169] Five IgM monomers form a complex with a J-chain to form a native IgM molecule. The J-chain is considered to facilitate polymerization of p chains before IgM is secreted from antibodyproducing cells. Sequences for the human IGJ gene are known in the art, for example, (IGMT Accession: J00256, X86355, M25625, AJ879487). The J chain establishes the disulfide bridges between IgM antibodies to form multimeric structures such as pentamers. See, for example, Sorensen et al. International Immunology, (2000), pages 19-27. While crystallization of IgM has proved to be notoriously challenging, Czajkowsky and Shao (PNAS 106(35): 14960-14965, 2009) published a homology-based structural model of IgM, based on the structure of the IgE Fc domain and the known disulfide pairings. The authors report that the human IgM pentamer is a mushroomshaped molecule with a flexural bias. The IgM heavy (p) chain contains five N-linked glycosylation sites: Asn-171, Asn-332, Asn-395, Asn-402 and Asn-563. In an IgM antibody where each binding unit is bivalent, the binding molecule itself can have 10 or 12 valencies.

[0170] The Kabat numbering system for the human IgM constant domain can be found in Kabat, et. al. “Tabulation and Analysis of Amino acid and nucleic acid Sequences of Precursors, V-F053-0202PCT / 25-116-WO-PCTRegions, C-Regions, J-Chain, T-Cell Receptors for Antigen, T-Cell Surface Antigens, b-2 Microglobulins, Major Histocompatibility Antigens, Thy-I, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-gamma Globulin, a-2 Macroglobulins, and Other Related Proteins,” U.S. Dept of Health and Human Services (1991). IgM constant regions can be numbered sequentially (i.e., amino acid #1 starting with the first amino acid of the constant region) or by using the Kabat numbering scheme.

[0171] A “full length IgM antibody heavy chain” is a polypeptide that includes, in N- terminal to C-terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CM1 or Cp1), an antibody heavy chain constant domain 2 (CM2 or Cp2), an antibody heavy chain constant domain 3 (CM3 or Cp3), and an antibody heavy chain constant domain 4 (CM4 or Cp4) that can include a tp, as indicated above.

[0172] In particular embodiments, each binding unit of a multimeric binding molecule as provided herein includes two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each including at least an IgM Cp4 domain and an IgM tp domain. In certain embodiments the IgM heavy chain constant regions can each further include an IgM Cp3 domain situated N-temninal to the IgM Cp4 and IgM tp domains.

[0173] In particular embodiments, the IgM heavy chain constant regions can each further include an IgM Cp2 domain situated N-terminal to the IgM Cp3 domain. Exemplary multimeric binding molecules provided herein include human IgM constant regions that include the wild-type human Cp2, Cp3, and Cp4-tp domains as follows:VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAE AKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWN SGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADV FVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNR VTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 164).

[0174] In certain IgM-derived multimeric binding molecules as provided herein each IgM constant region can include, instead of, or in addition to an IgM Cp2 domain, an IgG hinge region or functional variant thereof situated N-terminal to the IgM Cp3 domain. An exemplary variant human lgG1 hinge region amino acid sequence in which the cysteine at position 6 is substituted with serine is VEPKSSDKTHTCPPCPAP (SEQ ID NO: 165). An exemplary IgM constant region of this type includesthe variant human lgG1 hinge region fused to a multimerizing fragment of the human IgM constant region including the Cp3, Cp4, and tp domains, and includes the amino acid sequence:F053-0202PCT / 25-116-WO-PCTVEPKSSDKTHTCPPCPAPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNG EAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHR PDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPG RYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY(SEQ ID NO: 166).

[0175] Human IgM constant regions, and also certain non-human primate IgM constant regions, as provided herein typically include five (5) naturally-occurring asparagine (N)-linked glycosylation motifs or sites. As used herein “an N-linked glycosylation motif” includes the amino acid sequence N-X1-S / T, wherein N is asparagine, X1 is any amino acid except proline (P), and S / T is serine (S) or threonine (T). The glycan is attached to the nitrogen atom of the asparagine residue. See, e.g., Drickamer K, Taylor ME (2006), Introduction to Glycobiology (2nd ed.). Oxford University Press, USA. N-linked glycosylation motifs occur in the human IgM heavy chain constant regions of SEQ ID NO: 36 or SEQ ID NO: 100 starting at positions 46 (“N1”), 209 (“N2”), 272 (“N3”), 279 (“N4”), and 440 (“N5”). These five motifs are conserved in non-human primate IgM heavy chain constant regions, and four of the five are conserved in the mouse IgM heavy chain constant region. Each of these sites in the human IgM heavy chain constant region, except for N4, can be mutated to prevent glycosylation at that site, while still allowing IgM expression and assembly into a hexamer or pentamer.

[0176] The human IgM heavy chain constant region typically includes the amino acid sequence of SEQ ID NO: 36 (identical to, e.g., GenBank Accession Nos. pir||S37768, CAA47708.1, and CAA47714.1). Referring to this SEQ ID NO: 36, the human Cp1 region ranges from amino acid 5 to amino acid 102; the human Cp2 region ranges from amino acid 114 to amino acid 205, the human Cp3 region ranges from amino acid 224 to amino acid 319, the Cp4 region ranges from amino acid 329 to amino acid 430, and the tp ranges from amino acid 431 to amino acid 453.

[0177] In particular embodiments, an IgM heavy chain constant region includes the sequence: GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL GQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDS VTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTI SRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSA PMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLV MSDTAGTCY (SEQ ID NO: 100; (UniProt ID P01871)— allele IGHM*04). This sequence differs from SEQ ID NO: 36 by one amino acid at position 191.F053-0202PCT / 25-116-WO-PCT

[0178] Other forms of the human IgM constant region with minor sequence variations exist, including GenBank Accession Nos. P01871.4, CAB37838.1, and pir||MHHU. The amino acid substitutions, insertions, and / or deletions at positions corresponding to SEQ ID NO: 36 described herein can likewise be incorporated into alternate human IgM sequences, as well as into IgM constant region amino acid sequences of other species, e.g., those shown in FIG. 1 of PCT / US2019 / 020374.

[0179] In certain aspects, a variant human IgM constant region includes an amino acid substitution corresponding to the wild-type human IgM constant region at position P311, P313, R344, E345, S401, E402, and / or E403 of SEQ ID NO: 36. These positions correspond to the Kabat numbering system as follows: S401 of SEQ ID NO: 36 corresponds to S524 of Kabat; E402 of SEQ ID NO: 36 corresponds to E525 of Kabat; E403 of SEQ ID NO: 36 corresponds to E526 of Kabat; R344 of SEQ ID NO: 36 corresponds to R467 of Kabat; and E345 of SEQ ID NO: 36 corresponds to E468 of Kabat.

[0180] In particular embodiments, “corresponds to” means the designated position of SEQ ID NO: 36 and the amino acid in the sequence of the IgM constant region of any species which is homologous to the specified position. See FIG. 1 of PCT / US2019 / 020374.

[0181] In particular embodiments, P311 of SEQ ID NO: 36 can be substituted, e.g., with alanine (P311A), serine (P311S), or glycine (P311G) and / or P313 of SEQ ID NO: 36 can be substituted, e.g., with alanine (P313A), serine (P313S), or glycine (P313G). P311 and P313 of SEQ ID NO: 36 can be substituted with alanine (P311A) and serine (P313S), respectively as shown in the following sequence: (mutations in bold underline) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 167).

[0182] In certain aspects, S401 of SEQ ID NO: 36 can be substituted with any amino acid. In certain aspects, S401 of SEQ ID NO: 36 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPF053-0202PCT / 25-116-WO-PCTRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVAEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 168).

[0183] In certain aspects, E402 of SEQ ID NO: 36 can be substituted with any amino acid. In certain aspects, E402 of SEQ ID NO: 36 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSAEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 169).

[0184] In certain aspects, E403 of SEQ ID NO: 36 can be substituted with any amino acid. In certain aspects, E403 of SEQ ID NO: 36 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEAEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 170).

[0185] In certain aspects, R344 of SEQ ID NO: 36 can be substituted with any amino acid. In certain aspects, R344 of SEQ ID NO: 36 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLF053-0202PCT / 25-116-WO-PCTSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLAESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 171).

[0186] In certain aspects, E345 of SEQ ID NO: 36 can be substituted with any amino acid. In certain aspects, E345 of SEQ ID NO: 36 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRASATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 172).

[0187] As indicated, five IgM binding units can form a complex with a J-chain to form a pentameric IgM antibody. The precursor form of the human J-chain includes:MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLN NRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKC YTAVVPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 37). The signal peptide extends from amino acid 1 to amino acid 22 of SEQ ID NO: 37 and the mature human J-chain extends from amino acid 23 to amino acid 159 of SEQ ID NO: 37.

[0188] The mature human J-chain includes the amino acid sequence QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 38).

[0189] The term “J-chain” as used herein refers to the J-chain of native sequence IgM or IgA antibodies of any animal species. When specified, it can also refer to any functional fragment thereof, derivative thereof, and / or variant thereof, including a mature human J-chain amino acid sequence provided herein as SEQ ID NO: 38. A functional fragment, derivative, and / or variant of a J-chain has at least 90% sequence identity to the reference J-chain and retains the multimerizing function of the reference J-chain.

[0190] In certain aspects, the J-chain of the IgM antibody as provided herein includes an aminoF053-0202PCT / 25-116-WO-PCTacid substitution at the amino acid position corresponding to amino acid Y102, T103, N49 or S51 of SEQ ID NO: 38.

[0191] By “an amino acid corresponding to” a position of SEQ ID NO: 38 is meant the amino acid in the sequence of the J-chain of any species which is homologous to the referenced residue in the human J-chain. For example, the position corresponding to Y102 in SEQ ID NO: 38 is conserved in the J-chain amino acid sequences of at least 43 other species. The position corresponding to T103 in SEQ ID NO: 38 is conserved in the J-chain amino acid sequences of at least 37 other species. The positions corresponding to N49 and S51 in SEQ ID NO: 38 are conserved in the J-chain amino acid sequences of at least 43 other species. See FIG. 4 of U.S. Patent No. 9,951,134 and FIG. 2 of PCT / US2019 / 020374.

[0192] In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 38 can be substituted with any amino acid. In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 38 can be substituted with alanine (alanine substitution indicated by bold underline): QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCATYDRNKCYTA VPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 173),With serine (serine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCSTYDRNKCYTAWPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 174),Or with arginine (arginine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCRTYDRNKCYTA VPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 175).

[0193] In certain aspects, the amino acid corresponding to T103 of SEQ ID NO: 38 can be substituted with any amino acid. In a particular aspect, the amino acid corresponding to T103 of SEQ ID NO: 38 can be substituted with alanine as follows (alanine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCYAYDRNKCYTAVVPLVYGGETKMVETALTP DACYPD (SEQ ID NO: 176).

[0194] In certain aspects, the variant J-chain or functional fragment thereof of the IgM antibody as provided herein includes an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of SEQ ID NO: 38, provided that S51 is not substituted withF053-0202PCT / 25-116-WO-PCTthreonine (T), or wherein the J-chain includes amino acid substitutions at the amino acid positions corresponding to both amino acids N49 and S51 of SEQ ID NO: 38.

[0195] The amino acids corresponding to N49 and S51 of SEQ ID NO: 38 along with the amino acid corresponding to 150 of SEQ ID NO: 38 include an N-linked glycosylation motif in the J-chain. Accordingly, mutations at N49 and / or S51 (with the exception of a single threonine substitution at S51) can prevent glycosylation at this motif. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO: 38 can be substituted with any amino acid. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO: 38 can be substituted with alanine (A), glycine (G), threonine (T), serine (S) or aspartic acid (D). In a particular aspect the position corresponding to N49 of SEQ ID NO: 38 can be substituted with alanine (A). In a particular aspect the J-chain is a variant human J-chain and includes the amino acid sequence:QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNREAISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 177).

[0196] In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 38 can be substituted with any amino acid except threonine. In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 38 can be substituted with alanine (A) or glycine (G). In a particular aspect the position corresponding to S51 of SEQ ID NO: 38 can be substituted with alanine (A). In a particular aspect the variant J-chain or functional fragment thereof is a variant human J-chain and includes the amino acid sequence:EDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENIADPTSPLRTRFVYHLSDLC KKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDA CYPD (SEQ ID NO: 178).

[0197] Particular embodiments include a heterologous polypeptide (e.g., a single-domain antibody binding domain) fused to the J-chain or functional fragment thereof via a peptide linker, e.g., a peptide linker including at least 5 amino acids, but no more than 25 amino acids. In certain aspects, the peptide linker includes (GGGGS)n (SEQ ID NO: 84) wherein n is 1-5.

[0198] A single-domain antibody binding domain can be introduced into the J-chain at any location that allows the binding of the binding domain to its binding target without interfering with J-chain function or the function of an associated IgA, IgM, or hybrid IgG antibody. Insertion locations include at or near the C- terminus, at or near the N-terminus or at an internal location that, based on the three-dimensional structure of the J-chain, is accessible. In certain aspects, the antigen-binding domain can be introduced into the mature human J-chain of SEQ ID NO: 38F053-0202PCT / 25-116-WO-PCTbetween cysteine residues 92 and 101 of SEQ ID NO: 38. In a further aspect, the antigen-binding domain can be introduced into the human J-chain of SEQ ID NO: 38 at or near a glycosylation site. In a further aspect, the antigen-binding domain can be introduced into the human J-chain of SEQ ID NO: 38 within 10 amino acid residues from the C- terminus, or within 10 amino acids from the N-terminus.

[0199] In particular embodiments, the single-domain antibody is introduced into the native human J-chain sequence of SEQ ID NO: 38 by chemical or chemo-enzymatic derivatization. In particular embodiments, the single-domain antibody is introduced into the native human J-chain sequence of SEQ ID NO: 38 by a chemical linker. In some embodiments, the chemical linker is a cleavable or non-cleavable linker. In particular embodiments, the cleavable linker is a chemically labile linker or an enzyme-labile linker. In some embodiments, the linker is selected from the group including N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP), iminothiolane (IT), afunctional derivatives of imidoesters, active esters, aldehydes, bis-azido compounds, bis-diazonium derivatives, diisocyanates, and bis-active fluorine compounds. In particular embodiments, the modified J-chain is modified by insertion of an enzyme recognition site, and by post-translationally attaching a binding moiety at the enzyme recognition site through a peptide or non-peptide linker.

[0200] In certain aspects the modified J-chain can include the formula X[Ln]J or J[Ln]X, where J includes a mature native J-chain or functional fragment thereof, X includes a heterologous binding domain, and [Ln] is a linker sequence including n amino acids, where n is a positive integer from 1 to 100, 1 to 50, or 1 to 25. In certain aspects N is 5, 10, 15, or 20.

[0201] J-chains from the following species can also be used in certain embodiments: Pan troglodytes, Pongo abelii, Callithrix jacchus, Macaca mulatta, Papio Anubis, Saimiri boliviensis, Tupaia chinensis, Tursiops truncatus, Orcinus orca, Loxodonta Africana, Leptonychotes weddellii, Ceratotherium simum, Felis catus, Canis familiaris, Ailuropoda melanoleuca, Mustela furo, Equus caballus, Cavia porcellus, Camel us ferus, Capra hi reus, Chinchilla lanigera, Mesocricetus auratus, Ovis aries, Myotis lucifugus, Pantholops hodgsonii, Bos taurus, Mus musculus, Rattus norvegicus, Echinops telfairi, Oryctolagus cuniculus, Monodelphis domestica, Alligator mississippiensis, Chrysemys picta, Sarcophilus harrisii, Ornithorhynchus anatinus, Melopsittacus undulatus, Anas platyrhynchos, Gallus gallus, Meleagris gallopavo, Falco peregrinus, Zonotrichia albicollis, and Pteropus alecto.

[0202] In particular embodiments, the antibodies can multimerize by optionally including a multimerization domain. A “multimerization domain” is a domain that causes two or more proteinsF053-0202PCT / 25-116-WO-PCT(monomers) to interact with each other through covalent and / or non-covalent association(s). Multimerization domains present in proteins can result in protein interactions that form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units / monomers incorporated into the multimer.

[0203] In particular embodiments, the multimerization domain is a dimerization domain that allows binding of two complementary monomers to form a dimer. In particular embodiments, a dimerization and docking domain (DDD) can be derived from the cAMP-dependent protein kinase (PKA) regulatory subunits and can be paired with an anchoring domain (AD). The AD can be derived from a specific region found in various A-kinase anchoring proteins (AKAPs) that mediates association with the R subunits of PKA. Additional DDDs and ADs include: the 4-helix bundle type DDD (Newlon, etal. EMBO J. 2001; 20: 1651-1662; Newlon, etal. Nature Struct Biol.1999; 3: 222-227) domains obtained from p53, DCoH (pterin 4 a carbinolamine dehydratase / dimerization cofactor of hepatocyte nuclear factor 1 a (TCF1)) and HNF-1 (hepatocyte nuclear factor 1) (Rose, et al. Nature Struct Biol. 2000; 7: 744-748). Other AD sequences of potential use may be found in US 2003 / 0232420A1.

[0204] In particular embodiments, complementary binding domains can dimerize. In particular embodiments, the binding domain is a transmembrane polypeptide derived from a FCERI chain. In particular embodiments, an antibody or fragment thereof can include a part of a FCERI a chain and another antibody or fragment thereof can include a part of an FCERI [3 chain such that said FCERI chains spontaneously dimerize together to form a dimeric antibody (e.g., bispecific antibody). In particular embodiments, an antibody or fragment thereof can include a part of a FCERI a chain and another antibody or fragment thereof part of a FCERI y chain such that said FCERI chains spontaneously trimerize together to form a trimeric antibody, and in another embodiment the multi-domain binding molecule can include a part of FCERI a chain, a part of FCERI 3 chain and a part of FCERI y chain such that said FCERI chains spontaneously tetramerize together to form a tetrameric multi-domain binding molecule.

[0205] Leucine zippers are described in US 5932448; SH2 and SH3 are described in Vidal et al., Biochemistry, 43:7336- 44, 2004); PTB is described in Zhou et al., Nature, 378:584- 592, 1995); WW is described in Sudol Prog Biochys MoL Bio, 65:113-132, 1996; PDZ is described in Kim et al., Nature, 378: 85-88, 1995 and Komau et al., Science, 269:1737-1740, 1995; and WD40 is described in Hu etal., J Biol Chem., 273:33489- 33494, 1998.

[0206] Additional multimerization domains and systems are described in, for example, Hodneland, et al. Proc Natl Acd Sci USA. 2002; 99: 5048-5052; Arakawa et al., J Biol. Chem., 269:27833-27839, 1994; Radziejewski et al., Biochem, 32: 1350, 1993; W02012001647A2; USF053-0202PCT / 25-116-WO-PCT5821333; GenBank Accession no. AAF73912.1 (Nishi et al., Mol Cell Biol, 25: 2607-2621, 2005), the SH3 domain of IB1 from GenBank Accession no. AAD22543.1 (Kristensen el al., EMBO J., 25: 785-797, 2006), the PTB domain of human DOK-7 from GenBank Accession no. NP_005535.1 (Wagner et al., Cold Spring Harb Perspect Biol. 5: a008987, 2013), the PDZ-like domain of SATB1 from UniProt Accession No. Q01826 (Galande et al., Mol Cell Biol. Aug; 21: 5591-5604, 2001), the WD40 repeats of APAF from UniProt Accession No. 014727 (Jorgensen et al., 2009. PLOS One. 4(12):e8463), the PAS motif of the dioxin receptor from UniProt Accession No. I6L9E7 (Pongratzef al., Mol Cell Biol, 18:4079-4088, 1998) and the EF hand motif of parvalbumin from UniProt Accession No. P20472 (Jamalian et al., Int J Proteomics, 2014: 153712, 2014). C4b, dextrameric, and ferritin-based multimerization can be used.

[0207] In particular embodiments, complementary binding domains can be induced using a third molecule or chemical inducer. This method of dimerization requires that one antibody or fragment thereof include a chemical inducer of dimerization binding domain 1 (CBD1) and the second antibody or fragment thereof include the second chemical inducer of dimerization binding domain (CBD2), wherein CBD1 and CBD2 are capable of simultaneously binding to a chemical inducer of dimerization (CID). CBD1 may include a rapamycin binding domain of FK-binding protein 12 (FKBP12) and CBD2 may include a FKBP12-Rapamycin Binding (FRB) domain of mTOR.

[0208] (v) Expression of Recombinant Proteins. Antibodies and other proteins disclosed herein can be produced by recombinant expression. Recombinant polynucleotide constructs typically include an expression control sequence operably linked to the coding sequences of antibody chains, including naturally-associated or heterologous promoter regions. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the cross- reacting antibodies.

[0209] In particular embodiments, mammalian cells are used as a host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and non- antibody-producing myelomas including Sp2 / 0 and NS0. Preferably, the cells are nonhuman. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen etal., Immunol. Rev. 89:49 (1986)), and information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminatorF053-0202PCT / 25-116-WO-PCTsequences. In particular embodiments, expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, and bovine papillomavirus (see Co et al., J. Immunol. 1992, 148:1149).

[0210] Once expressed, antibodies can be purified according to standard procedures of the art, including high-performance liquid chromatography (HPLC) purification, column chromatography, gel electrophoresis and the like (see generally, Scopes, Protein Purification (Springer- Verlag, NY, 1982)).

[0211] In particular embodiments, antibodies are formed using the Daedalus expression system as described in Pechman et al. (Am J Physiol 294: R1234-R1239, 2008). The Daedalus system utilizes inclusion of minimized ubiquitous chromatin opening elements in transduction vectors to reduce or prevent genomic silencing and to help maintain the stability of decigram levels of expression. This system can bypass tedious and time-consuming steps of other protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg / l of correctly folded and post-translationally modified, endotoxin-free protein of up to 70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications. Bandaranayake et al., Nucleic Acids Res., 39(21) 2011. In some instances, purification by chromatography may not be needed due to the purity of manufacture according to the methods described herein.

[0212] (vi) Antibody Conjugates. Anti-BCMA conjugates include binding domains disclosed herein linked to another molecule, other than an additional binding domain. Examples of antibody conjugates (e.g., anti-BCMA conjugates) include antibody immunotoxins, antibody-drug conjugates (ADCs), antibody-detectable label conjugates, antibody radioisotope conjugates, and antibody-particle conjugates.

[0213] Antibody immunotoxins include an anti-BCMA binding domain disclosed herein conjugated to one or more cytotoxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof). A toxin can be any agent that is detrimental to cells. Frequently used plant toxins are divided into two classes: (1) holotoxins (or class II ribosome inactivating proteins), such as ricin, abrin, mistletoe lectin, and modeccin, and (2) hemitoxins (class I ribosome inactivating proteins), such as pokeweed antiviral protein (PAP), saporin, Bryodin 1, bouganin, and gelonin. Commonly used bacterial toxins include diphtheria toxin (DT) and Pseudomonas exotoxin (PE). Kreitman, Current Pharmaceutical Biotechnology 2:313-325 (2001). The toxin may be obtained from essentially any source and can be a syntheticF053-0202PCT / 25-116-WO-PCTor a natural product.

[0214] Immunotoxins with multiple (e.g., four) cytotoxins per binding domain can be prepared by partial reduction of the binding domain with an excess of a reducing reagent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 30 min, then the buffer can be exchanged by elution through SEPHADEX G-25 resin with 1 mM DTPA (diethylene triamine penta-acetic acid) in Dulbecco’s phosphate-buffered saline (DPBS). The eluent can be diluted with further DPBS, and the thiol concentration of the binding domain can be measured using 5,5'-dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, for example 5-fold, of a linker-cytotoxin conjugate can be added at 4°C. for 1 hr, and the conjugation reaction can be quenched by addition of a substantial excess, for example 20-fold, of cysteine. The resulting immunotoxin mixture can be purified on SEPHADEX G-25 equilibrated in PBS to remove unreacted linker-cytotoxin conjugate, desalted if desired, and purified by size-exclusion chromatography. The resulting immunotoxin can then be sterile filtered, for example, through a 0.2 pm filter, and can be lyophilized if desired for storage.

[0215] In particular embodiments, antibody-drug conjugates refer to targeted molecules which combine properties of both antibodies and cytotoxic drugs (e.g., chemotherapeutic drugs) by targeting potent cytotoxic drugs to antigen-expressing cells (Teicher, B. A. (2009) Current Cancer Drug Targets 9:982-1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, P. J. and Senter P. D. (2008) The Cancer Jour. 14(3): 154-169; Chari, R. V. (2008) Acc. Chem. Res. 41:98-107). See also Kamath & Iyer (Pharm Res.32(11): 3470-3479, 2015), which describes considerations for the development of antibody-drug conjugates. The drug moiety (D) of an antibody-drug conjugate may include any compound, moiety or group that has a cytotoxic or cytostatic effect. To prepare antibody-drug conjugates, linker-cytotoxin conjugates can be made by conventional methods analogous to those described by Doronina etal. (Bioconjugate Chem. 17: 114-124, 2006). Exemplary drugs include actinomycin D, anthracycline, auristatin, calicheamicin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1 -dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, maytansinoid (including monomethyl auristatin E [MMAE]; vedotin), mithramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine (PBD), taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, vincristine, and stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.

[0216] ADC compounds of the disclosure include those with anti-BCMA activity. In particularF053-0202PCT / 25-116-WO-PCTembodiments, the ADC compounds include an antibody conjugated, i.e. , covalently attached, to the drug moiety. In particular embodiments, the antibody is covalently attached to the drug moiety through a linker. A linker can include any chemical moiety that is capable of linking an antibody, antibody fragment (e.g., antigen binding fragments which are also referred to as binding fragments) or functional equivalent to another moiety, such as a drug moiety. Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Alternatively, linkers can be substantially resistant to cleavage (e.g., stable linker or noncleavable linker). In some aspects, the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid-based linker. The ADCs selectively deliver an effective dose of a drug to cells expressing BCMA (e.g., cancer cells) whereby greater selectivity, i.e. a lower efficacious dose, may be achieved while increasing the therapeutic index (“therapeutic window”).

[0217] To prepare ADCs, linker-drug conjugates can be made by conventional methods analogous to those described by Doronina et al. (Bioconjugate Chem. 17: 114-124, 2006). Antibody-drug conjugates with multiple (e.g., four) drugs per antibody can be prepared by partial reduction of the antibody with an excess of a reducing reagent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 30 min, then the buffer can be exchanged by elution through SEPHADEX G-25 resin with 1 mM DTPA in Dulbecco’s phosphate-buffered saline (DPBS). The eluent can be diluted with further DPBS, and the thiol concentration of the antibody can be measured using 5,5'-dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, for example 5-fold, of a linker-drug conjugate can be added at 4°C. for 1 hr, and the conjugation reaction can be quenched by addition of a substantial excess, for example 20-fold, of cysteine. The resulting ADC mixture can be purified on SEPHADEX G-25 equilibrated in PBS to remove unreacted linker-drug conjugate, desalted if desired, and purified by size-exclusion chromatography. The resulting ADC can then be sterile filtered, for example, through a 0.2 pm filter, and can be lyophilized if desired for storage.

[0218] Antibody-detectable label conjugates include an anti-BCMA binding domain linked to a detectable label. Detectable labels can include any suitable label or detectable group detectable by, for example, optical, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. In particular embodiments, detectable labels can include fluorescent labels, chemiluminescent labels, spectral colorimetric labels, enzymatic labels, and affinity tags.

[0219] Fluorescent labels can be particularly useful in cell staining, identification, imaging, and isolation uses. Exemplary fluorescent labels include blue fluorescent proteins (e.g. eBFP, eBFP2,F053-0202PCT / 25-116-WO-PCTAzurite, mKalamal, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g. GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen)), CopGFP, AceGFP, avGFP, ZsGreenl, Oregon Green™(Thermo Fisher Scientific)); Luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato); red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red™ (Thermo Fisher Scientific)); far red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and tandem conjugates.

[0220] Exemplary fluorescence labels include rhodamine, phycoerythrin, and fluorescein.

[0221] Chemiluminescent labels can include lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, or oxalate ester.

[0222] Spectral colorimetric labels can include colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

[0223] Exemplary enzyme labels include horseradish peroxidase, hydrolases, and alkaline phosphatase. Enzymatic labels can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes can include malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[0224] Affinity tags can include, for example, His tag (HHHHHH (SEQ ID NO: 179)), Flag tag (DYKDDDD (SEQ ID NO: 180), Xpress tag (DLYDDDDK (SEQ ID NO: 181)), Avi tag (GLNDIFEAQKIEWHE (SEQ ID NO: 182)), Calmodulin binding peptide (CBP) tag (KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO: 183)), Polyglutamate tag (EEEEEE (SEQ ID NO: 184)), HA tag (YPYDVPDYA (SEQ ID NO: 185)), Myc tag (EQKLISEEDL (SEQ ID NO: 186)), Strep tag (WRHPQFGG (SEQ ID NO: 187)), STREP® tag II (WSHPQFEK (SEQ ID NO: 188); IBA Institut fur Bioanalytik, Germany; see, e.g., US 7,981,632), Softag 1 (SLAELLNAGLGGS (SEQ ID NO: 189)), Softag 3 (TQDPSRVG (SEQ ID NO: 190)), and V5 tag (GKPIPNPLLGLDST (SEQ ID NO: 191)).

[0225] Antibody-radioisotope conjugates include an anti-BCMA binding domain linked to a radioisotope for use in nuclear medicine. Nuclear medicine refers to the diagnosis and / orF053-0202PCT / 25-116-WO-PCTtreatment of conditions by administering radioactive isotopes (radioisotopes or radionuclides) to a subject. Therapeutic nuclear medicine is often referred to as radiation therapy or radioimmunotherapy (RIT).

[0226] Examples of radioactive isotopes that can be conjugated to antibodies or binding fragments thereof of the present disclosure include actinium-225, iodine- 131, arsenic-211, iodine-131, indium-111 , yttrium-90, and lutetium-177, as well as alpha-emitting radionuclides such as astatine-211 or bismuth-212 or bismuth-213. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin™ (DEC Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the disclosure.

[0227] Examples of radionuclides that are useful for radiation therapy include225Ac and227Th.225Ac is a radionuclide with the half-life of ten days. As225Ac decays the daughter isotopes221Fr,213Bi, and209Pb are formed.227Th has a half-life of 19 days and forms the daughter isotope223Ra. Additional examples of useful radioisotopes include228Ac,111Ag,124Am,74As,211At,209At,194Au,128Ba,7Be,206Bi,245Bk,246Bk,76Br,11C,14C,47Ca,254Cf,242Cm,51Cr,67Cu,153Dy,157Dy,159Dy,165Dy,166Dy,171Er,250Es,254Es,147Eu,157Eu,52Fe,59Fe,251Fm,252Fm,253Fm,66Ga,72Ga,146Gd,153Gd,68Ge,3H,170Hf,171Hf,193Hg,193mHg,160mHo,130l,131l,135l,114mln,185lr,42K,43K,76Kr,79Kr,81mKr,132La,262Lr,169Lu,174ml_u,176mLu,257Md,260Md,28Mg,52Mn,90Mo,24Na,95Nb,138Nd,57Ni,66Ni,234Np,150,1820s,189mOs,191Os,32P,201Pb,101Pd,143Pr,191Pt,243Pu,225Ra,81Rb,188Re,105Rh,211Rn,103Ru,35S,44Sc,72Se,153Sm,125Sn,91Sr,173Ta,154Tb,127Te,234Th,45Ti,166Tm,230U,237U,240U,48V,178W,181W,188W,125Xe,127Xe,133Xe,133mXe,135Xe,85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, and / or97Zr. Radioisotopes can be used as a type of detectable label called a radiolabel. In particular embodiments, a radioisotope includes131l,90Y, and / or211At. In particular embodiments, a radioisotope is selected that does not emit daughter radionuclides that cause organ toxicity.

[0228] Antibody-particle conjugates include an antibody linked to a particle. In particular embodiments, particles include microparticles, nanoparticles, nanoshells, nanobeads, microbeads, or nanodots. Particles can include, for example, latex beads, polystyrene beads, fluorescent beads, and / or colored beads, and can be made from organic matter and / or inorganic matter.

[0229] In particular embodiments, an antibody as disclosed herein can be linked to a conjugate by any method known in the art. In particular embodiments, the constant region can be modified to allow for site specific conjugation. Such techniques include the use of naturally occurring or engineered cysteine residues, disulfide bridges, poly-histidine sequences, glycoengineering tags,F053-0202PCT / 25-116-WO-PCTand transglutaminase recognition sequences. Antibody fragments can also be modified for sitespecific conjugation, see for example, Kim et al., Mol Cancer Ther 2008;7(8).

[0230] (vii) Compositions and Formulations for Administration. Any of the binding domains described herein (e.g., antibodies, multi-domain binding molecules, antibody conjugates) in any exemplary format can be formulated alone or in combination into compositions for administration to subjects. Additionally, nucleic acids encoding the antibodies can also be formulated into compositions for administration (e.g., nucleic acids encapsulated within nanoparticles (e.g., liposomes or polymer-based nanoparticles) and / or as partof a vector delivery system (e.g., a viral vector or plasmid). Binding domains (e.g., antibodies, multi-domain binding molecules, antibody conjugates) and / or nucleic acids encoding antibodies are collectively referred to herein as “active ingredients”. Certain examples may include formulations. Formulations include cells genetically modified to express a binding domain disclosed herein (e.g., recombinant receptor) within a pharmaceutically-acceptable carrier.

[0231] Salts and / or pro-drugs of the active ingredients can also be used.

[0232] A pharmaceutically-acceptable salt includes any salt that retains the activity of the active ingredient and is acceptable for pharmaceutical use. A pharmaceutically-acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.

[0233] Suitable pharmaceutically-acceptable acid addition salts can be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids.

[0234] Suitable pharmaceutically-acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, arginine and procaine.

[0235] A prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage or by hydrolysis of a biologically labile group.

[0236] Exemplary generally used pharmaceutically-acceptable carriers include any and all absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and / or delivery vehicles. Exemplary carriers include saline, buffered saline, physiological saline, water, Hanks'F053-0202PCT / 25-116-WO-PCTsolution, Ringer's solution, Normosol-R (Abbott Labs), Plasma-Lyte A® (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof.

[0237] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

[0238] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0239] An exemplary chelating agent is EDTA (ethylene-diamine-tetra-acetic acid).

[0240] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0241] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0242] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the active ingredient or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on therapeutic weight.

[0243] The compositions and / or formulations disclosed herein can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The formulations and / or compositions disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intrauterine, intraplacental, intramuscular, intravesicular, oral, and / or subcutaneous administration.

[0244] For injection, compositions and / or formulations can be formulated as aqueous solutions,F053-0202PCT / 25-116-WO-PCTsuch as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can include formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the composition can be in lyophilized and / or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0245] For oral administration, the compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. For oral solid compositions such as powders, capsules and tablets, suitable excipients include binders (gum tragacanth, acacia, cornstarch, gelatin), fillers such as sugars, e.g., lactose, sucrose, mannitol and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and / or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents can be added, such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms can be sugar-coated or enteric-coated using standard techniques. Flavoring agents, such as peppermint, oil of Wintergreen, cherry flavoring, orange flavoring, etc. can also be used.

[0246] Compositions can be formulated as an aerosol. In particular embodiments, the aerosol is provided as part of an anhydrous, liquid or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers can also be used with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, a dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may also be formulated including a powder mix of active ingredient and a suitable powder base such as lactose or starch.

[0247] Compositions can also be formulated as depot preparations. Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0248] Additionally, compositions and / or formulations can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers including at least one active ingredient. Various sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release systems may, depending on their chemical nature, release one or more active ingredients following administration for a few weeks up to overF053-0202PCT / 25-116-WO-PCT100 days. Depot preparations can be administered by injection; parenteral injection; instillation; or implantation into soft tissues, a body cavity, or occasionally into a blood vessel with injection through fine needles.

[0249] Depot compositions can include a variety of bioerodible polymers including poly(lactide), poly(glycolide), poly(caprolactone) and poly(lactide)-co(glycolide) (PLG) of desirable lactide:glycolide ratios, average molecular weights, polydispersities, and terminal group chemistries. Blending different polymer types in different ratios using various grades can result in characteristics that borrow from each of the contributing polymers.

[0250] The use of different solvents (for example, dichloromethane, chloroform, ethyl acetate, triacetin, N-methyl pyrrolidone, tetrahydrofuran, phenol, or combinations thereof) can alter microparticle size and structure in order to modulate release characteristics. Other useful solvents include water, ethanol, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (IPA), ethyl benzoate, and benzyl benzoate.

[0251] Exemplary release modifiers can include surfactants, detergents, internal phase viscosity enhancers, complexing agents, surface active molecules, co-solvents, chelators, stabilizers, derivatives of cellulose, (hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68 / F127), polysorbates, Span® (Croda Americas, Wilmington, Delaware), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Delaware), sucrose acetate isobutyrate (SAIB), salts, and buffers.

[0252] Excipients that partition into the external phase boundary of microparticles or nanoparticles such as surfactants including polysorbates, dioctylsulfosuccinates, poloxamers, PVA, can also alter properties including particle stability and erosion rates, hydration and channel structure, interfacial transport, and kinetics in a favorable manner.

[0253] Additional processing of the disclosed sustained release depot compositions can utilize stabilizing excipients including mannitol, sucrose, trehalose, and glycine with other components such as polysorbates, P As, and dioctylsulfosuccinates in buffers such as Tris, citrate, or histidine. A freeze-dry cycle can also be used to produce very low moisture powders that reconstitute to similar size and performance characteristics of the original suspension.

[0254] In particular embodiments, the compositions include active ingredients of at least 0.1% w / v or w / w of the composition; at least 1% w / v or w / w of composition; at least 10% w / v or w / w of composition; at least 20% w / v or w / w of composition; at least 30% w / v or w / w of composition; at least 40% w / v or w / w of composition; at least 50% w / v or w / w of composition; at least 60% w / v or w / w of composition; at least 70% w / v or w / w of composition; at least 80% w / v or w / w of composition; at least 90% w / v or w / w of composition; at least 95% w / v or w / w of composition; orF053-0202PCT / 25-116-WO-PCTat least 99% w / v or w / w of composition.

[0255] In certain examples, cells are genetically modified to express a binding domain (e.g., antibody) disclosed herein, for example, as part of a recombinant receptor (for example, a CAR or eTCR). In these embodiments, genetically modified cells can be prepared as formulations for delivery in buffers such as Hanks' solution, Ringer's solution, or physiological saline. Cells can be genetically modified using methods known in the art. Exemplary targeted genetic engineering approaches include the use of CRISPR / Cas nuclease systems, zinc finger nucleases (ZFNs), and / or transcription activator like effector nucleases (TALENs). In particular embodiments, the cells are immune cells. In particular embodiments, the immune cells include T cells. In particular embodiments, the T cells include CD8+ and / or CD4+ T cells.

[0256] Therapeutically effective amounts of cells within formulations can be greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1010cells, or greater than 1011cells.

[0257] In particular embodiments, cells are in a formulation volume of a liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Hence, the density of administered cells is typically greater than 104cells / ml, 105cells / ml, 106cells / ml, 107cells / ml, or 108cells / ml.

[0258] In certain examples, compositions include a secondary treatment. Examples of secondary treatments are described elsewhere herein.

[0259] Any composition or formulation disclosed herein can advantageously include any other pharmaceutically-acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration. Exemplary pharmaceutically-acceptable carriers are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, compositions and formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U.S. FDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0260] (viii) Kits. Also provided herein are kits including at least one binding domain or sequences encoding at least one binding domain disclosed herein. Kits may be formed with components to practice, for example, the methods described herein. In particular embodiments, the kit includes an anti-BCMA binding domain, a multi-domain binding molecule, an antibody conjugate, a recombinant receptor, or sequences encoding an anti-BCMA binding domain, a multi-domain binding molecule, an antibody conjugate, or a recombinant receptor as described herein. In particular embodiments, the kit includes cells expressing a binding domain or composition to modify cells to express a binding domain. In particular embodiments, the kit includes cellsF053-0202PCT / 25-116-WO-PCTexpressing a recombinant receptor or composition to modify cells to express a recombinant receptor. The kit may include material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and / or other material useful in sample processing, washing, or conducting any other step of the method described herein.

[0261] The kit according to the present disclosure may also include instructions for carrying out the method. Instructions included in the kit of the present disclosure may be affixed to packaging material or may be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site which provides instructions.

[0262] (ix) Methods of Use. Methods disclosed herein include treating subjects (e.g., humans, veterinary animals (dogs, cats, reptiles, birds) livestock (e.g., horses, cattle, goats, pigs, chickens) and research animals (e.g., monkeys, rats, mice, fish) with compositions and / or formulations disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0263] An “effective amount” is the amount of a composition or formulation necessary to result in a desired physiological change in a subject. For example, an effective amount can provide an immunogenic effect. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically-significant effect in an in vitro assay, an animal model, clinical study, or clinical study relevant to the assessment of an condition’s development, progression, and / or resolution, as well as the effects of the condition. An immunogenic composition can be provided in an effective amount, wherein the effective amount stimulates an immune response.

[0264] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a condition to be treated or displays only early signs or symptoms of the condition to be treated such that treatment is administered for the purpose of diminishing, preventing, or decreasing the risk of developing the condition. Thus, a prophylactic treatment functions as a preventative treatment against a condition. In particular embodiments, prophylactic treatments reduce, delay, or prevent the worsening of a condition.

[0265] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of a condition and is administered to the subject for the purpose of reducingF053-0202PCT / 25-116-WO-PCTthe severity or progression of the condition.

[0266] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0267] In particular embodiments, therapeutically effective amounts provide anti-cancer effects. Anti-cancer effects include a decrease in the number of cancer cells, decrease in the number of metastases, a decrease in tumor volume, an increase in life expectancy, induced chemo- or radiosensitivity in cancer cells, inhibited angiogenesis near cancer cells, inhibited cancer cell proliferation, inhibited tumor growth, prevented or reduced metastases, prolonged subject life, reduced cancer-associated pain, and / or reduced relapse or re-occurrence of cancer following treatment.

[0268] In particular embodiments, therapeutically effective amounts induce an immune response. The immune response can be against a BCMA-expressing cancer cell.

[0269] The term “BCMA-expressing cell” or "BCMA-positive cell" refers to a cell that expresses BCMA on its surface. The term " BCMA -positive cancer cell" refers to a cancer cell that expresses BCMA on its surface. In some embodiments, expression of BCMA on the cell surface is determined, for example, using antibodies to BCMA in a method such as immunohistochemistry, FACS, etc. Alternatively, BCMA mRNA expression is considered to correlate to BCMA expression on the cell surface and can be determined by, for example, in situ hybridization and / or RT-PCR (including quantitative RT-PCR).

[0270] Examples of BCMA-related disorders that can be treated with compositions and / or formulations disclosed herein include hematological malignancies such as multiple myeloma, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), or Hodgkin lymphoma..

[0271] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes an IC50 as determined in cell culture against a particular target. Such information can be used to more accurately determine useful doses in subjects of interest.

[0272] The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of disease, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0273] Useful doses of active ingredients within compositions range from, for example, 0.1 to 5 pg / kg or from 0.5 to 1 pg / kg. In other examples, a dose can include 1 pg / kg, 15 pg / kg, 30 pg / kg,F053-0202PCT / 25-116-WO-PCT50 pg / kg, 55 pg / kg, 70 pg / kg, 90 pg / kg, 150 pg / kg, 350 pg / kg, 500 pg / kg, 750 pg / kg, 1000 pg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg, 2000 mg / kg, 3000 mg / kg, 4000 mg / kg, 5000 mg / kg or more.

[0274] Exemplary doses of cell-based formulations can include 104to 109cells / kg body weight, or 103to 1011cells / kg body weight. Therapeutically effective amounts to administer can include greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 101° cells, or greater than 1011cells.

[0275] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly). In particular embodiments, therapeutically effective amounts can be achieved by administering repeated doses during the course of a treatment regimen. In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA-approved treatment protocol.

[0276] The compositions and formulations described herein can be administered by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration can include intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intrarectal, topical, intrathecal, intramuscular, oral, subcutaneous, and / or sublingual administration.

[0277] In certain embodiments, compositions and / or formulations are administered to a subject in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities (or secondary treatments), or in combination or alternation with any other compound or therapy that the healthcare provider deems beneficial for the patient. The combination and / or alternation therapy can be therapeutic, adjunctive, or palliative.

[0278] In particular embodiments, other relevant treatment modalities include chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation.

[0279] Chemotherapy includes the administration of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents; alkyl sulfonates; aziridines; ethyleniminesF053-0202PCT / 25-116-WO-PCTand methylamelamines; nitrogen mustards; nitrosureas; antibiotics; anti-metabolites); folic acid analogues; purine analogs; pyrimidine analogs; androgens; anti-adrenals; folic acid replenisher; taxoids; platinum analogs I; topoisomerase inhibitor; retinoic acid derivatives; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and anti-androgens and pharmaceutically acceptable salts, acids or derivatives of any of the above. Combinations of chemotherapeutic agents are also administered where appropriate, including, CHOP, i.e., Cyclophosphamide (Cytoxan®), Doxorubicin (hydroxydoxorubicin), Vincristine (Oncovin®), and Prednisone.

[0280] In some embodiments, the chemotherapeutic agent is administered at the same time or within one week after the administration of the compositions and / or formulations described herein. In other embodiments, the chemotherapeutic agent is administered from 1 to 4 weeks or from 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the compositions and / or formulations. In other embodiments, the chemotherapeutic agent is administered at least 1 month before administering the compositions and / or formulations. In some embodiments, the methods further include administering two or more chemotherapeutic agents.

[0281] In additional embodiments, the compositions and / or formulations described herein can be administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include steroids and glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDS). Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists, (e.g., etanercept (EN BREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors and adhesion molecule inhibitors. The biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofm) and intramuscular) and minocycline.

[0282] In certain embodiments, the compositions and / or formulations described herein are administered in conjunction with another cytokine (e.g., in addition to the pro-inflammatory cytokine expressed by the genetic construct). “Cytokine” as used herein is meant to refer to proteins released by one cell population that act on another cell as intercellular mediators.F053-0202PCT / 25-116-WO-PCTExamples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; plateletgrowth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-1 and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferonalpha, beta, and - gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte- macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL- 1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-I I, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.

[0283] The Exemplary Embodiments below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0284] (x) Exemplary Embodiments.1. A chimeric antigen receptor (CAR) including:an extracellular component including a single-chain variable fragment (scFv) that binds B-cell maturation antigen (BCMA), the scFv including the sequence of SEQ ID NO: 39 or SEQ ID NO: 40;a transmembrane domain including a CD28 transmembrane region; and an intercellular component including an effector domain of CD28 and / or an effector domain of CD3 .2. A binding domain that binds B-cell maturation antigen (BCMA), the binding domain including a variable heavy chain including a complementarity determining region (CDR) heavy (1-1)1, a CDRH2, and a CDRH3 and a variable light including a CDR light (L)1, a CDRL2 and a CDRL3, wherein:the CDRH1 includes the sequence of SEQ ID NO: 3, the CDRH2 includes the sequence of SEQ ID NO: 4, and the CDRH3 includes the sequence of SEQ ID NO: 5, theF053-0202PCT / 25-116-WO-PCTCDRL1 includes the sequence of SEQ ID NO: 6, the CDRL2 includes the sequence WAS, and the CDRL3 includes the sequence of SEQ ID NO: 7, according to IMGT;the CDRH1 includes the sequence of SEQ ID NO: 8, the CDRH2 includes the sequence of SEQ ID NO: 9, and the CDRH3 includes the sequence of SEQ ID NO: 10, the CDRL1 includes the sequence of SEQ ID NO: 11 , the CDRL2 includes the sequence of SEQ ID NO: 12, and the CDRL3 includes the sequence of SEQ ID NO: 7, according to Kabat;the CDRH1 includes the sequence of SEQ ID NO: 13, the CDRH2 includes the sequence of SEQ ID NO: 14, and the CDRH3 includes the sequence of SEQ ID NO: 10, the CDRL1 includes the sequence of SEQ ID NO: 11 , the CDRL2 includes the sequence of SEQ ID NO: 12, and the CDRL3 includes the sequence of SEQ ID NO: 7, according to Chothia;the CDRH1 includes the sequence of SEQ ID NO: 15, the CDRH2 includes the sequence of SEQ ID NO: 16, and the CDRH3 includes the sequence of SEQ ID NO: 5, the CDRL1 includes the sequence of SEQ ID NO: 11 , the CDRL2 includes the sequence of SEQ ID NO: 17, and the CDRL3 includes the sequence of SEQ ID NO: 7, according to North; orthe CDRH1 includes the sequence of SEQ ID NO: 18, the CDRH2 includes the sequence of SEQ ID NO: 19, and the CDRH3 includes the sequence of SEQ ID NO: 20, the CDRL1 includes the sequence of SEQ ID NO: 21, the CDRL2 includes the sequence of SEQ ID NO: 22, and the CDRL3 includes the sequence of SEQ ID NO: 23, according to Contact.The binding domain of embodiment 2, whereinthe variable heavy chain includes at least 90% sequence identity to the sequence of SEQ ID NO: 24 and the variable light chain includes at least 90% sequence identity to the sequence as of SEQ ID NO: 25.The binding domain of embodiment 2, whereinthe variable heavy chain includes the sequence of SEQ ID NO: 24 and the variable light chain includes the sequence of SEQ ID NO: 25.The binding domain of any of embodiments 2-4, whereinthe variable heavy chain is encoded by a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 102 and the variable light chain is encoded by a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 103.The binding domain of any of embodiments 2-5, wherein the binding domain is linked to aF053-0202PCT / 25-116-WO-PCThuman constant region.The binding domain of embodiment 6, wherein the human constant region includes a human light chain constant region and / or a human heavy chain constant region.The binding domain of embodiment 7, wherein the human light chain constant region includes a human IgK light chain constant region or a human IgA light chain constant region.The binding domain of embodiments 7 or 8, wherein the human heavy chain constant region includes an IgG, IgM, IgD, IgA, or IgE heavy chain constant region.The binding domain of any of embodiments 7-9, wherein the human heavy chain constant region includes an IgG heavy chain constant region.The binding domain of any of embodiments 6-10, wherein the human constant region includes an Fc modification.A nucleotide encoding the binding domain of any of embodiments 2-11.The nucleotide of embodiment 12, wherein the nucleotide includes the sequence of SEQ ID NO: 102 and SEQ ID NO: 103.A single-chain variable fragment (scFv) including the binding domain of embodiment 2. The scFv of embodiment 14, in a VL-VH orientation.The scFv of embodiments 14 or 15, wherein a linker of the scFv includes a Gly-Ser linker. The scFv of embodiment 16, wherein the Gly-Ser linker is (GlyxSery)n wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.The scFv of any of embodiments 14-17, wherein a linker of the scFv includes the sequence of SEQ ID NO: 99.The scFv of any of embodiments 14-18, wherein the scFv includes a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 39 or SEQ ID NO: 40. The scFv of any of embodiments 14-19, wherein the scFv includes the sequence of SEQ ID NO: 39 or SEQ ID NO: 40.A nucleotide encoding an scFv of any of embodiments 14-20.The nucleotide of embodiment 21, wherein the nucleotide includes the sequence of SEQ ID NO: 192 and SEQ ID NO: 193.A recombinant receptor that, when expressed by a cell, includes an extracellular component including a binding domain of any of embodiments 2-11.The recombinant receptor of embodiment 23, wherein the binding domain of embodiment 2 includes the scFv of embodiment 14.F053-0202PCT / 25-116-WO-PCTThe recombinant receptor of embodiments 23 or 24, wherein the recombinant receptor includes a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a hybrid thereof.The recombinant receptor of embodiment 25, wherein the CAR includes an intracellular component linked to the extracellular component by a transmembrane domain.The recombinant receptor of embodiment 26, wherein the intracellular component includes an effector domain including: 4-1 BB (CD137), CD3y, CD35, CD3E, CD3 , CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, 0X40, ROR2, SLAMF1, TCRa, TCR , TRIM, Wnt, Zap70, or a combination thereof.The recombinant receptor of embodiments 26 or 27, wherein the intracellular component includes an effector domain of CD28 and / or an effector domain of CD3 .The recombinant receptor of embodiment 28, wherein the intracellular component includes the sequence of SEQ ID NO: 63 or a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 63.The recombinant receptor of any of embodiments 26-29, wherein the transmembrane domain includes a transmembrane region of: the a, p or chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.The recombinant receptor of any of embodiments 26-30, wherein the transmembrane domain includes a CD28 transmembrane region.The recombinant receptor of embodiment 31, wherein the CD28 transmembrane region includes the sequence of SEQ ID NO: 55 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 55.The recombinant receptor of any of embodiments 26-32, wherein the extracellular component of the CAR further includes a spacer region.The recombinant receptor of embodiment 33, wherein the spacer region includes a long hinge.The recombinant receptor of embodiment 34, wherein the long hinge includes the sequence of SEQ ID NO: 42 or a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 42.The recombinant receptor of any of embodiments 26-35, including the sequence of SEQ ID NO: 82 or SEQ ID NO: 83 or a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 82 or SEQ ID NO: 83.The recombinant receptor of any of embodiments 25-36, wherein the eTCR includes aF053-0202PCT / 25-116-WO-PCTconstant alpha domain (Ca) and a constant beta domain (Cp).The recombinant receptor of embodiment 37, wherein the binding domain of embodiment 1 is linked to the Cadomain or the Cp domain.The recombinant receptor of embodiments 37 or 38, wherein the binding domain of embodiment 1 is linked to the Cadomain and the C domain.A nucleotide encoding the recombinant receptor of any of embodiments 23-39.The nucleotide of embodiment 40, wherein the nucleotide includes the sequence of SEQ ID NO: 195 or 196 or has at least 90% sequence identity to the sequence of SEQ ID NO: 195 or 196.A multi-domain binding molecule including at least two binding domains, wherein at least one binding domain of the at least two binding domains includes the binding domain of any of embodiments 2-11.The multi-domain binding molecule of embodiment 42, wherein the multi-domain binding molecule includes an immune cell engaging molecule.The multi-domain binding molecule of embodiment 43, wherein the immune cell engaging molecule activates a B cell, T cell, natural killer (NK) cell, or macrophage.The multi-domain binding molecule of embodiment 44, wherein the T cell is a CD3 T cell, a CD4 T cell, a CD8 T cell, a central memory T cell, an effector memory T cell, and / or a naive T cell.The multi-domain binding molecule of any of embodiments 43-45, wherein a binding domain of the immune cell engaging molecule binds CD3, CD28, CD8, NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1, CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2, Toll-like receptors 1-9, IL-4Ra, or MARCO.The multi-domain binding molecule of any of embodiments 42-46, wherein the at least two binding domains comprise at least two copies of the binding domain of embodiment 1. The multi-domain binding molecule of embodiment 47, wherein the at least two copies are joined by a protein linker.The multi-domain binding molecule of embodiment 48, wherein the protein linker is a Gly-Ser linker.The multi-domain binding molecule of embodiment 49, wherein the Gly-Ser linker is (GlyxSery)nwherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.The multi-domain binding molecule of any of embodiments 42-50, including 2, 3, 4, 5, 6,F053-0202PCT / 25-116-WO-PCT7, 8, 9, or 10 copies of the binding domain of embodiment 1.The multi-domain binding molecule of any of embodiments 42-52, wherein the multidomain binding molecule is a dimer, trimer, tetramer, pentamer, hexamer, or heptamer. The multi-domain binding molecule of embodiment 42, wherein the at least two binding domains are linked to an Fc region of an antibody.The multi-domain binding molecule of embodiment 53, wherein the Fc region is an IgA Fc region or an IgM Fc region.The multi-domain binding molecule of embodiment 54, wherein the Fc region includes a multimerizing fragment of the IgA Fc region or a multimerizing fragment of the IgM Fc region.The multi-domain binding molecule of embodiment 55, wherein the multimerizing fragment of the IgA Fc region includes an IgA tailpiece.The multi-domain binding molecule of embodiments 55 or 56, wherein the multimerizing fragment of the IgA Fc region includes an IgA CA3 domain and an IgA tailpiece.The multi-domain binding molecule of any of embodiments 55-57, wherein the multimerizing fragment of the IgA Fc region includes an IgA CA2 domain, an IgA CA3 domain, and an IgA tailpiece.The multi-domain binding molecule of any of embodiments 55-58, wherein the multimerizing fragment of the IgA Fc region includes an IgA CA1 domain, an IgA CA2 domain, an IgA CA3 domain, and an IgA tailpiece.The multi-domain binding molecule of any of embodiments 55-59, wherein the multimerizing fragment of the IgM Fc region includes an IgM tailpiece.The multi-domain binding molecule of any of embodiments 55-60, wherein the multimerizing fragment of the IgM Fc region includes a Cp4 domain and an IgM tailpiece. The multi-domain binding molecule of any of embodiments 55-61, wherein the multimerizing fragment of the IgM Fc region includes a Cp3 domain, a Cp4 domain, and an IgM tailpiece.The multi-domain binding molecule of any of embodiments 55-62, wherein the multimerizing fragment of the IgM Fc region includes a Cp2 domain, a Cp3 domain, a C 4 domain, and an IgM tailpiece.The multi-domain binding molecule of any of embodiments 55-63, wherein the multimerizing fragment of the IgM Fc region includes a Cp1 domain, a Cp2 domain, a C 3 domain, a Cp4 domain, and an IgM tailpiece.A conjugate including the binding domain of any of embodiments 2-11 linked to anF053-0202PCT / 25-116-WO-PCTimmunotoxin, a drug, a detectable label, a radioisotope, or a particle.The conjugate of embodiment 65, wherein the immunotoxin includes a plant toxin or bacterial toxin.The conjugate of embodiment 66, wherein the plant toxin includes ricin, abrin, mistletoe lectin, modeccin, pokeweed antiviral protein, saporin, Bryodin 1, bouganin, or gelonin. The conjugate of embodiments 66 or 67, wherein the bacterial toxin includes diphtheria toxin or Pseudomonas exotoxin.The conjugate of any of embodiments 65-68, wherein the drug includes a cytotoxic drug. The conjugate of embodiment 69, wherein the cytotoxic drug includes actinomycin D, anthracycline, auristatin, calicheamicin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1 -dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, maytansinoid, mithramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine, taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, or vincristine.The conjugate of any of embodiments 65-70, wherein the detectable label includes a fluorescent label, a chemiluminescent label, a spectral colorimetric label, an enzymatic label, or an affinity tag.The conjugate of embodiment 70, wherein the radioisotope includes228Ac,111Ag,124Am,74As,211At,209At,194Au,128Ba,7Be,206Bi,245Bk,246Bk,76Br,11C,14C,47Ca,254Cf,242Cm,51Cr,67Cu,153Dy,157Dy,159Dy,165Dy,166Dy,171Er,250Es,254Es,147Eu,157Eu,52Fe,59Fe,251Fm,252Fm,253Fm,66Ga,72Ga,146Gd,153Gd,68Ge,3H,170Hf,171Hf,193Hg,193mHg,160mHo,130l,131l,135l,114mln,185lr,42K,43K,76Kr,79Kr,81mKr,132La,262Lr,169Lu,174ml_u,176mLu,257Md,260Md,28Mg,52Mn,90Mo,24Na,95Nb,138Nd,57Ni,66Ni,234Np,150,1820s,189mOs,191Os,32P,201Pb,101Pd,143Pr,191Pt,243Pu,225Ra,81Rb,188Re,105Rh,211Rn,103Ru,35S,44Sc,72Se,153Sm,125Sn,91Sr,173Ta,154Tb,127Te,234Th,45Ti,166Tm,230U,237U,240U,48V,178W,181W,188W,125Xe,127Xe,133Xe,133mXe,135Xe,85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, or97Zr.The conjugate of any of embodiments 65-72, wherein the radioisotope does not emit daughter radionuclides.A cell genetically modified to express the binding domain of any of embodiments 2-11. The cell of embodiment 74, wherein the cell is an immune cell.The cell of embodiment 75, wherein the immune cell is a T cell, B cell, natural killer cell,F053-0202PCT / 25-116-WO-PCTor macrophage.A composition including the binding domain of any of embodiments 2-11 or the nucleic acid of embodiments 12 or 13; and a pharmaceutically-acceptable carrier.A formulation including a cell genetically modified to express the binding domain of embodiment 2; and a pharmaceutically-acceptable carrier.A kit including the binding domain of any of embodiments 2-11, the scFv or embodiment 14, the recombinant receptor of embodiment 23, the multi-domain binding molecule of embodiment 42, the conjugate of embodiment 65, the cell of embodiment 74, the composition of embodiment 77, the formulation of embodiment 78, and / or the nucleotide of embodiments 12, 21, and / or 40.A method of providing an immune response against BCMA-expressing cells in a subject in need thereof including administering a therapeutically effective amount of the composition of embodiment 77 or the formulation of embodiment 78 to the subject thereby providing an immune response against BCMA-expressing cells in the subject.The method of embodiment 80, wherein the BCMA-expressing cells are cancer cells. The method of embodiments 80 or 81, wherein the immune response results in killing of the cancer cells.The method of embodiment 81 , wherein the cancer cells comprise multiple myeloma cells, acute B-lymphoblastic leukemia cells, non-Hodgkin lymphoma (NHL) cells, or Hodgkin lymphoma cells.The method of any of embodiments 80-83, wherein the administering a therapeutically effective amount includes administering intravesically, intravenously, intradermally, intraarterially, intraparenterally, intranodally, intralymphaticaly, intraperitoneally, intralesionally, intraprostaticaly, intravaginally, intrarectally, topically, intrathecally, intramuscularly, or subcutaneously.A method of treating a subject with a BCMA-related disorder including administering a therapeutically effective amount of the composition of embodiment 77 or the formulation of embodiment 78 to the subject, thereby treating the subject.The method of embodiment 85, wherein the BCMA-related disorder includes a hematological malignancy.The method of embodiment 86, wherein the hematological malignancy includes multiple myeloma, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), or Hodgkin lymphoma.The method of any of embodiments 85-87, wherein the administering a therapeuticallyF053-0202PCT / 25-116-WO-PCTeffective amount includes administering intravesically, intravenously, intradermally, intraarterially, intraparenterally, intranodally, intralymphaticaly, intraperitoneally, intralesionally, intraprostaticaly, intravaginally, intrarectally, topically, intrathecally, intramuscularly, or subcutaneously.

[0285] (xi) Closing Paragraphs. The nucleic acid and amino acid sequences provided herein are shown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 C.F.R. §1.831-1.835 and set forth in WIPO Standard ST.26 (implemented on July 1, 2022). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate.

[0286] Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0287] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Vai) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (nonpolar): Proline (Pro), Ala, Vai, Leu, lie, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Vai, Leu, and lie; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.F053-0202PCT / 25-116-WO-PCT

[0288] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: lie (+4.5); Vai (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glutamate (-3.5); Gin (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0289] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0290] As detailed in US 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Vai (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0291] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of an encoded product to a statistically-significant degree.

[0292] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.F053-0202PCT / 25-116-WO-PCT

[0293] “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, HI-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0294] Variants also include nucleic acid molecules that hybridize under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 pg / ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in loweredF053-0202PCT / 25-116-WO-PCTstringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCI; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 pg / ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g., 5XSSC). Variations in the above conditions may be accomplished through the inclusion and / or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0295] "Binds" refers to an association of a binding domain (of, for example, an anti-BCMA binding domain) to its cognate binding molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M’1, while not significantly associating with any other molecules or components in a relevant environment sample. “Specifically binds” is also referred to as “binds” herein. Binding domains may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity" binding domains refer to those binding domains with a Ka of at least 107M’1, at least 108M’1, at least 109M’1, at least 1010M’1, at least 1011M-1, at least 1012M’1, or at least 1013M’1. In particular embodiments, "low affinity" binding domains refer to those binding domains with a Ka of up to 107M’1, up to 106M-1, up to 105M-1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10-5M to 10'13M). In certain embodiments, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka (equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off-rate (Koff) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, et a / ., 1949, Ann. N.Y. Acad. Sci. 51:660; and US 5,283,173, US 5,468,614, or the equivalent).

[0296] Unless otherwise indicated, the practice of the present disclosure can employ conventionalF053-0202PCT / 25-116-WO-PCTtechniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Sambrook, etal. Molecular Cloning: A Laboratory Manual, 4th Edition (2012); F. M. Ausubel, etal. eds., Current Protocols in Molecular Biology, (2003); the series Methods In Enzymology (Academic Press, Inc.); Behlke, et al., Polymerase Chain Reaction: Theory and Technology (2019); Greenfield, ed. Antibodies, A Laboratory Manual, Second Edition (2014); and Capes-Davis and R. I. Freshney, eds. Freshney's Culture of Animal Cells 8th Edition (2021).

[0297] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in binding between a disclosed binding domain and its epitope.

[0298] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% ofF053-0202PCT / 25-116-WO-PCTthe stated value; or ±1% of the stated value.

[0299] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0300] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0301] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0302] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.F053-0202PCT / 25-116-WO-PCT

[0303] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0304] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0305] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0306] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

F053-0202PCT / 25-116-WO-PCTCLAIMSWhat is claimed is:

1. A chimeric antigen receptor (CAR) comprising:an extracellular component comprising a single-chain variable fragment (scFv) that binds B-cell maturation antigen (BC A), the scFv comprising the sequence of SEQ ID NO: 39 or SEQ ID NO: 40;a transmembrane domain comprising a CD28 transmembrane region; and an intercellular component comprising an effector domain of CD28 and / or an effector domain of CD3 .

2. A binding domain that binds B-cell maturation antigen (BCMA), the binding domain comprising a variable heavy chain comprising a complementarity determining region (CDR) heavy (H)1, a CDRH2, and a CDRH3 and a variable light comprising a CDR light (L)1, a CDRL2 and a CDRL3, wherein:the CDRH1 comprises the sequence of SEQ ID NO: 3, the CDRH2 comprises the sequence of SEQ ID NO: 4, and the CDRH3 comprises the sequence of SEQ ID NO: 5, the CDRL1 comprises the sequence of SEQ ID NO: 6, the CDRL2 comprises the sequence WAS, and the CDRL3 comprises the sequence of SEQ ID NO: 7, according to IMGT;the CDRH1 comprises the sequence of SEQ ID NO: 8, the CDRH2 comprises the sequence of SEQ ID NO: 9, and the CDRH3 comprises the sequence of SEQ ID NO: 10, the CDRL1 comprises the sequence of SEQ ID NO: 11, the CDRL2 comprises the sequence of SEQ ID NO: 12, and the CDRL3 comprises the sequence of SEQ ID NO: 7, according to Kabat;the CDRH1 comprises the sequence of SEQ ID NO: 13, the CDRH2 comprises the sequence of SEQ ID NO: 14, and the CDRH3 comprises the sequence of SEQ ID NO: 10, the CDRL1 comprises the sequence of SEQ ID NO: 11, the CDRL2 comprises the sequence of SEQ ID NO: 12, and the CDRL3 comprises the sequence of SEQ ID NO: 7, according to Chothia;the CDRH1 comprises the sequence of SEQ ID NO: 15, the CDRH2 comprises the sequence of SEQ ID NO: 16, and the CDRH3 comprises the sequence of SEQ ID NO: 5, the CDRL1 comprises the sequence of SEQ ID NO: 11, the CDRL2 comprises the sequence of SEQ ID NO: 17, and the CDRL3 comprises the sequence of SEQ ID NO: 7, according to North; orthe CDRH1 comprises the sequence of SEQ ID NO: 18, the CDRH2 comprisesF053-0202PCT / 25-116-WO-PCTthe sequence of SEQ ID NO: 19, and the CDRH3 comprises the sequence of SEQ ID NO: 20, the CDRL1 comprises the sequence of SEQ ID NO: 21, the CDRL2 comprises the sequence of SEQ ID NO: 22, and the CDRL3 comprises the sequence of SEQ ID NO: 23, according to Contact.

3. The binding domain of claim 2, whereinthe variable heavy chain comprises at least 90% sequence identity to the sequence of SEQ ID NO: 24 and the variable light chain comprises at least 90% sequence identity to the sequence as of SEQ ID NO: 25.

4. The binding domain of claim 2, whereinthe variable heavy chain comprises the sequence of SEQ ID NO: 24 and the variable light chain comprises the sequence of SEQ ID NO: 25.

5. The binding domain of claim 2, whereinthe variable heavy chain is encoded by a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 102 and the variable light chain is encoded by a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 103.

6. The binding domain of claim 2, wherein the binding domain is linked to a human constant region.

7. The binding domain of claim 6, wherein the human constant region comprises a human light chain constant region and / or a human heavy chain constant region.

8. The binding domain of claim 7, wherein the human light chain constant region comprises a human IgK light chain constant region or a human IgA light chain constant region.

9. The binding domain of claim 7, wherein the human heavy chain constant region comprises an IgG, IgM, IgD, IgA, or IgE heavy chain constant region.

10. The binding domain of claim 7, wherein the human heavy chain constant region comprises an IgG heavy chain constant region.

11. The binding domain of claim 6, wherein the human constant region comprises an Fc modification.

12. A nucleotide encoding the binding domain of claim 2.

13. The nucleotide of claim 12, wherein the nucleotide comprises the sequence of SEQ ID NO: 102 and SEQ ID NO: 103.

14. A single-chain variable fragment (scFv) comprising the binding domain of claim 2.

15. The scFv of claim 14, in a VL-VH orientation.

16. The scFv of claim 14, wherein a linker of the scFv comprises a Gly-Ser linker.

17. The scFv of claim 16, wherein the Gly-Ser linker is (GlyxSery)nwherein x and y areF053-0202PCT / 25-116-WO-PCTindependently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

18. The scFv of claim 14, wherein a linker of the scFv comprises the sequence of SEQ ID NO:99.

19. The scFv of claim 14, wherein the scFv comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 39 or SEQ ID NO: 40.

20. The scFv of claim 14, wherein the scFv comprises the sequence of SEQ ID NO: 39 or SEQ ID NO: 40.

21. A nucleotide encoding an scFv of claim 14.

22. The nucleotide of claim 21, wherein the nucleotide comprises the sequence of SEQ ID NO: 192 and SEQ ID NO: 193.

23. A recombinant receptor that, when expressed by a cell, comprises an extracellular component comprising a binding domain of claim 2.

24. The recombinant receptor of claim 23, wherein the binding domain of claim 2 comprises the scFv of claim 14.

25. The recombinant receptor of claim 23, wherein the recombinant receptor comprises a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a hybrid thereof.

26. The recombinant receptor of claim 25, wherein the CAR comprises an intracellular component linked to the extracellular component by a transmembrane domain.

27. The recombinant receptor of claim 26, wherein the intracellular component comprises an effector domain comprising: 4-1 BB (CD137), CD3y, CD36, CD3c, CD3 , CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, 0X40, ROR2, SLAMF1, TCRa, TCR , TRIM, Wnt, Zap70, or a combination thereof.

28. The recombinant receptor of claim 26, wherein the intracellular component comprises an effector domain of CD28 and / or an effector domain of CD3 .

29. The recombinant receptor of claim 28, wherein the intracellular component comprises the sequence of SEQ ID NO: 63 or a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 63.

30. The recombinant receptor of claim 26, wherein the transmembrane domain comprises a transmembrane region of: the a, [3 or chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.

31. The recombinant receptor of claim 26, wherein the transmembrane domain comprises aF053-0202PCT / 25-116-WO-PCTCD28 transmembrane region.

32. The recombinant receptor of claim 31, wherein the CD28 transmembrane region comprises the sequence of SEQ ID NO: 55 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 55.

33. The recombinant receptor of claim 26, wherein the extracellular component of the CAR further comprises a spacer region.

34. The recombinant receptor of claim 33, wherein the spacer region comprises a long hinge.

35. The recombinant receptor of claim 34, wherein the long hinge comprises the sequence of SEQ ID NO: 42 or a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 42.

36. The recombinant receptor of claim 26, comprising the sequence of SEQ ID NO: 82 or SEQ ID NO: 83 or a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 82 or SEQ ID NO: 83.

37. The recombinant receptor of claim 25, wherein the eTCR comprises a constant alpha domain (Ca) and a constant beta domain (Cp).

38. The recombinant receptor of claim 37, wherein the binding domain of claim 1 is linked to the Ca domain or the C domain.

39. The recombinant receptor of claim 37, wherein the binding domain of claim 1 is linked to the Cadomain and the Cp domain.

40. A nucleotide encoding the recombinant receptor of claim 23.

41. The nucleotide of claim 40, wherein the nucleotide comprises the sequence of SEQ ID NO: 195 or 196 or has at least 90% sequence identity to the sequence of SEQ ID NO: 195 or 196.

42. A multi-domain binding molecule comprising at least two binding domains, wherein at least one binding domain of the at least two binding domains comprises the binding domain of claim 2.

43. The multi-domain binding molecule of claim 42, wherein the multi-domain binding molecule comprises an immune cell engaging molecule.

44. The multi-domain binding molecule of claim 43, wherein the immune cell engaging molecule activates a B cell, T cell, natural killer (NK) cell, or macrophage.

45. The multi-domain binding molecule of claim 44, wherein the T cell is a CD3 T cell, a CD4 T cell, a CD8 T cell, a central memory T cell, an effector memory T cell, and / or a naive T cell.

46. The multi-domain binding molecule of claim 43, wherein a binding domain of the immuneF053-0202PCT / 25-116-WO-PCTcell engaging molecule binds CD3, CD28, CD8, NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1, CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2, Toll-like receptors 1-9, IL-4Ra, or MARCO.

47. The multi-domain binding molecule of claim 42, wherein the at least two binding domains comprise at least two copies of the binding domain of claim 1.

48. The multi-domain binding molecule of claim 47, wherein the at least two copies are joined by a protein linker.

49. The multi-domain binding molecule of claim 48, wherein the protein linker is a Gly-Ser linker.

50. The multi-domain binding molecule of claim 49, wherein the Gly-Ser linker is (GlyxSery)nwherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.

51. The multi-domain binding molecule of claim 42, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the binding domain of claim 1.

52. The multi-domain binding molecule of claim 42, wherein the multi-domain binding molecule is a dimer, trimer, tetramer, pentamer, hexamer, or heptamer.

53. The multi-domain binding molecule of claim 42, wherein the at least two binding domains are linked to an Fc region of an antibody.

54. The multi-domain binding molecule of claim 53, wherein the Fc region is an IgA Fc region or an IgM Fc region.

55. The multi-domain binding molecule of claim 54, wherein the Fc region comprises a multimerizing fragment of the IgA Fc region or a multimerizing fragment of the IgM Fc region.

56. The multi-domain binding molecule of claim 55, wherein the multimerizing fragment of the IgA Fc region comprises an IgA tailpiece.

57. The multi-domain binding molecule of claim 55, wherein the multimerizing fragment of the IgA Fc region comprises an IgA CA3 domain and an IgA tailpiece.

58. The multi-domain binding molecule of claim 55, wherein the multimerizing fragment of the IgA Fc region comprises an IgA CA2 domain, an IgA CA3 domain, and an IgA tailpiece.

59. The multi-domain binding molecule of claim 55, wherein the multimerizing fragment of the IgA Fc region comprises an IgA CA1 domain, an IgA CA2 domain, an IgA CA3 domain, and an IgA tailpiece.

60. The multi-domain binding molecule of claim 55, wherein the multimerizing fragment of theF053-0202PCT / 25-116-WO-PCTIgM Fc region comprises an IgM tailpiece.

61. The multi-domain binding molecule of claim 55, wherein the multimerizing fragment of the IgM Fc region comprises a Cp4 domain and an IgM tailpiece.

62. The multi-domain binding molecule of claim 55, wherein the multimerizing fragment of the IgM Fc region comprises a Cp3 domain, a Cp4 domain, and an IgM tailpiece.

63. The multi-domain binding molecule of claim 55, wherein the multimerizing fragment of the IgM Fc region comprises a Cp2 domain, a Cp3 domain, a Cp4 domain, and an IgM tailpiece.

64. The multi-domain binding molecule of claim 55, wherein the multimerizing fragment of the IgM Fc region comprises a Cp1 domain, a Cp2 domain, a Cp3 domain, a Cp4 domain, and an IgM tailpiece.

65. A conjugate comprising the binding domain of claim 2 linked to an immunotoxin, a drug, a detectable label, a radioisotope, or a particle.

66. The conjugate of claim 65, wherein the immunotoxin comprises a plant toxin or bacterial toxin.

67. The conjugate of claim 66, wherein the plant toxin comprises ricin, abrin, mistletoe lectin, modeccin, pokeweed antiviral protein, saporin, Bryodin 1 , bouganin, or gelonin.

68. The conjugate of claim 66, wherein the bacterial toxin comprises diphtheria toxin or Pseudomonas exotoxin.

69. The conjugate of claim 65, wherein the drug comprises a cytotoxic drug.

70. The conjugate of claim 69, wherein the cytotoxic drug comprises actinomycin D, anthracycline, auristatin, calicheamicin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1 -dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, maytansinoid, mithramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine, taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, or vincristine.

71. The conjugate of claim 65, wherein the detectable label comprises a fluorescent label, a chemiluminescent label, a spectral colorimetric label, an enzymatic label, or an affinity tag.

72. The conjugate of claim 70, wherein the radioisotope comprises228Ac,111Ag,124Am,74As,211At,209At,194Au,128Ba,7Be,206Bi,245Bk,246Bk,75Br,11C,14C,47Ca,254Cf,242Cm,51Cr,67Cu,153Dy,157Dy,159Dy,165Dy,166Dy,171Er,250Es,254Es,147Eu,157Eu,52Fe,59Fe,251Fm,252Fm,253Fm,66Ga,72Ga,146Gd,153Gd,68Ge,3H,170Hf,171Hf,193Hg,193mHg,160mHo,130l,F053-0202PCT / 25-116-WO-PCT131l,135l,114mln,185lr,42K,43K,76Kr,79Kr,81mKr,132La,262Lr,169Lu,174mLu,176mLu,257Md,260Md,28Mg,52Mn,90Mo,24Na,95Nb,138Nd,57Ni,66Ni,234Np,150,1820s,189mOs,1910s,32P,201Pb,101Pd,143Pr,191Pt,243Pu,225Ra,81Rb,188Re,105Rh,211Rn,103Ru,35S,44Sc,72Se,153Sm,125Sn,91Sr,173Ta,154Tb,127Te,234Th,45Ti,166Tm,230U,237U,240U,48V,178W,181W,188W,125Xe,127Xe,133Xe,133mXe,135Xe,85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, or97Zr.

73. The conjugate of claim 65, wherein the radioisotope does not emit daughter radionuclides.

74. A cell genetically modified to express the binding domain of claim 2.

75. The cell of claim 74, wherein the cell is an immune cell.

76. The cell of claim 75, wherein the immune cell is a T cell, B cell, natural killer cell, or macrophage.

77. A composition comprising the binding domain of claim 2 or the nucleic acid of claim 12;and a pharmaceutically-acceptable carrier.

78. A formulation comprising a cell genetically modified to express the binding domain of claim 2; and a pharmaceutically-acceptable carrier.

79. A kit comprising the binding domain of claim 2, the scFv or claim 14, the recombinant receptor of claim 23, the multi-domain binding molecule of claim 42, the conjugate of claim 65, the cell of claim 74, the composition of claim 77, the formulation of claim 78, and / or the nucleotide of claims 12, 21, and / or 40.

80. A method of providing an immune response against BCMA-expressing cells in a subject in need thereof comprising administering a therapeutically effective amount of the composition of claim 77 or the formulation of claim 78 to the subject thereby providing an immune response against BCMA-expressing cells in the subject.

81. The method of claim 80, wherein the BCMA-expressing cells are cancer cells.

82. The method of claim 80, wherein the immune response results in killing of the cancer cells.

83. The method of claim 81, wherein the cancer cells comprise multiple myeloma cells, acute B-lymphoblastic leukemia cells, non-Hodgkin lymphoma (NHL) cells, or Hodgkin lymphoma cells.

84. The method of claim 80, wherein the administering a therapeutically effective amount comprises administering intravesically, intravenously, intradermally, intraarterially, intraparenterally, intranodally, intralymphaticaly, intraperitoneally, intralesionally, intraprostaticaly, intravaginally, intrarectally, topically, intrathecally, intramuscularly, or subcutaneously.

85. A method of treating a subject with a BCMA-related disorder comprising administering aF053-0202PCT / 25-116-WO-PCTtherapeutically effective amount of the composition of claim 77 or the formulation of claim 78 to the subject, thereby treating the subject.

86. The method of claim 85, wherein the BCMA-related disorder comprises a hematological malignancy.

87. The method of claim 86, wherein the hematological malignancy comprises multiple myeloma, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), or Hodgkin lymphoma.

88. The method of claim 85, wherein the administering a therapeutically effective amount comprises administering intravesically, intravenously, intradermally, intraarterially, intraparenterally, intranodally, intralymphaticaly, intraperitoneally, intralesionally, intraprostaticaly, intravaginally, intrarectally, topically, intrathecally, intramuscularly, or subcutaneously.

Images