Baff-r bispecific t-cell engager antibody

Abstract

The present invention provides recombinant proteins (e.g., bispecific antibodies) capable of binding to BAFF-R. The recombinant proteins (e.g., bispecific antibodies) provided herein are particularly useful for the treatment of cancer and autoimmune diseases.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. provisional application 62 / 782,317 filed December 19, 2018, the entire text of which is incorporated herein by reference for all purposes.

[0003] Reference to the "sequence list," table, or computer program list appendix submitted as an ASCII file

[0004] The sequence list recorded in file 048440-715001WO_SEQUENCE_LISTING_ST25 was written on December 12, 2019, on a machine format IBM-PC, MS Windows operating system with 45,056 bytes, and is cited herein by reference. Background Technology

[0005] Antibody therapy is one of the most successful immunotherapies available in hospitals for treating hematological malignancies. A representative example is rituximab, which targets CD20 to induce cytotoxic effects against B-cell lymphoma. However, a major drawback associated with rituximab is the emergence of rituximab resistance, which is thought to be caused by the downregulation of CD20, preventing the antibody from binding to target cells. Therefore, there is a need in the industry for new therapeutic antibodies targeting alternative specific targets on malignant cells. The problem to be solved

[0006] The present invention provides a composition of BAFF-R bispecific antibodies and a method of using the same, thereby solving these and other problems in the industry. means of solving the problem

[0007] In one embodiment, a recombinant protein is provided, comprising: (i) a first antibody region capable of binding to an effector cell ligand; and (ii) a second antibody region comprising a light chain variable domain and a heavy chain variable domain. The light chain variable domain comprises CDR L1 presented in SEQ ID NO. 1, CDR L2 presented in SEQ ID NO. 2, and CDR L3 presented in SEQ ID NO. 3. The heavy chain variable domain comprises CDR H1 presented in SEQ ID NO. 4, CDR H2 presented in SEQ ID NO. 5, and CDR H3 presented in SEQ ID NO. 6.

[0008] In one embodiment, a recombinant protein is provided, comprising: (i) a first antibody region capable of binding to an effector cell ligand; and (ii) a second antibody region comprising a light chain variable domain and a heavy chain variable domain. The light chain variable domain comprises CDR L1 presented in SEQ ID NO. 38, CDR L2 presented in SEQ ID NO. 39, and CDR L3 presented in SEQ ID NO. 40. The heavy chain variable domain comprises (b) CDR H1 presented in SEQ ID NO. 41, CDR H2 presented in SEQ ID NO. 42, and CDR H3 presented in SEQ ID NO. 43.

[0009] In one embodiment, a pharmaceutical composition is provided comprising a therapeutically effective amount of recombinant protein and a pharmaceutically acceptable excipient, including an embodiment provided herein.

[0010] In one embodiment, a method for treating cancer in a subject requiring treatment is provided, comprising administering a therapeutically effective amount of a recombinant protein, including an embodiment thereof described herein, to the subject, thereby treating the cancer in said subject.

[0011] In another aspect, a method for treating an autoimmune disease in a subject requiring treatment is provided, comprising administering a therapeutically effective amount of a recombinant protein, including an embodiment thereof described herein, to the subject, thereby treating the autoimmune disease in said subject. Brief explanation of the drawing

[0012] FIGS. 1a and 1b. Illustrate the generation of a BAFF-R bispecific antibody (BAFF-R BiTE, a recombinant protein including embodiments thereof provided herein) by Knob-Hall technology. FIG. 1a. Schematic diagram of BAFF-R BiTE. FIG. 1b. Reduced and non-reduced gel states for BAFF-R BiTE (H90Bi). The control group included a control / unrelated BiTE (Control Bi, 4-4Bi) and a humanized BAFF-R antibody (e.g., H90 Ab is also referred to herein as "humanized C90", and is also referred to as "humanized C90-1", "humanized C90-2", "humanized C90-3", "C90", "C90-1", "C90-2", or "C90-3" depending on the structural frame sequence). Fig. 2. Binding specificity of bispecific antibodies. FACS histograms of wild-type mouse fibroblast L cells, engineered BAFF-R expressing L cells (B2D L cells), and T cells isolated from a healthy donor, bound to BAFF-R / CD3 bispecific antibody (H90 BiTE). Fig. 3. Specific cytotoxicity of the bispecific antibody against BAFF-R-expressing L cells. The plot represents the percentage of specific cytotoxicity calculated from the chromium release assay. The BAFF-R / CD3 bispecific antibody (H90 BiTE) was incubated with chromium-labeled target wild-type (left) or BAFF-R-expressing L cells (right) along with effector CD8 T cells isolated from a healthy single donor. The ratio of effector cells to isolated CD8 T cells was 10:1. The incubation time was 4 hours. Fig. 4. Cytotoxicity of bispecific antibodies against malignant B cell lines. The plot represents the percentage of specific cytotoxicity calculated from the chromium release assay against various malignant B cell lines as indicated. The ratio of effector cells to isolated CD8 T cells was 10:1. The incubation time was 4 hours. Fig. 5. Dose-dependent cytotoxicity of bispecific antibodies. The plot shows the percentage of specific cytotoxicity calculated by the chromium release assay for JeKo-1 mantle cell lymphoma and normal isolated B cells. The ratio of effector cells to isolated CD8 T cells was 10:1. The culture time was 4 hours. Fig. 6. In vivo Study Schedule. Time schedule of the treatment schedule of inoculating NSG mice with tumors and treating with BAFF-R BiTE. The schematic diagram shows the treatment schedule of the Z-138 MCL tumor model with administration at time points indicated by the horizontal line. Bioluminescence imaging is performed weekly, and mice are observed for up to 90 days after tumor inoculation. Fig. 7. Z-138 model treated with BAFF-R BiTE. Intravenous (IV) tumor inoculation with Z-138 MCL on day 0 (5 x 10 4 Bioluminescence imaging of the NSG mouse experimental group (n = 5 / experimental group) after dog cells / mouse. The experimental group (BAFF-R BiTE 300 μg) was 300 μg BAFF-R BiTE + 5 x 10 6 Dogs received T cells. The control group received 5 x 10 from the same donor. 6 Dogs were administered either T cells or saline (PBS). FIGS. 8a to 8f. FACS plots of BAFF-R BiTE antibody staining showing binding to maternal L cells (Fig. 8a) compared to BAFF-R positive L cells (B2D) (Fig. 8b) or donor T cells (Fig. 8c). Staining included a combination of secondary antibody and BAFF-R humanized antibody as controls for L cells, and a combination of secondary and CD3 antibodies as controls for T cells. Specific details for implementing the invention

[0013] I. Definition

[0014] Although various embodiments and aspects of the present invention have been illustrated and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided merely as examples. Without departing from the invention, numerous modifications, changes, and substitutions will come to mind for those skilled in the art. It should be understood that various alternatives to the embodiments of the invention described herein may be used to practice the invention.

[0015] Section titles used herein are for compositional purposes only and should not be construed as limiting the technical gist described. All documents or parts of documents cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are cited in their entirety for any purpose.

[0016] "Nucleic acid" refers to a nucleotide (e.g., deoxyribonucleotide or ribonucleotide) and a polymer in a single-, double-, or multi-stranded form or as a complement thereof; or a nucleoside (e.g., deoxyribonucleotide or ribonucleotide). In embodiments, "nucleic acid" does not include a nucleoside. The terms "polynucleotide," "oligonucleotide," "oligo," etc. refer to a linear nucleotide sequence in the ordinary, conventional sense. The term "nucleoside" refers to a glycosylamine containing a nucleobase and a pentose sugar (ribose or deoxyribose) in the ordinary, conventional sense. Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. The term "nucleotide" refers to a single unit of a polynucleotide, i.e., a monomer, in the ordinary, conventional sense. A nucleotide may be a ribonucleotide, a deoxyribonucleotide, or a modified version thereof. Examples of polynucleotides considered herein include single and double-stranded DNA, single and double-stranded RNA, and hybrid molecules having mixtures of single and double-stranded DNA and RNA. Nucleic acids, for example, examples of polynucleotides considered herein include any type of RNA, e.g., mRNA, siRNA, miRNA, and guide RNA, and any type of DNA, genomic DNA, plasmid DNA, and minicircle DNA, as well as fragments thereof. In the context of polynucleotides, the term "duplex" refers to a double strand in the ordinary, conventional sense. Nucleic acids may be linear or branched. For example, a nucleic acid may be a linear chain of nucleotides, or a nucleic acid may be branched, for example, thereby comprising one or more arms or branches of nucleotides. Optionally, branched nucleic acids branch repeatedly to form higher-order structures such as dendrimers.

[0017] Additionally, the above term encompasses known nucleotide analogs or modified backbone residues or linkers that have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to a reference nucleotide. Examples of such analogs include, but are not limited to, phosphoramidate, phosphorodithioate (also known as phosphorothioate having a double-bonded sulfur group replacing oxygen in a phosphate), phosphorodithioate, phosphonocarboxylic acid, phosphonocarboxylate, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or phosphodiester derivatives comprising an O-methylphosphoramidate linker (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), as well as modifications to nucleotide bases, such as 5-methylcytidine or pseudouridine; and includes peptide nucleic acid backbones and linkers. Other analog nucleic acids include those having a positive backbone, a non-ionic backbone, modified sugars, and a non-ribose backbone (e.g., phosphorodiamidate morpholino oligo or locked nucleic acid (LNA) as known in the industry), including those described in U.S. Patents US 5,235,033 and US 5,034,506 and Chapters 6 and 7 of ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, Revised Edition. Additionally, nucleic acids containing one or more carboxylic cyclic sugars are also included within one definition of nucleic acid. Modification of the ribose-phosphate backbone may be performed for various reasons, for example, to increase the stability and half-life of these molecules in a physiological environment or as probes on a biochip.A mixture of naturally occurring nucleic acids and analogs can be prepared, or alternatively, a mixture of different nucleic acid analogs and a mixture of naturally occurring nucleic acids and analogs can be prepared. In an embodiment, the linkers between nucleotides in DNA are phosphodiesters, phosphodiester derivatives, or a combination of both.

[0018] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as these amino acids that have been later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphorine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids—namely, hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. These analogs possess the same basic chemical structure as naturally occurring amino acids, although they have a modified R group (e.g., norleucine) or a modified peptide backbone. Amino acid mimics refer to chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids. The terms "non-naturally occurring amino acid" and "non-natural amino acid" refer to amino acid analogs, synthetic amino acids, and amino acid mimics that are not found in nature.

[0019] Amino acids may be referred to herein by three-character symbols commonly known herein or by one-character symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Likewise, nucleotides may be referred to by their commonly accepted single-character codes.

[0020] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to a specific nucleic acid sequence, a "conservatively modified variant" refers to a nucleic acid that encodes the same or essentially identical amino acid sequence. Due to the degenerate nature of the genetic code, many nucleic acid sequences will encode any given protein. For example, codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at any position where alanine is specified by a codon, the codon can be changed to any corresponding codon described without altering the encoded polypeptide. Such nucleic acid variations are "silencing variants," which are a type of conservatively modified variant. All nucleic acid sequences in this document that encode polypeptides also describe all possible silencing variants of the nucleic acid. Those skilled in the art will recognize that each codon of the nucleic acid (with the exception of AUG, which is generally the only codon for methionine, and TGG, which is generally the only codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, each silencing variant of the nucleic acid encoding the polypeptide is contained in each described sequence.

[0021] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single amino acid or a small percentage of amino acids within the encoded sequence constitute a "conservatively modified variant," wherein the alteration results in a substitution of amino acids with chemically similar amino acids. Tables of conservative substitutions providing functionally similar amino acids are widely known in the art. Such conservatively modified variants are added to, but are not excluded from, the polymorphic variants, interspecies homologs, and alleles of the present invention.

[0022] Each of the following eight groups contains amino acids that are conservative substitutions for one another:

[0023] 1) Alanine (A), Glycine (G);

[0024] 2) Aspartic acid (D), glutamic acid (E);

[0025] 3) Asparagine (N), Glutamine (Q);

[0026] 4) Arginine (R), Lysine (K);

[0027] 5) Isoleucine (I), leucine (L), methionine (M), valine (V);

[0028] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0029] 7) Serine (S), Threonine (T); and

[0030] 8) Cysteine ​​(C), Methionine (M)

[0031] (See, for example, Creighton, Proteins (1984)).

[0032] The “percentage of sequence identity” is determined by comparing two optimally aligned sequences against a comparison window, wherein a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to calculate the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying this result by 100 to calculate the percentage of sequence identity.

[0033] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "identical" percentage refers to two or more sequences or subsequences having a specific proportion of identical or identical amino acid residues or nucleotides (i.e., 60% identity relative to the entire polypeptide sequence of the present invention or individual domains of the polypeptide of the present invention, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity relative to a specific region, e.g., the entire polypeptide sequence of the present invention or individual domains of the polypeptide of the present invention) when compared and aligned with maximum correspondence relative to a comparison window or a specified region using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Furthermore, these sequences are referred to as "substantially identical." This definition may also refer to the complement of the test sequence. Optionally, the identity extends beyond a region of at least about 50 nucleotides in length, more preferably a region of 100 to 500 or 1000 or more nucleotides in length. The present invention comprises a polypeptide substantially identical to any of SEQ ID NOs 7 to 12 or SEQ ID NOs 44 to 49.

[0034] For sequence comparison, typically one sequence acts as a reference sequence to be compared with a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are defined. Default program parameters can be used, or alternative parameters can be specified. Next, the sequence comparison algorithm calculates the percentage of sequence identity for the test sequence compared to the reference sequence based on the program parameters.

[0035] The “comparison window” as used herein comprises, for example, a whole-length sequence in which the sequence can be compared with an equal number of reference sequences at adjacent positions after two sequences have been optimally aligned, or a reference for any number of segments at adjacent positions selected from a group consisting of 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides. Sequence alignment methods for comparison are widely known in the art. Optimal sequence alignment for comparison is, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman (1970) Adv. Appl. Math. 2: 482c), or by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch (1970) J. Mol. Biol. 48: 443), by Pearson and Lipman's similarity search method (Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85: 2444), by computerized execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA of the Wisconsin Genetics Software Package, Genetics Computing Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (e.g., Ausubel et al ..., see Current Protocols in Molecular Biology (supplemented in 1995)) may be implemented.

[0036] An example of an algorithm suitable for determining the percentages of sequence identity and sequence similarity is Altschul et al. (1977) Nuc. Acids Res. 25: 3389-3402 and Altschul et al.These are the BLAST and BLAST 2.0 algorithms described in (1990) J. Mol. Biol. 215: 403-410, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). These algorithms involve first identifying high-score sequence pairs (HSPs) by identifying short words of length W in the query sequence, which match or satisfy a positive threshold score T when aligned with words of the same length in the database sequence. T is the neighbor word score threshold (Altschul et al.These are referred to as ( , above). These initial neighbor word hits act as seeds to initiate a search for longer HSPs containing them. Word hits expand in both directions along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The expansion of word hits in each direction stops when the cumulative alignment score drops from its maximum reach value to a quantified X, when the cumulative score becomes zero or less due to alignment of one or more negative score residues, or when it reaches the end of the sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and a two-strand comparison as defaults. For amino acid sequences, the BLASTP program uses a word length of 3, an expectation (E) of 10, a BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915), an alignment (B) of 50, an expectation (E) of 10, M = 5, N = -4, and a two-strand comparison as defaults.

[0037] In addition, the BLAST algorithm performs a statistical analysis of similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which represents the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, in a comparison between a test nucleic acid and a reference nucleic acid, if the minimum sum probability is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0038] An indication that two nucleic acid sequences or polypeptides are substantially identical is, as described below, that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with the antibody generated against the polypeptide encoded by the second nucleic acid. Thus, the polypeptide is typically substantially identical to the second polypeptide, and, for example, the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that two molecules or their complements hybridize with each other under strict conditions, as described below. Additionally, another indication that two nucleic acid sequences are substantially identical is that the sequences can be amplified using the same primer.

[0039] An amino acid residue of an antibody is "corresponds" to a given residue when it occupies the same essential structural position as a given residue within the antibody. For example, a selected residue in a comparison antibody corresponds to position 48 (according to the Kavat numbering system described herein) when it occupies the same essential spatial or structural association as evaluated using applicable methods in the industry for Kavat position 48. For example, the comparison antibody may be aligned to maximum sequence homology with the antibody provided herein, and a position in the aligned comparison antibody aligned with Kavat position 48 may be determined to correspond thereto. Alternatively, instead of (or in addition to) the primary sequence alignment as described above, a three-dimensional structural alignment may also be used, for example, where the structure of the comparison antibody is aligned to maximum correspondence with the antibody provided herein and the overall structure is compared. In such cases, an amino acid occupying the same essential position as Kavat position 48 in the structural model may be referred to as corresponding.

[0040] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues, wherein the polymer may be conjugated to a moiety not composed of amino acids. The terms apply to amino acid polymers in which one or more amino acid residues are synthetic chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. "Fusion protein" refers to a chimeric protein encoding two or more separate protein sequences expressed by recombination as a single moiety. "Recombinant protein" refers to a protein that may be produced from the expression of recombinant DNA.

[0041] The terms "peptidyl" and "peptidyl moiety" refer to monovalent peptides.

[0042] Antibodies are large, complex molecules with a complex internal structure (molecular weight ~150,000 Da or about 1,320 amino acids). Natural antibody molecules contain two identical polypeptide chain pairs, each having one light chain and one heavy chain. Next, each light chain and heavy chain consists of two regions: a variable ("V") region involved in binding to target antigens and a constant ("C") region that interacts with other components of the immune system. The variable regions of the light and heavy chains combine in three-dimensional space to form a variable region that binds to antigens (e.g., receptors on the cell surface). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called complementarity determining regions ("CDRs"). In the antibody variable domain, six CDRs (three from the light chain and three from the heavy chain) come together in three-dimensional space to form the actual antibody binding site (paratope) that docks with the target antigen (epitope). The location and length of the CDR are clearly Kabat, E. et al. It was defined by Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1983, 1987. The variable region not included in the CDR is called the structural frame ("FR"), which forms the environment of the CDR.

[0043] As provided herein, "antibody variant" refers to a polypeptide capable of binding to an antigen and comprising one or more structural domains (e.g., light chain variable domain, heavy chain variable domain) of an antibody or a fragment thereof. Non-limiting examples of antibody variants include monodomain antibodies or nanobodyes, monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgG, scFv, bispecific diacetate, trispecific triacetate, scFv-Fc, miniacetate, IgNAR, V-NAR, hcIgG, VhH, or phpticetate. As provided herein, "pepticetate" refers to a peptide moiety attached (via a covalent or non-covalent linker) to the Fc domain of an antibody. Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camels. A general description of antibodies from camels and their variable regions and methods for their production, isolation and use can be found in International Patent Applications WO 97 / 49805 and WO 97 / 49805, the full text of which is cited herein by reference for all purposes. Likewise, antibodies from cartilaginous fish and their variable regions and methods for their production, isolation and use can be found in International Patent Application WO 2005 / 118629, the full text of which is cited herein by reference for all purposes.

[0044] As provided herein, the terms “Bispecific T cell engager (BiTE),” “BiTe,” or “Bispecific antibody” are used in the conventional sense widely known in the art and refer to a bispecific recombinant protein capable of binding to two different antigens, for example, simultaneously. In contrast to traditional monoclonal antibodies, a BiTE antibody consists of two independently different antibody regions (e.g., two single-chain variable fragments (scFv)), each of which binds to a different antigen. One antibody region can engage with effector cells (e.g., T cells) by binding to an effector cell-specific antigen (e.g., CD3 molecule), and the remaining antibody region can bind to target cells (e.g., cancer cells or autoimmune reactive cells) through a cell surface antigen (e.g., BAFF-R) expressed by said target cells. The binding of BiTE to two antigens will link effector cells (e.g., T cells) to target cells (e.g., tumor cells) and activate effector cells (e.g., T cells) through effector cell-specific antigen signaling (e.g., CD3 signaling). Next, the activated effector cells (e.g., T cells) will exert cytotoxic activity against target cells (e.g., tumor cells).

[0045] The terms "CDR L1," "CDR L2," and "CDR L3" provided herein refer to complementarity determining regions (CDR) 1, 2, and 3 of the variable light chain (L) of the antibody. In an embodiment, the variable light chain provided herein comprises CDR L1, CDR L2, and CDR L3 in the direction from the N-terminus to the C-terminus. Likewise, the terms "CDR H1," "CDR H2," and "CDR H3" provided herein refer to complementarity determining regions (CDR) 1, 2, and 3 of the variable heavy chain (H) of the antibody. In an embodiment, the variable heavy chain provided herein comprises CDR H1, CDR H2, and CDR H3 in the direction from the N-terminus to the C-terminus.

[0046] The terms "FR L1," "FR L2," "FR L3," and "FR L4" provided herein are used according to their common meaning in the art and refer to frame regions (FR) 1, 2, 3, and 4 of the variable light chain (L) of the antibody. In an embodiment, the variable light chain provided herein comprises FR L1, FR L2, FR L3, and FR L4 in the direction from the N-terminus to the C-terminus. The terms "FR H1," "FR H2," "FR H3," and "FR H4" provided herein are used according to their common meaning in the art and refer to frame regions (FR) 1, 2, 3, and 4 of the variable heavy chain (H) of the antibody. In an embodiment, the variable heavy chain provided herein comprises FR H1, FR H2, FR H3, and FR H4 in the direction from the N-terminus to the C-terminus.

[0047] The term "antibody" is used according to the meaning commonly known in the industry. Antibodies exist, for example, as intact immunoglobulins or as many well-characterized fragments produced by digestion by various peptidases. Thus, for example, pepsin digests antibodies under the disulfide bonds of the hinge region, and the antibody itself, by means of disulfide bonds V H -C H1 It produces F(ab)'2, a dimer of Fab, the light chain connected to it. F(ab)'2 can be reduced under mild conditions to cleave the disulfide bond at the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab that is part of the hinge region (see Fundamental Immunology (Paul ed., 3rd ed., 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, those skilled in the art may [describe] these fragments chemically or using recombinant DNA methodologies freshlyIt will be understood that they can be synthesized. Accordingly, as used herein, the term antibody also includes antibody fragments produced by modification of whole antibodies or newly synthesized antibodies using recombinant DNA methodology (e.g., single-stranded Fv) or antibodies identified using phage display libraries (e.g., see McCafferty et al., Nature 348: 552-554 (1990)).

[0048] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kD) and one "heavy chain" (about 50 to 70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids responsible primarily for antigen recognition. The terms variable light chain (VL) or light chain variable region and variable heavy chain (VH) or heavy chain variable region refer to these light chain and heavy chain regions, respectively. As used herein, the terms variable light chain (VL) and light chain variable region may be used interchangeably. As used herein, the terms variable heavy chain (VH) and heavy chain variable region may be used interchangeably. The Fc (i.e., fragment crystallizable region) is the "base" or "tail" of the immunoglobulin and typically consists of two heavy chains that contribute to two or three constant domains, depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response to a given antigen. The Fc region also binds to various cellular receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

[0049] A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, typically linked by a short linker peptide of 10 to about 25 amino acids. The linker is generally glycine-rich for flexibility, but may also be serine or threonine-rich for solubility. The linker may link the N-terminus of the VH to the C-terminus of the VL, or vice versa.

[0050] As provided herein, the term "antigen" refers to a molecule capable of binding to the antibody binding domain provided herein. As provided herein, the "antigen binding domain" is a region of an antibody that binds to an antigen (epitope). As described above, the antigen binding domain generally consists of one constant domain and one variable domain of each heavy chain and light chain (CH, CL, VH, and VL, respectively). A paratope or antigen binding site is formed at the N-terminus of the antigen binding domain. The two variable domains of the antigen binding domain typically bind to the epitope on the antigen.

[0051] For the production of monoclonal or polyclonal antibodies, any technique known in the art may be used (e.g., see Kohler & Milstein, Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96, Monoclonal Antibodies and Cancer Therapy (1985)). "Monoclonal" antibodies (mAbs) refer to antibodies derived from a single clone. The production technique for single-chain antibodies (US Patent US 4,946,778) may be applied to produce antibodies against the polypeptide of the present invention. Additionally, humanized antibodies may be expressed using other organisms, such as genetically modified mice or other mammals. Alternatively, phage display techniques may be used to identify antibodies and dimeric Fab fragments that specifically bind to selected antigens (e.g., McCafferty et al. , Nature 348: 552-554 (1990); marks et al. , see Biotechnology 10: 779-783 (1992)).

[0052] The epitope of an mAb is the antigenic region to which the mAb binds. When two antibodies competitively inhibit (block) binding to an antigen, they bind to the same or overlapping epitopes. That is, one antibody with a 1×, 5×, 10×, 20×, or 100× excess inhibits the binding of the remaining antibody by at least 30%, preferably 50%, 75%, 90%, or even 99%, as measured by a competitive binding assay (e.g., Junghans et al., see Cancer Res. 50: 1495 (1990)). Alternatively, if all amino acid mutations of the antigen that reduce or eliminate the binding of one antibody essentially reduce or eliminate the binding of the other antibody, the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody, the two antibodies have overlapping epitopes.

[0053] The "antibody region" as provided herein refers to a monovalent or multivalent protein moiety that forms part of a protein, including embodiments provided herein, and is capable of binding to an antigen (epitope). The antibody region provided herein may include a domain of an antibody or a fragment thereof (e.g., Fab). Thus, the antibody region may include a light chain variable domain (VL) and / or a heavy chain variable domain (VH). In an embodiment, the antibody region provided herein includes a light chain variable (VL) domain. In an embodiment, the antibody region is a light chain variable (VL) domain.

[0054] As described above, the "light chain variable (VL) domain" provided herein refers to a variable region of the light chain of an antibody, antibody variant, or fragment thereof. Likewise, the "heavy chain variable (VH) domain" provided herein refers to a variable region of the heavy chain of an antibody, antibody variant, or fragment thereof. The light chain variable domain and the heavy chain variable domain together form a paratope that binds to an antigen (epitope). The paratope or antigen binding site is formed at the N-terminus of the antibody, antibody variant, or fragment thereof. In an embodiment, the light chain variable (VL) domain comprises CDR L1, CDR L2, CDR L3 and FR L1, FR L2, FR L3, and FR L4 (framework regions) of the antibody light chain. In an embodiment, the heavy chain variable (VH) domain comprises CDR H1, CDR H2, CDR H3 and FR H1, FR H2, FR H3, and FR H4 (framework regions) of the antibody heavy chain. In an embodiment, the light chain variable (VL) domain and the light chain constant (CL) domain form part of the antibody light chain. In an embodiment, the heavy chain variable (VH) domain and the heavy chain constant (CH1) domain form part of the antibody heavy chain. In an embodiment, the heavy chain variable (VH) domain and one or more heavy chain constant (CH1, CH2, or CH3) domains form part of the antibody heavy chain. Thus, in an embodiment, the light chain variable (VL) domain forms part of the antibody. In an embodiment, the heavy chain variable (VH) domain forms part of the antibody. In an embodiment, the light chain variable (VL) domain forms part of the therapeutic antibody. In an embodiment, the heavy chain variable (VH) domain forms part of the therapeutic antibody. In an embodiment, the light chain variable (VL) domain forms part of the human antibody. In an embodiment, the heavy chain variable (VH) domain forms part of the human antibody. In an embodiment, the light chain variable (VL) domain forms part of the human antibody. In an embodiment, the heavy chain variable (VH) domain forms part of the humanized antibody.In an embodiment, the light chain variable (VL) domain forms part of a chimeric antibody. In an embodiment, the heavy chain variable (VH) domain forms part of a chimeric antibody. In an embodiment, the light chain variable (VL) domain forms part of an antibody fragment. In an embodiment, the heavy chain variable (VH) domain forms part of an antibody fragment. In an embodiment, the light chain variable (VL) domain forms part of an antibody variant. In an embodiment, the heavy chain variable (VH) domain forms part of an antibody variant. In an embodiment, the light chain variable (VL) domain forms part of Fab. In an embodiment, the heavy chain variable (VH) domain forms part of Fab. In an embodiment, the light chain variable (VL) domain forms part of scFv. In an embodiment, the heavy chain variable (VH) domain forms part of scFv. In an embodiment, the light chain variable (VL) domain forms part of a bispecific antibody. In an embodiment, the heavy chain variable (VH) domain forms part of the bispecific antibody.

[0055] The term "isolated," when applied to proteins, means that the protein is essentially free of other associated cellular components in its natural state. It may be in a dry or aqueous solution, but is preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemical techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The protein, which is the dominant species present in the product, is substantially purified. The term "purified" means that the protein essentially produces a single band on the electrophoretic gel. Specifically, this means that the protein is at least 85%, more preferably at least 95%, and most preferably at least 99% pure.

[0056] When referring to proteins or peptides, the terms “specifically (or selectively) binding” or “specifically (or selectively) immunoreactive” to antibodies refer to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biological agents. Thus, under specified immunoassay conditions, the specified antibody binds to the specific protein at least twice the background level and does not substantially bind to other proteins present in the sample in significant amounts. Typically, the specific or selective reaction will be at least twice the background level of the background signal or noise, and more typically 10 to 100 times or more the background level.

[0057] The “humanized antibody” is a genetically engineered antibody in which at least one CDR (or a functional fragment thereof) from a mouse antibody (“donor antibody”, which may be from a rat, hamster, or other non-human species) is conjugated onto a human antibody (“recipient antibody”). In an embodiment, one or more mouse CDRs are conjugated (e.g., all six mouse CDRs are conjugated). The sequence of the recipient antibody may be, for example, a mature human antibody sequence (or a fragment thereof), a common sequence of the human antibody sequence (or a fragment thereof), or a germline region sequence (or a fragment thereof). Thus, the humanized antibody may be an antibody having one or more CDRs from the donor antibody and a variable region frame (FR). The FR may form part of the constant region and / or variable region within the human antibody. Additionally, to maintain high binding affinity, amino acids in the human recipient sequence may be replaced with corresponding amino acids from the donor sequence, for example, where (1) an amino acid is in the CDR, or (2) an amino acid is in the human frame region (e.g., an amino acid immediately adjacent to one of the CDRs). See U.S. Patents US 5,530,101 and US 5,585,089, referenced herein, for detailed instructions on the production of humanized antibodies. While humanized antibodies often incorporate all six CDRs from mouse antibodies (e.g., as defined by Kavat, but often also include the hypervariable loop H1 as defined by Chotia), they may also be made with fewer mouse CDRs and / or less than the complete mouse CDR sequence (e.g., Pascalis et al ., J. Immunol. 169: 3076, 2002; Vajdos et al. , Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al. , Mol. Immunol. 36: 1079-1091, 1999; Tamura et al., Journal of Immunology, 164: 1432-1441, 2000).

[0058] Typically, the humanized antibody provided herein may comprise (i) a light chain variable region and human variable region frame comprising at least one CDR (often three CDRs) from a mouse antibody (also referred to herein as mouse CDR); and (ii) a heavy chain variable region comprising at least one CDR (often three CDRs) from a mouse antibody and a human variable region frame (FR). The light chain and heavy chain variable region frame (FR) may each be a mature human antibody variable region frame sequence (or a fragment thereof), a germline variable region frame sequence (or a fragment thereof) (combined with a J region sequence), or a common sequence of the human antibody variable region frame sequence (or a fragment thereof). In an embodiment, the humanized antibody comprises the light chain variable region described in (i) and the heavy chain variable region described in (ii) together with the light chain human constant region and the heavy chain human constant region.

[0059] Other approaches to designing humanized antibodies also include the methods of the aforementioned U.S. Patents US 5,530,101 and US 5,585,089, e.g., “superhumanization” (Tan et al. (see J. Immunol. 169: 1119, 2002 and U.S. Patent US 6,881,557) or the method of Studnicak et al. (Studnicak et al.It can be used to achieve the same results as in Protein Eng. 7: 805, 1994). Additionally, other approaches to produce genetically engineered and immunogenic mAbs include, for example, Vaswami et al., Annals of Allergy, Asthma and Immunology 81: 105, 1998; Roguska et al., Protein Eng. 9: 895, 1996; and “reshaping,” “hyperchimerization,” and veneering / resurfacement as described in U.S. Patents US 6,072,035 and US 5,639,641.

[0060] "Ligand" refers to a preparation capable of binding to a receptor molecule (e.g., antibody), such as a polypeptide or other molecule.

[0061] "Contact" is used in its ordinary, general sense and refers to a process that allows at least two distinct species (e.g., chemical compounds including biomolecules or cells) to come into sufficient proximity to react, interact, or make physical contact. However, it should be understood that the resulting reaction product may be produced from a reaction between added reagents, or directly from an intermediate from one or more added reagents that may be produced in a reaction mixture.

[0062] The term “contacting” may include allowing two species to react, interact, or come into physical contact, wherein said two species may be, for example, antibodies or BAFF-R proteins as described herein. In an embodiment, contacting includes, for example, allowing a humanized antibody described herein to interact with BAFF-R.

[0063] As used herein, "cell" refers to a cell that performs metabolic or other functions sufficient to preserve or replicate its genomic DNA. Cells may be identified by methods widely known in the art, including, for example, the presence of an intact membrane, staining by a specific dye, the ability to produce offspring, or, in the case of germ cells, the ability to combine with a second germ cell to produce viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, e.g., mammals, insects (e.g., spodoptera), and human cells.

[0064] As defined herein, the terms “inhibition,” “to inhibit,” “inhibiting,” etc., in relation to protein-inhibitor interactions, mean a negative effect (e.g., reduction) on the activity or function of a protein compared to the activity or function of the protein in the absence of an inhibitor. In an embodiment, inhibition refers to a reduction of a disease or symptoms of a disease (e.g., cancer or autoimmune disease). Accordingly, inhibition includes at least partially, partially or entirely blocking stimulation, reducing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signaling or enzymatic activity or the amount of a protein. Similarly, “inhibitor” is a compound or protein that inhibits activity by, for example, binding, partially or entirely blocking, reducing, preventing, delaying, inactivating, desensitizing, or down-regulating activity.

[0065] The terms “disease” or “pathological condition” refer to the condition or health condition of a patient or subject that can be treated by the compound, pharmaceutical composition, or method provided herein. In an embodiment, the disease is cancer (e.g., lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma). The disease may be an autoimmune disease, an inflammatory disease, cancer, an infectious disease, a metabolic disease, an embryological disease, a cardiovascular disease, a liver disease, an intestinal disease, an endocrine disease, a neurological disease, or other disease.

[0066] As used herein, the term "cancer" refers to all types of cancer, neoplasms, or malignant tumors found in mammals, including leukemia, lymphoma, melanoma, neuroendocrine tumors, carcinomas, and sarcomas. Exemplary cancers that can be treated by the compounds, pharmaceutical compositions, or methods provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, renal cancer, multiple myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple-negative, ER-positive, ER-negative, chemotherapy-resistant, Herceptin-resistant, HER2-positive, doxorubicin-resistant, tamoxifen-resistant, ductal carcinoma, lobular carcinoma, primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung carcinoma, squamous cell lung carcinoma, adenocarcinoma, giant cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma pleomorphic, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, It includes ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma.Additional examples include thyroid cancer, endocrine cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, gastric cancer, uterine cancer or medulloblastoma, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma pleomorphic, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumor, cancer, malignant pancreatic insulinoma, malignant carcinoid, urinary tract and bladder cancer, premature cutaneous lesion, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical cancer, neoplasm of endocrine or exocrine pancreas, medullary thyroid cancer, thyroid Includes medullary carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, papillary Paget's disease, phyllodes tumor, lobular carcinoma, ductal carcinoma, pancreatic astrocytoma, hepatoastrocytoma, or prostate cancer.

[0067] The term "leukemia" broadly refers to a progressive malignant disease of the blood-forming organs, generally characterized by the distorted proliferation and development of leukocytes and their precursors in the blood or bone marrow. Leukemia is generally clinically classified based on (1) the duration and characteristics of the acute or chronic disease; (2) the type of cells involved, such as bone marrow (myeloid), lymph (lymphoid), or monocytes; and (3) an increase or non-increase in the number of abnormal cells in the leukemic or non-leukemic (sub-leukemic) blood. Exemplary leukemias that can be treated by the compounds, pharmaceutical compositions, or methods provided herein are, for example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukocytic leukemia, basophilic leukemia, blastocyst leukemia, bovine leukemia, chronic myeloid leukemia, cutaneous leukemia, embryonic leukemia, eosinophilic leukemia, growth leukemia, hair cell leukemia, hematopoietic stem cell leukemia, hemoblastic leukemia, histocytic leukemia, stem cell leukemia, acute mononuclear leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphoplastic leukemia, lymphocytic leukemia, lymphosarcomatous leukemia, mast cell leukemia, megakaryocytic Includes leukemia, myeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Negeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, leader cell leukemia, Schilling leukemia, stem cell leukemia, sub-leukemic leukemia, or undifferentiated cell leukemia.

[0068] As used herein, the terms “metastasis,” “metastatic,” and “metastatic cancer” may be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ to another non-adjacent organ or part of the body. Cancer arises from a site of origin, e.g., the breast, which is referred to as the primary tumor, e.g., primary breast cancer. Some cancer cells from the primary tumor or site of origin acquire the ability to penetrate and infiltrate surrounding normal tissues in a local area, and / or the ability to penetrate the walls of the lymphatic or vascular systems to circulate through the system to other parts and tissues of the body. A second clinically detectable tumor formed from cancer cells of the primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to the original tumor. Thus, in the case of lung cancer metastasizing to the breast, the secondary tumor in the breast area consists of abnormal lung cells rather than abnormal breast cancer cells. The secondary tumor in the breast is referred to as metastatic lung cancer. Therefore, the term metastatic cancer refers to a disease in which the subject has a primary tumor or has one or more secondary tumors. The term non-metastatic cancer, or cancer that is not metastatic, refers to a disease in which the subject has a primary tumor but does not have one or more secondary tumors. For example, metastatic lung cancer refers to a disease in which the subject has a primary lung tumor or has a history of a primary lung tumor and has one or more secondary tumors at a second or multiple locations, for example, in the breast.

[0069] In the context of a substance or substance activity or function associated with a disease (e.g., cancer (e.g., leukemia, lymphoma, B-cell lymphoma, or multiple myeloma)), the term "associated" or "related to" means that the disease (e.g., cancer (e.g., leukemia, lymphoma, B-cell lymphoma, or multiple myeloma) is caused (wholly or partially) by the substance or substance activity or function, or that the symptoms of the disease are caused (wholly or partially) by it.

[0070] As used herein, the term "autoimmune disease" refers to a disease or pathological condition in which the immune system of a subject exhibits an abnormal immune response to a substance that does not normally induce an immune response in a healthy subject. Examples of autoimmune diseases that can be treated by the compounds, pharmaceutical compositions, or methods described herein include acute infectious encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic dysfunction, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune purpura thrombocytopenia (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal or neuronal neuropathy, Vallo's disease, Behcet's disease, bullous pemphigus-like disease, cardiomyopathy, Castleman's disease, celiac disease. Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, pemphigus scari / benign mucosal pemphigus scari, Crohn's disease, Cogan syndrome, cold agglutination sickness, congenital heart block, Coxsackie's cardiomyopathy, CREST disease, true mixed cryoglobulinemia, demyelinating neuropathy, herpetic dermatitis, dermatomyositis, Debick's disease (ocular neuritis), discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrotic alveolitis, giant cell arteritis (transient arteritis), giant cell cardiomyopathy, glomerulonephritis, Goodpascher syndrome, granulomatosis with polyangiitis (GPA) (previously (also known as Wegener's granulomatosis), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, gestational herpes, hypogammaglobulinemia, idiopathic purpura, thrombocytopenia (ITP), IgA nephropathy, IgG4-related scleroderma,Immunomodulatory lipoprotein, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes (Type 1 diabetes), pediatric myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytolytic vasculitis, lichen planus, lichen sclerosus, mungoid conjunctivitis, linear IgA disease (LAD), lupus (SLE), Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Muren ulcer, Mooka-Haberman disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, ophthalmic neuritis (Deviq's disease), neutropenia, pituitary pemphigoid-like vaginosis, optic neuritis, relapsing rheumatism, PANDAS (Streptococcus-associated pediatric autoimmune neuropsychiatric disorder), adrenal neoplasmic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsons-Needy-Turner syndrome, peripheral Uveitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosum, type 1, type 2, and type 3 autoimmune polygland syndrome, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrene, erythrocyte dysplasia vera, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dyskinesia, Reiter's syndrome, recurrent polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, rigid man syndrome, Includes subacute bacterial cardiomyopathy (SBE), Susac syndrome, sympathetic ophthalmitis, Takayasu's arteritis, transient arteritis / giant cell arteritis, thrombocytopenia purpura (TTP), Tolosa-Hunt syndrome, transmyelitis, diabetes mellitus type 1, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, bullous dermatitis, vitiligo, or Wegener's granulomatosis (i.e., granulomatosis with polyangiitis (GPA)).

[0071] "B cell" or "B lymphocyte" refers to the standard usage in the industry. B cells are lymphocytes, a type of white blood cell, that develop into antibody-producing plasma cells ("mature B cells"). "Immature B cells" are cells capable of developing into mature B cells. Generally, pro-B cells undergo immunoglobulin heavy chain rearrangement to become pre-B cells, and then undergo immunoglobulin light chain rearrangement to become immature B cells. Immature B cells include T1 and T2 B cells.

[0072] "T cells" or "T lymphocytes," as used herein, are a type of lymphocyte (a subtype of leukocyte) that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of T cell receptors on their cell surfaces. T cells include, for example, natural killer T (NKT) cells, cytotoxic T lymphocytes (CTL), regulatory T (Treg) cells, and helper T cells. Other types of T cells can be distinguished using T cell detectors.

[0073] "Memory T cells" are T cells that have previously encountered, responded to their ethnic antigens during a previous infection, or encountered cancer or a previous vaccination. In a second encounter with an ethnic antigen, memory T cells can proliferate (divide) to trigger a more rapid and powerful immune response than when the immune system first responded to the pathogen.

[0074] "Regulatory T cells" or "suppressor T cells" are lymphocytes that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases.

[0075] As provided herein, the term "effector cell ligand" refers to a cell surface molecule expressed on effector cells of the immune system (e.g., cytotoxic T cells, helper T cells, B cells, natural killer cells). When a first antibody region binds to an effector cell ligand expressed on an effector cell, the effector cell is activated and can perform its function (e.g., selective killing or eradication of malignant cells, infected cells, or otherwise unhealthy cells). In an embodiment, the effector cell ligand is the CD3 protein. In an embodiment, the effector cell ligand is the CD16 protein. In an embodiment, the effector cell ligand is the CD32 protein. In an embodiment, the effector cell ligand is the NKp46 protein. As provided herein, the first antibody region may be an antibody, an antibody variant, a fragment of an antibody, or a fragment of an antibody variant.

[0076] As referred to herein, “CD3 protein” comprises any recombination or natural form of differentiation cluster 3 (CD3) protein, or a CD3 complex mediating signal transduction, and comprises variants or homologs thereof that retain CD3 complex activity (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to CD3). In some embodiments, said variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a continuous sequence of 50, 100, 150, or 200 amino acids) compared to the natural CD3 protein in the CD3 complex.

[0077] As referred to herein, "CD16 protein" comprises any recombinant or naturally occurring form of differentiation cluster 16 (CD16) protein, also known as low-affinity immunoglobulin gamma Fc region receptor III-A, or variants or homologues thereof that retain CD16 activity (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to CD16). In some embodiments, said variants or homologues have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a continuous sequence of 50, 100, 150, or 200 amino acids) compared to the naturally occurring CD16 protein. In an embodiment, the CD16 protein is substantially identical to the protein identified by uniprotot reference number P08637, or to a variant or homologue having substantial identity therewith.

[0078] As referred to herein, “CD32 protein” comprises any recombinant or naturally occurring form of differentiation cluster 32 (CD32) protein, also known as low-affinity immunoglobulin gamma Fc region receptor II-A, or variants or homologues thereof that retain CD16 activity (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to CD32). In some embodiments, said variants or homologues have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a continuous sequence of 50, 100, 150, or 200 amino acids) compared to the naturally occurring CD32 protein. In an embodiment, the CD32 protein is substantially identical to the protein identified by uniprot reference number P12318, or to a variant or homologue having substantial identity therewith.

[0079] As referred to herein, "NKp46 protein" comprises any recombinant or naturally occurring form of NKp46 protein, also known as natural cytotoxicity trigger receptor 1, or variants or homologues thereof that retain NKp46 activity (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to NKp46). In some embodiments, said variants or homologues have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a continuous sequence of 50, 100, 150, or 200 amino acids) compared to the naturally occurring NKp46 protein. In an embodiment, the NKp46 protein is substantially identical to the protein identified by uniprot reference number O76036, or to a variant or homologue having substantial identity with it.

[0080] "BAFF-R," "BAFF receptor," or "BAFF-R protein" as referred to herein include any recombinant or naturally occurring form of the B-cell activator receptor (BAFF-R), also known as tumor necrosis factor receptor superfamily member 13C (TNFRSF13C), or variants or homologues thereof that retain BAFF-R activity (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to BAFF-R). In some embodiments, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a continuous sequence of 50, 100, 150, or 200 amino acids) compared to the naturally occurring BAFF-R protein. In an embodiment, the BAFF-R protein is substantially identical to the protein identified by Uniprot reference number Q96RJ3, or a variant or homologue having substantial identity therewith. In an embodiment, the BAFF-R protein is substantially identical to the protein identified by Uniprot reference number Q9D8D0, or a variant or homologue having substantial identity therewith. In an embodiment, the BAFF-R protein is substantially identical to the protein identified by NCBI reference number GI:16445027, or a variant or homologue having substantial identity therewith. In an embodiment, the BAFF-R protein is substantially identical to the protein identified by NCBI reference number GI:16306481, or to a variant or homologue having substantial identity with it.

[0081] II. Recombinant Protein Composition

[0082] In particular, the present invention provides a recombinant protein (e.g., a bispecific antibody) capable of binding to BAFF-A and an antigen (e.g., CD3) expressed on effector cells. The recombinant protein (e.g., a bispecific antibody) comprises (i) a first antibody region capable of binding to an effector cell ligand (e.g., CD3); and (ii) a second antibody region capable of binding to a second antibody antigen (e.g., human BAFF-R protein), and binds to the human BAFF-R protein with high affinity and specificity. Simultaneous binding of the recombinant protein (e.g., a bispecific antibody) provided herein to an antigen on effector cells (e.g., CD3 on effector T cells) and to BAFF-R on B cells induces apoptosis of effector cells (e.g., T cells) and malignant or autoreactive B cells. Accordingly, the compositions and methods provided herein may be used, in particular, for the treatment of cancer (e.g., B-cell malignancies) or autoimmune diseases. For the purposes of the present invention, a second antibody region including an embodiment provided herein comprises any BAFF-R specific antibody described in published international patent application PCT / US2017 / 036178, which is incorporated herein by reference for all purposes. For example, the second antibody region may comprise any humanized BAFF-R antibody referred herein as “H90 Ab”, “humanized C90”, “humanized C90-1”, “humanized C90-2”, “humanized C90-3”, “C90”, “C90-1”, “C90-2”, or “C90-3”.

[0083] In one embodiment, a recombinant protein is provided, comprising: (i) a first antibody region capable of binding to an effector cell ligand (e.g., CD3); and (ii) a second antibody region comprising a light chain variable domain and a heavy chain variable domain. The light chain variable domain comprises CDR L1 presented in SEQ ID NO. 1, CDR L2 presented in SEQ ID NO. 2, and CDR L3 presented in SEQ ID NO. 3. The heavy chain variable domain comprises CDR H1 presented in SEQ ID NO. 4, CDR H2 presented in SEQ ID NO. 5, and CDR H3 presented in SEQ ID NO. 6. In an embodiment, the second antibody region binds to a BAFF-R protein or a fragment thereof. Where a CDR demonstrated herein is "presented" by a sequence number (e.g., sequence numbers 1, 2, 3, 4, 5, 6, 38, 38, 40, 41, 42, 43), the CDR has the sequence referred to by the said sequence number. Generally, where a sequence is presented as the sequence of a specific sequence number, said sequence has the sequence of the sequence referred to by said sequence number.

[0084] The recombinant protein provided herein may comprise light chain and heavy chain variable regions derived from different antibody clones (e.g., C90-1, C90-2, C90-3, C55-1, C55-2, C55-3), which include the same CDR and different FRs (e.g., C90-1, C90-2 and C90-3) or different CDRs (e.g., CDR from the C90 clone and CDR from the C55 clone). The second antibody region of the recombinant protein including embodiments provided herein may be scFv. Where the second antibody region is scFv, the second antibody region may be designated as H90, C90-1, C90-2, or C90-3, and includes CDR L1 presented in SEQ ID NO. 1, CDR L2 presented in SEQ ID NO. 2, CDR L3 presented in SEQ ID NO. 3, CDR H1 presented in SEQ ID NO. 4, CDR H2 presented in SEQ ID NO. 5, and CDR H3 presented in SEQ ID NO. 6. In an embodiment, the second antibody region is scFv and may be designated as C55-1, C55-2, or C55-3, and includes CDR L1 presented in SEQ ID NO. 38, CDR L2 presented in SEQ ID NO. 39, CDR L3 presented in SEQ ID NO. 40, CDR H1 presented in SEQ ID NO. 41, CDR H2 presented in SEQ ID NO. 42, and CDR H3 presented in SEQ ID NO. 43.

[0085] In an embodiment, the light chain variable domain comprises the sequence of SEQ ID NO. 7, SEQ ID NO. 8, or SEQ ID NO. 9. In an embodiment, the light chain variable domain comprises the sequence of SEQ ID NO. 7. In an embodiment, the light chain variable domain comprises the sequence of SEQ ID NO. 8. In an embodiment, the light chain variable domain comprises the sequence of SEQ ID NO. 9. In an embodiment, the light chain variable domain is the sequence of SEQ ID NO. 7. In an embodiment, the light chain variable domain is the sequence of SEQ ID NO. 8. In an embodiment, the light chain variable domain is the sequence of SEQ ID NO. 9.

[0086] In an embodiment, the heavy chain variable domain comprises the sequence of SEQ ID NO. 10, SEQ ID NO. 11, or SEQ ID NO. 12. In an embodiment, the heavy chain variable domain comprises the sequence of SEQ ID NO. 10. In an embodiment, the heavy chain variable domain comprises the sequence of SEQ ID NO. 11. In an embodiment, the heavy chain variable domain comprises the sequence of SEQ ID NO. 12. In an embodiment, the heavy chain variable domain is the sequence of SEQ ID NO. 10. In an embodiment, the heavy chain variable domain is the sequence of SEQ ID NO. 11. In an embodiment, the heavy chain variable domain is the sequence of SEQ ID NO. 12.

[0087] In an embodiment, the light chain variable domain comprises FR L1 presented in SEQ ID NO. 13, FR L2 presented in SEQ ID NO. 14, FR L3 presented in SEQ ID NO. 15, and FR L4 presented in SEQ ID NO. 16. In an embodiment, the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 25, FR H2 presented in SEQ ID NO. 26, FR H3 presented in SEQ ID NO. 27, and FR H4 presented in SEQ ID NO. 28.

[0088] In an embodiment, the light chain variable domain comprises FR L1 presented in SEQ ID NO. 17, FR L2 presented in SEQ ID NO. 18, FR L3 presented in SEQ ID NO. 19, and FR L4 presented in SEQ ID NO. 20. In an embodiment, the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 29, FR H2 presented in SEQ ID NO. 30, FR H3 presented in SEQ ID NO. 31, and FR H4 presented in SEQ ID NO. 32.

[0089] In an embodiment, the light chain variable domain comprises FR L1 presented in SEQ ID NO. 21, FR L2 presented in SEQ ID NO. 22, FR L3 presented in SEQ ID NO. 23, and FR L4 presented in SEQ ID NO. 24. In an embodiment, the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 33, FR H2 presented in SEQ ID NO. 34, FR H3 presented in SEQ ID NO. 35, and FR H4 presented in SEQ ID NO. 36.

[0090] In an embodiment, the second antibody region comprises the light chain variable region of SEQ ID NO. 7 and the heavy chain variable region of SEQ ID NO. 10. In an embodiment, the second antibody region is C90-1. In a further embodiment, the second antibody region comprises the light chain variable region of SEQ ID NO. 7 and the heavy chain variable region of SEQ ID NO. 10.

[0091] In an embodiment, the second antibody region comprises the light chain variable region of SEQ ID NO. 8 and the heavy chain variable region of SEQ ID NO. 11. In an embodiment, the second antibody region is C90-2. In a further embodiment, the second antibody region comprises the light chain variable region of SEQ ID NO. 8 and the heavy chain variable region of SEQ ID NO. 11.

[0092] In an embodiment, the second antibody region comprises the light chain variable region of SEQ ID NO. 9 and the heavy chain variable region of SEQ ID NO. 12. In an embodiment, the second antibody region is C90-3. In a further embodiment, the second antibody region comprises the light chain variable region of SEQ ID NO. 9 and the heavy chain variable region of SEQ ID NO. 12.

[0093] In one embodiment, a recombinant protein is provided, comprising: (i) a first antibody region capable of binding to an effector cell ligand; and (ii) a second antibody region comprising (a) a light chain variable domain and a heavy chain variable domain. The light chain variable domain comprises CDR L1 presented in SEQ ID NO. 38, CDR L2 presented in SEQ ID NO. 39, and CDR L3 presented in SEQ ID NO. 40. The heavy chain variable domain comprises CDR H1 presented in SEQ ID NO. 41, CDR H2 presented in SEQ ID NO. 42, and CDR H3 presented in SEQ ID NO. 43.

[0094] In an embodiment, the light chain variable domain comprises the sequence of SEQ ID NO. 44, SEQ ID NO. 45, or SEQ ID NO. 46. In an embodiment, the light chain variable domain comprises the sequence of SEQ ID NO. 44. In an embodiment, the light chain variable domain comprises the sequence of SEQ ID NO. 45. In an embodiment, the light chain variable domain comprises the sequence of SEQ ID NO. 46. In an embodiment, the light chain variable domain is the sequence of SEQ ID NO. 44. In an embodiment, the light chain variable domain is the sequence of SEQ ID NO. 45. In an embodiment, the light chain variable domain is the sequence of SEQ ID NO. 46.

[0095] In an embodiment, the heavy chain variable domain comprises the sequence of SEQ ID NO. 47, SEQ ID NO. 48, or SEQ ID NO. 49. In an embodiment, the heavy chain variable domain comprises the sequence of SEQ ID NO. 47. In an embodiment, the heavy chain variable domain comprises the sequence of SEQ ID NO. 48. In an embodiment, the heavy chain variable domain comprises the sequence of SEQ ID NO. 49. In an embodiment, the heavy chain variable domain is the sequence of SEQ ID NO. 47. In an embodiment, the heavy chain variable domain is the sequence of SEQ ID NO. 48. In an embodiment, the heavy chain variable domain is the sequence of SEQ ID NO. 49.

[0096] In an embodiment, the light chain variable domain comprises FR L1 presented in SEQ ID NO. 50, FR L2 presented in SEQ ID NO. 51, FR L3 presented in SEQ ID NO. 52, and FR L4 presented in SEQ ID NO. 53. In an embodiment, the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 62, FR H2 presented in SEQ ID NO. 63, FR H3 presented in SEQ ID NO. 64, and FR H4 presented in SEQ ID NO. 65.

[0097] In an embodiment, the light chain variable domain comprises FR L1 presented in SEQ ID NO. 54, FR L2 presented in SEQ ID NO. 55, FR L3 presented in SEQ ID NO. 56, and FR L4 presented in SEQ ID NO. 57. In an embodiment, the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 66, FR H2 presented in SEQ ID NO. 67, FR H3 presented in SEQ ID NO. 68, and FR H4 presented in SEQ ID NO. 69.

[0098] In an embodiment, the light chain variable domain comprises FR L1 presented in SEQ ID NO. 58, FR L2 presented in SEQ ID NO. 59, FR L3 presented in SEQ ID NO. 60, and FR L4 presented in SEQ ID NO. 61. In an embodiment, the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 70, FR H2 presented in SEQ ID NO. 71, FR H3 presented in SEQ ID NO. 72, and FR H4 presented in SEQ ID NO. 73.

[0099] In an embodiment, the second antibody region comprises the light chain variable region of SEQ ID NO. 44 and the heavy chain variable region of SEQ ID NO. 47. In an embodiment, the second antibody region is C55-1. In a further embodiment, the second antibody region comprises the light chain variable region of SEQ ID NO. 44 and the heavy chain variable region of SEQ ID NO. 47.

[0100] In an embodiment, the second antibody region comprises the light chain variable region of SEQ ID NO. 45 and the heavy chain variable region of SEQ ID NO. 48. In an embodiment, the second antibody region is C55-2. In a further embodiment, the second antibody region comprises the light chain variable region of SEQ ID NO. 45 and the heavy chain variable region of SEQ ID NO. 48.

[0101] In an embodiment, the second antibody region comprises the light chain variable region of SEQ ID NO. 46 and the heavy chain variable region of SEQ ID NO. 49. In an embodiment, the second antibody region is C55-3. In a further embodiment, the second antibody region comprises the light chain variable region of SEQ ID NO. 46 and the heavy chain variable region of SEQ ID NO. 49.

[0102] The positions of CDR and FR are in the Kabat numbering system (Kabat et al.It can be defined by Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, US Government Printing Office (1991). Similarly, the positions occupied by individual residues within the light or heavy chain of an antibody can be defined by the Kavat numbering system. Thus, the positions of residues required for binding within the light and heavy chains of a humanized antibody can be defined by the positions of residues according to the Kavat numbering system widely known in the art. As described above, the humanized antibody may be an antibody having a CDR from a donor antibody (e.g., mouse) and a variable region frame (FR) from a human antibody. The frame region (FR) is referred to as holding the CDR in place in the humanized antibody. Proceeding from the amino-terminus, these regions are designated as FR L1, FR L2, FR L3, and FR L4 for the light chain, and as FR H1, FR H2, FR H3, and FR H4 for the heavy chain, respectively. Surprisingly, the present invention provides a recombinant protein (e.g., a bispecific antibody) comprising one or more residues within a frame region important for epitope binding in a second antibody region. Frame region residues involved in (or important for) epitope binding (e.g., BAFF-R binding) are referred to herein as binding frame region residues. Binding frame region residues may be present in the frame region of a light chain variable domain (i.e., FR L1, FR L2, FR L3, FR L4) or in the frame region of a heavy chain variable domain (i.e., FR H1, FR H2, FR H3, FR H4). Binding frame region residues present in the FR L3 region of the light chain are referred to herein as FR L3 binding frame region residues. Accordingly, binding frame region residues present in the FR H3 region of the heavy chain are referred to herein as FR H3 binding frame region residues.

[0103] In an embodiment, the second antibody region comprises at least one binding structure frame region residue. In an embodiment, the light chain variable domain comprises at least one binding structure frame region residue. In an embodiment, the light chain variable domain comprises one or more FR L1, FR L2, FR L3, or FR L4 binding structure frame region residues. In an embodiment, the light chain variable domain comprises one or more FR L1 binding structure frame region residues. In an embodiment, the light chain variable domain comprises one or more FR L2 binding structure frame region residues. In an embodiment, the light chain variable domain comprises one or more FR L3 binding structure frame region residues. In an embodiment, the light chain variable domain comprises one or more FR L4 binding structure frame region residues. In an embodiment, the heavy chain variable domain comprises one or more FR H1, FR H2, FR H3, or FR H4 binding structure frame region residues. In an embodiment, the heavy chain variable domain comprises one or more FR H1 binding structure frame region residues. In an embodiment, the heavy chain variable domain comprises one or more FR H2 bonding structure frame region residues. In an embodiment, the heavy chain variable domain comprises one or more FR H3 bonding structure frame region residues. In an embodiment, the heavy chain variable domain comprises one or more FR H4 bonding structure frame region residues.

[0104] In an embodiment, the light chain variable domain comprises at least one bonding structured frame region residue (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more residues), and the heavy chain variable domain comprises one or more bonding structured frame region residues (e.g., 1, 2, 3, It includes 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 or more residues. The positions of the binding structure frame region residues within the antibody or antibody region can be defined by a Kavat numbering system similar to the positions of the CDR residues.

[0105] In an embodiment, the light chain variable domain comprises serine at a position corresponding to KAVATS position 7. In an embodiment, the light chain variable domain comprises proline at a position corresponding to KAVATS position 8. In an embodiment, the light chain variable domain comprises valine at a position corresponding to KAVATS position 15. In an embodiment, the light chain variable domain comprises threonine at a position corresponding to KAVATS position 22. In an embodiment, the light chain variable domain comprises glutamine at a position corresponding to KAVATS position 24. In an embodiment, the light chain variable domain comprises glycine at a position corresponding to KAVATS position 41. In an embodiment, the light chain variable domain comprises lysine at a position corresponding to KAVATS position 42. In an embodiment, the light chain variable domain comprises alanine at a position corresponding to KAVATS position 43. In an embodiment, the light chain variable domain comprises proline at a position corresponding to KAVATS position 44. In an embodiment, the light chain variable domain comprises threonine at a position corresponding to KAVATS position 56. In an embodiment, the light chain variable domain comprises threonine at a position corresponding to Kavat position 72. In an embodiment, the light chain variable domain comprises phenylalanine at a position corresponding to Kavat position 73. In an embodiment, the light chain variable domain comprises glutamine at a position corresponding to Kavat position 79. In an embodiment, the light chain variable domain comprises valine at a position corresponding to Kavat position 104.

[0106] In an embodiment, the light chain variable domain comprises serine at the position corresponding to kabat position 7, proline at the position corresponding to kabat position 8, valine at the position corresponding to kabat position 15, threonine at the position corresponding to kabat position 22, glutamine or serine at the position corresponding to kabat position 24, glycine at the position corresponding to kabat position 41, lysine at the position corresponding to kabat position 42, alanine or threonine at the position corresponding to kabat position 43, proline at the position corresponding to kabat position 44, threonine at the position corresponding to kabat position 56, threonine at the position corresponding to kabat position 72, phenylalanine or lysine at the position corresponding to kabat position 73, glutamine at the position corresponding to kabat position 79, or valine at the position corresponding to kabat position 104.

[0107] In an embodiment, the light chain variable domain comprises serine at the position corresponding to kabat position 7, proline at the position corresponding to kabat position 8, valine at the position corresponding to kabat position 15, threonine at the position corresponding to kabat position 22, glutamine or serine at the position corresponding to kabat position 24, glycine at the position corresponding to kabat position 41, lysine at the position corresponding to kabat position 42, alanine or threonine at the position corresponding to kabat position 43, proline at the position corresponding to kabat position 44, threonine at the position corresponding to kabat position 56, threonine at the position corresponding to kabat position 72, phenylalanine or lysine at the position corresponding to kabat position 73, glutamine at the position corresponding to kabat position 79, and valine at the position corresponding to kabat position 104.

[0108] In an embodiment, the light chain variable domain comprises a binding structure frame region residue that is serine at the position corresponding to kabat position 7, proline at the position corresponding to kabat position 8, valine at the position corresponding to kabat position 15, threonine at the position corresponding to kabat position 22, glutamine or serine at the position corresponding to kabat position 24, glycine at the position corresponding to kabat position 41, lysine at the position corresponding to kabat position 42, alanine or threonine at the position corresponding to kabat position 43, proline at the position corresponding to kabat position 44, threonine at the position corresponding to kabat position 56, threonine at the position corresponding to kabat position 72, phenylalanine or lysine at the position corresponding to kabat position 73, glutamine at the position corresponding to kabat position 79, or valine at the position corresponding to kabat position 104.

[0109] In an embodiment, the heavy chain variable domain comprises threonine or alanine at a position corresponding to kabat position 10. In an embodiment, the heavy chain variable domain comprises lysine at a position corresponding to kabat position 11. In an embodiment, the heavy chain variable domain comprises valine at a position corresponding to kabat position 12. In an embodiment, the heavy chain variable domain comprises threonine at a position corresponding to kabat position 15. In an embodiment, the heavy chain variable domain comprises threonine at a position corresponding to kabat position 19. In an embodiment, the heavy chain variable domain comprises threonine at a position corresponding to kabat position 23. In an embodiment, the heavy chain variable domain comprises proline at a position corresponding to kabat position 41. In an embodiment, the heavy chain variable domain comprises alanine at a position corresponding to kabat position 44. In an embodiment, the heavy chain variable domain comprises proline or threonine at a position corresponding to kabat position 61. In an embodiment, the heavy chain variable domain contains arginine at a position corresponding to KAVATS position 66. In an embodiment, the heavy chain variable domain contains threonine at a position corresponding to KAVATS position 70. In an embodiment, the heavy chain variable domain contains lysine at a position corresponding to KAVATS position 75. In an embodiment, the heavy chain variable domain contains valine at a position corresponding to KAVATS position 79. In an embodiment, the heavy chain variable domain contains threonine at a position corresponding to KAVATS position 81. In an embodiment, the heavy chain variable domain contains methionine at a position corresponding to KAVATS position 82. In an embodiment, the heavy chain variable domain contains asparagine at a position corresponding to KAVATS position 82B. In an embodiment, the heavy chain variable domain contains methionine at a position corresponding to KAVATS position 82C. In an embodiment, the heavy chain variable domain contains proline at a position corresponding to KAVATS position 84. In an example embodiment, the heavy chain variable domain contains valine at a position corresponding to kabat position 85.In an embodiment, the heavy chain variable domain comprises lysine at a position corresponding to kabat position 108. In an embodiment, the heavy chain variable domain comprises valine at a position corresponding to kabat position 109.

[0110] In an embodiment, the heavy chain variable domain comprises threonine or alanine at the position corresponding to kabat position 10, lysine at the position corresponding to kabat position 11, valine at the position corresponding to kabat position 12, threonine at the position corresponding to kabat position 15, threonine at the position corresponding to kabat position 19, threonine at the position corresponding to kabat position 23, proline at the position corresponding to kabat position 41, alanine at the position corresponding to kabat position 44, serine or threonine at the position corresponding to kabat position 61, alanine at the position corresponding to kabat position 66, threonine at the position corresponding to kabat position 70, lysine at the position corresponding to kabat position 75, valine at the position corresponding to kabat position 79, threonine or lysine at the position corresponding to kabat position 81, methionine at the position corresponding to kabat position 82, asparagine at the position corresponding to kabat position 82B, and kabat position It includes methionine at the position corresponding to 82C, proline at the position corresponding to 84 Kavat positions, valine at the position corresponding to 85 Kavat positions, lysine at the position corresponding to 108 Kavat positions, or valine at the position corresponding to 109 Kavat positions.

[0111] In an embodiment, the heavy chain variable domain comprises threonine or alanine at the position corresponding to kabat position 10, lysine at the position corresponding to kabat position 11, valine at the position corresponding to kabat position 12, threonine at the position corresponding to kabat position 15, threonine at the position corresponding to kabat position 19, threonine at the position corresponding to kabat position 23, proline at the position corresponding to kabat position 41, alanine at the position corresponding to kabat position 44, serine or threonine at the position corresponding to kabat position 61, alanine at the position corresponding to kabat position 66, threonine at the position corresponding to kabat position 70, lysine at the position corresponding to kabat position 75, valine at the position corresponding to kabat position 79, threonine or lysine at the position corresponding to kabat position 81, methionine at the position corresponding to kabat position 82, asparagine at the position corresponding to kabat position 82B, and kabat position It includes methionine at the position corresponding to 82C, proline at the position corresponding to 84 Kavat positions, valine at the position corresponding to 85 Kavat positions, lysine at the position corresponding to 108 Kavat positions, and valine at the position corresponding to 109 Kavat positions.

[0112] In an embodiment, the heavy chain variable domain comprises threonine or alanine at the position corresponding to kabat position 10, lysine at the position corresponding to kabat position 11, valine at the position corresponding to kabat position 12, threonine at the position corresponding to kabat position 15, threonine at the position corresponding to kabat position 19, threonine at the position corresponding to kabat position 23, proline at the position corresponding to kabat position 41, alanine at the position corresponding to kabat position 44, serine or threonine at the position corresponding to kabat position 61, alanine at the position corresponding to kabat position 66, threonine at the position corresponding to kabat position 70, lysine at the position corresponding to kabat position 75, valine at the position corresponding to kabat position 79, threonine or lysine at the position corresponding to kabat position 81, methionine at the position corresponding to kabat position 82, asparagine at the position corresponding to kabat position 82B, and kabat position It includes binding structure frame region residues that are methionine at the position corresponding to 82C, proline at the position corresponding to 84 Kavat positions, valine at the position corresponding to 85 Kavat positions, lysine at the position corresponding to 108 Kavat positions, or valine at the position corresponding to 109 Kavat positions.

[0113] A recombinant protein (e.g., a bispecific antibody) comprising an embodiment thereof provided herein may comprise one or more Fab' fragments. A recombinant protein (e.g., a bispecific antibody) comprising an embodiment thereof provided herein may comprise a first antibody region and a second antibody region, wherein the first antibody region may be a first Fab' fragment or the second antibody region may be a second Fab' fragment. Where the recombinant protein (e.g., a bispecific antibody) provided herein comprises a Fab' fragment, the recombinant protein may comprise a first antibody binding region and a second antibody binding region, each independently comprising a heavy chain (e.g., including constant and variable regions) and a light chain (e.g., including constant and variable regions). The Fab' fragment comprises a humanized heavy chain (e.g., including constant and variable regions) and a humanized light chain (e.g., including constant and variable regions). Accordingly, in an embodiment, the first antibody region is the first Fab' fragment, or the second antibody region is the second Fab' fragment. In an embodiment, the first antibody region is the first Fab' fragment. In an embodiment, the second antibody region is the second Fab' fragment. In an embodiment, the first antibody region is the first Fab' fragment, and the second antibody region is the second Fab' fragment.

[0114] The first or second antibody region may be the first scFv or the second scFv, respectively. A single-chain antibody comprises a variable light chain and a variable heavy chain. Those skilled in the art will immediately recognize that, in contrast to immunoglobulin antibodies which comprise two identical polypeptide chain pairs, each having one light chain and one heavy chain, a single-chain antibody comprises a single light chain and a single heavy chain. Each light chain and heavy chain is further composed of two regions: a variable ("V") region (i.e., the variable light chain and variable heavy chain) involved in binding to a target antigen, and a constant ("C") region that interacts with other components of the immune system. The variable light chain and variable heavy chain of a single-chain antibody may be connected via a linker peptide. In an embodiment, the linker peptide comprises the sequence presented in SEQ ID NO. 37. In an embodiment, the linker peptide is the sequence presented in SEQ ID NO. 37. Examples of linker peptides for single-chain antibodies are described in Bird, RE, Hardman, KD, Jacobson, JW, Johnson, S., Kaufman, BM, Lee, SM, Lee, T., Pope, SH, Riordan, GS and Whitlow, M. (1988). Methods for preparing scFv antibodies have been described. Huse et al., Science 246: 1275-1281 (1989); Ward et al., Nature 341: 544-546 (1989); and Vaughan et al.,See Nature Biotech. 14: 309-314 (1996). Briefly, mRNA is isolated from B cells of immunized animals, and cDNA is prepared. cDNA is amplified using primers specific to the variable domains of the heavy and light chains of immunoglobulin. The PCR product is purified, and the nucleic acid sequences are linked. If a linker peptide is required, the nucleic acid sequence encoding the peptide is inserted between the heavy and light chain nucleic acid sequences. The nucleic acid encoding scFv is inserted into a vector and expressed in appropriate host cells.

[0115] In an embodiment, the first antibody region is a first single-chain variable fragment, or the second antibody region is a second single-chain variable fragment. In an embodiment, the first antibody region is a first single-chain variable fragment. In an embodiment, the second antibody region is a second single-chain variable fragment. In an embodiment, the first antibody region is a first single-chain variable fragment, and the second antibody region is a second single-chain variable fragment.

[0116] The ability of an antibody to bind to a specific epitope (e.g., BAFF-R) is equal to the equilibrium dissociation constant (K D It can be explained by ). The equilibrium dissociation constant (K defined herein). D ) is the ratio of the dissociation rate (K-off) to the binding rate (K-on) of the second antibody region capable of binding to the BAFF-R protein. This is the formula K D = K-off / K-on is explained by. Thus, in the embodiment, the second antibody region has an equilibrium dissociation constant (K) of less than about 5 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of less than about 4.5 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of less than about 4 nM. DIt can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of less than about 3.5 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of less than about 3 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of less than about 2.5 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of less than about 2 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of less than about 1.5 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of less than about 1 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of less than about 0.5 nM. D It can bind to the BAFF-R protein.

[0117] In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of about 0.5 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of about 1 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of about 1.5 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of about 2 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of about 2.5 nM. DIt can bind to the BAFF-R protein. In an embodiment, the third antibody region has an equilibrium dissociation constant of about 2 nM (K D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of about 3.5 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of about 4 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of about 4.5 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of about 5 nM. D It can bind to the BAFF-R protein. In an embodiment, the second antibody region has an equilibrium dissociation constant (K) of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 nM. D It can bind to the BAFF-R protein.

[0118] In one embodiment, K of less than about 4 nM D A recombinant protein capable of binding to BAFF-R is provided. In another embodiment, K of less than about 4 nM is provided. D A recombinant protein that binds to BAFF-R is provided. In an embodiment, the antibody does not induce BAFF-R activity.

[0119] The recombinant protein provided herein comprises a first antibody region capable of binding to an effector cell ligand. In an embodiment, the effector cell ligand is a CD3 protein. In an embodiment, the first antibody region comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 74, CDR L2 presented in SEQ ID NO. 75, and CDR L3 presented in SEQ ID NO. 76; and (b) a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 77, CDR H2 presented in SEQ ID NO. 78, and CDR H3 presented in SEQ ID NO. 79.

[0120] In an embodiment, the first antibody region comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 81, CDR L2 presented in SEQ ID NO. 82, and CDR L3 presented in SEQ ID NO. 83; and (b) a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 84, CDR H2 presented in SEQ ID NO. 85, and CDR H3 presented in SEQ ID NO. 86.

[0121] In an embodiment, the first antibody region is a first single-strand variable fragment (scFv). In an embodiment, the first scFv comprises the sequence of SEQ ID NO. 80 or SEQ ID NO. 87. In an embodiment, the first scFv comprises the sequence of SEQ ID NO. 80. In an embodiment, the first scFv comprises the sequence of SEQ ID NO. 87. In an embodiment, the first scFv is the sequence of SEQ ID NO. 80 or SEQ ID NO. 87. In an embodiment, the first scFv is the sequence of SEQ ID NO. 80. In an embodiment, the first scFv is the sequence of SEQ ID NO. 87.

[0122] In an embodiment, the second antibody region binds to the BAFF-R protein. In an embodiment, the BAFF-R protein is the human BAFF-R protein. In an embodiment, the BAFF-R protein is encoded by a nucleic acid sequence identified by NCBI gene ID number 115650. In an embodiment, the BAFF-R protein forms part of a cell. In an embodiment, the BAFF-R protein is expressed on the cell surface. In an embodiment, the cell is a lymphoid cell. In an embodiment, the cell is a B cell. In an embodiment, the cell is a cancer cell. In an embodiment, the cancer cell is a lymphoma cell.

[0123] In one embodiment, the recombinant protein comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 74, CDR L2 presented in SEQ ID NO. 75, and CDR L3 presented in SEQ ID NO. 76; and (b) a first antibody region comprising a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 77, CDR H2 presented in SEQ ID NO. 78, and CDR H3 presented in SEQ ID NO. 79, and a second antibody region comprising a light chain variable domain having the sequence of SEQ ID NO. 7 and a heavy chain variable domain having the sequence of SEQ ID NO. 10.

[0124] In one embodiment, the recombinant protein comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 74, CDR L2 presented in SEQ ID NO. 75, and CDR L3 presented in SEQ ID NO. 76; and (b) a first antibody region comprising a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 77, CDR H2 presented in SEQ ID NO. 78, and CDR H3 presented in SEQ ID NO. 79, and a second antibody region comprising a light chain variable domain having the sequence of SEQ ID NO. 8 and a heavy chain variable domain having the sequence of SEQ ID NO. 11.

[0125] In one embodiment, the recombinant protein comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 74, CDR L2 presented in SEQ ID NO. 75, and CDR L3 presented in SEQ ID NO. 76; and (b) a first antibody region comprising a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 77, CDR H2 presented in SEQ ID NO. 78, and CDR H3 presented in SEQ ID NO. 79, and a second antibody region comprising a light chain variable domain having the sequence of SEQ ID NO. 9 and a heavy chain variable domain having the sequence of SEQ ID NO. 12.

[0126] In one embodiment, the recombinant protein comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 81, CDR L2 presented in SEQ ID NO. 82, and CDR L3 presented in SEQ ID NO. 83; and (b) a first antibody region comprising a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 84, CDR H2 presented in SEQ ID NO. 85, and CDR H3 presented in SEQ ID NO. 86, and a second antibody region comprising a light chain variable domain having the sequence of SEQ ID NO. 7 and a heavy chain variable domain having the sequence of SEQ ID NO. 10.

[0127] In one embodiment, the recombinant protein comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 81, CDR L2 presented in SEQ ID NO. 82, and CDR L3 presented in SEQ ID NO. 83; and (b) a first antibody region comprising a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 84, CDR H2 presented in SEQ ID NO. 85, and CDR H3 presented in SEQ ID NO. 86, and a second antibody region comprising a light chain variable domain having the sequence of SEQ ID NO. 8 and a heavy chain variable domain having the sequence of SEQ ID NO. 11.

[0128] In one embodiment, the recombinant protein comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 81, CDR L2 presented in SEQ ID NO. 82, and CDR L3 presented in SEQ ID NO. 83; and (b) a first antibody region comprising a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 84, CDR H2 presented in SEQ ID NO. 85, and CDR H3 presented in SEQ ID NO. 86, and a second antibody region comprising a light chain variable domain having the sequence of SEQ ID NO. 9 and a heavy chain variable domain having the sequence of SEQ ID NO. 12.

[0129] In one embodiment, the recombinant protein comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 74, CDR L2 presented in SEQ ID NO. 75, and CDR L3 presented in SEQ ID NO. 76; and (b) a first antibody region comprising a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 77, CDR H2 presented in SEQ ID NO. 78, and CDR H3 presented in SEQ ID NO. 79, and a second antibody region comprising a light chain variable domain having the sequence of SEQ ID NO. 44 and a heavy chain variable domain having the sequence of SEQ ID NO. 47.

[0130] In one embodiment, the recombinant protein comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 74, CDR L2 presented in SEQ ID NO. 75, and CDR L3 presented in SEQ ID NO. 76; and (b) a first antibody region comprising a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 77, CDR H2 presented in SEQ ID NO. 78, and CDR H3 presented in SEQ ID NO. 79, and a second antibody region comprising a light chain variable domain having the sequence of SEQ ID NO. 45 and a heavy chain variable domain having the sequence of SEQ ID NO. 48.

[0131] In one embodiment, the recombinant protein comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 74, CDR L2 presented in SEQ ID NO. 75, and CDR L3 presented in SEQ ID NO. 76; and (b) a first antibody region comprising a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 77, CDR H2 presented in SEQ ID NO. 78, and CDR H3 presented in SEQ ID NO. 79, and a second antibody region comprising a light chain variable domain having the sequence of SEQ ID NO. 46 and a heavy chain variable domain having the sequence of SEQ ID NO. 49.

[0132] In one embodiment, the recombinant protein comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 81, CDR L2 presented in SEQ ID NO. 82, and CDR L3 presented in SEQ ID NO. 83; and (b) a first antibody region comprising a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 84, CDR H2 presented in SEQ ID NO. 85, and CDR H3 presented in SEQ ID NO. 86, and a second antibody region comprising a light chain variable domain having the sequence of SEQ ID NO. 44 and a heavy chain variable domain having the sequence of SEQ ID NO. 47.

[0133] In one embodiment, the recombinant protein comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 81, CDR L2 presented in SEQ ID NO. 82, and CDR L3 presented in SEQ ID NO. 83; and (b) a first antibody region comprising a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 84, CDR H2 presented in SEQ ID NO. 85, and CDR H3 presented in SEQ ID NO. 86, and a second antibody region comprising a light chain variable domain having the sequence of SEQ ID NO. 45 and a heavy chain variable domain having the sequence of SEQ ID NO. 48.

[0134] In one embodiment, the recombinant protein comprises (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 81, CDR L2 presented in SEQ ID NO. 82, and CDR L3 presented in SEQ ID NO. 83; and (b) a first antibody region comprising a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 84, CDR H2 presented in SEQ ID NO. 85, and CDR H3 presented in SEQ ID NO. 86, and a second antibody region comprising a light chain variable domain having the sequence of SEQ ID NO. 46 and a heavy chain variable domain having the sequence of SEQ ID NO. 49.

[0135] In one embodiment, the recombinant protein comprises a first antibody region having the sequence of SEQ ID NO. 80 and a second antibody region having a light chain variable domain having the sequence of SEQ ID NO. 7 and a heavy chain variable domain having the sequence of SEQ ID NO. 10.

[0136] In one embodiment, the recombinant protein comprises a first antibody region having the sequence of SEQ ID NO. 80 and a second antibody region having a light chain variable domain having the sequence of SEQ ID NO. 8 and a heavy chain variable domain having the sequence of SEQ ID NO. 11.

[0137] In one embodiment, the recombinant protein comprises a first antibody region having the sequence of SEQ ID NO. 80 and a second antibody region having a light chain variable domain having the sequence of SEQ ID NO. 9 and a heavy chain variable domain having the sequence of SEQ ID NO. 12.

[0138] In one embodiment, the recombinant protein comprises a first antibody region having the sequence of SEQ ID NO. 87 and a second antibody region having a light chain variable domain having the sequence of SEQ ID NO. 7 and a heavy chain variable domain having the sequence of SEQ ID NO. 10.

[0139] In one embodiment, the recombinant protein comprises a first antibody region having the sequence of SEQ ID NO. 87 and a second antibody region having a light chain variable domain having the sequence of SEQ ID NO. 8 and a heavy chain variable domain having the sequence of SEQ ID NO. 11.

[0140] In one embodiment, the recombinant protein comprises a first antibody region having the sequence of SEQ ID NO. 87 and a second antibody region having a light chain variable domain having the sequence of SEQ ID NO. 9 and a heavy chain variable domain having the sequence of SEQ ID NO. 12.

[0141] In one embodiment, the recombinant protein comprises a first antibody region having the sequence of SEQ ID NO. 80 and a second antibody region having a light chain variable domain having the sequence of SEQ ID NO. 44 and a heavy chain variable domain having the sequence of SEQ ID NO. 47.

[0142] In one embodiment, the recombinant protein comprises a first antibody region having the sequence of SEQ ID NO. 80 and a second antibody region having a light chain variable domain having the sequence of SEQ ID NO. 45 and a heavy chain variable domain having the sequence of SEQ ID NO. 48.

[0143] In one embodiment, the recombinant protein comprises a first antibody region having the sequence of SEQ ID NO. 80 and a second antibody region having a light chain variable domain having the sequence of SEQ ID NO. 46 and a heavy chain variable domain having the sequence of SEQ ID NO. 49.

[0144] In one embodiment, the recombinant protein comprises a first antibody region having the sequence of SEQ ID NO. 87 and a second antibody region comprising a light chain variable domain having the sequence of SEQ ID NO. 44 and a heavy chain variable domain having the sequence of SEQ ID NO. 47.

[0145] In one embodiment, the recombinant protein comprises a first antibody region having the sequence of SEQ ID NO. 87 and a second antibody region having a light chain variable domain having the sequence of SEQ ID NO. 45 and a heavy chain variable domain having the sequence of SEQ ID NO. 48.

[0146] In one embodiment, the recombinant protein comprises a first antibody region having the sequence of SEQ ID NO. 87 and a second antibody region having a light chain variable domain having the sequence of SEQ ID NO. 46 and a heavy chain variable domain having the sequence of SEQ ID NO. 49.

[0147] III. Pharmaceutical Composition

[0148] The formulations of the present invention (e.g., the recombinant proteins provided herein) are often administered as active therapeutic agents, i.e., as pharmaceutical compositions comprising various other pharmaceutically acceptable components. See Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania, 1980. The preferred form depends on the intended mode of administration and therapeutic application. Additionally, the composition may include pharmaceutically acceptable non-toxic carriers or diluents depending on the desired formulation, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. Additionally, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0149] The composition may be administered for therapeutic or prophylactic treatment. In therapeutic application, the composition is administered to a patient suffering from a disease (e.g., cancer) in a “therapeutic effective dose.” The effective dose for such use depends on the severity of the disease and the patient’s general health condition. Single or multiple administrations of the composition may be administered depending on the dose and frequency required and tolerated by the patient. For the purposes of the present invention, “patient” or “subject” includes both humans and other animals, particularly mammals. Thus, the method is applicable to both human therapeutic and veterinary applications. In a preferred embodiment, the patient is a mammal, preferably a primate, and in the most preferred embodiment, the patient is a human.

[0150] Formulations suitable for oral administration may consist of (a) an effective amount of the antibody provided herein suspended in a liquid solution, e.g., water, saline, or a diluent such as PEG 400; (b) capsules, shakes, or tablets containing a predetermined amount of the active ingredient, each as a liquid, solid, granule, or gelatin; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. Tablet forms may comprise one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, humectants, preservatives, flavorings, dyes, disintegrants, and pharmaceutically qualified carriers. Lozenge forms may contain an active ingredient in flavoring agents, e.g., sucrose, as well as Pastille, which contains an active ingredient in sucrose or an inert base such as gelatin and glycerin, and acacia emulsions, gels, etc., which contain a carrier known in the industry in addition to the active ingredient.

[0151] Additionally, the pharmaceutical composition may include large, slowly metabolized macromolecules such as proteins, polysaccharides like chitosan, polylactic acid, polyglycolic acid, and copolymers (e.g., latex-functionalized cephalosporose™, agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). Furthermore, these carriers may act as immunostimulators (i.e., adjuvants).

[0152] Formulations suitable for rectal administration include, for example, suppositories composed of nucleic acid packaged with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. Additionally, it is possible to use gelatin rectal capsules composed of a combination of selected compounds and a base, for example, liquid triglycerides, polyethylene glycol, and paraffin hydrocarbons.

[0153] For example, formulations suitable for parenteral administration, such as via intra-articular (in the joint), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include water-soluble and non-water-soluble isotonic sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient, and water-soluble and non-water-soluble sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In the practice of the present invention, the composition may be administered orally, topically, intraperitoneally, intravesically, or intrathecally, for example by intravenous infusion. Parenteral administration, oral administration, and intravenous administration are preferred methods of administration. Formulations of the compound may be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials.

[0154] Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the types previously described. In vitroCells transduced by nucleic acids for therapy may also be administered intravenously or parenterally as described above.

[0155] The pharmaceutical formulation is preferably in a unit dose form. In this form, the formulation is subdivided into unit doses containing an appropriate amount of the active ingredient. The unit dose form may be a packaged formulation, and the packaging includes individually measured amounts of formulation, such as packet tablets, capsules, and powders placed in vials or ampoules. Additionally, the unit dose form may be the capsule, tablet, shake, or lozenge itself, or any appropriate number of these in a packaged form. If desired, the composition may also contain other eligible therapeutic agents.

[0156] Combined administration considers co-administration using separate formulations or a single pharmaceutical formulation, and sequential administration in any order, preferably with a period during which both (or all) active agents simultaneously exert biological activity.

[0157] The effective amount of the composition provided herein depends on many different factors, including the means of administration, the target site, the patient's physiological condition, whether the patient is a human or an animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. However, for guidance, a person skilled in the art will immediately recognize an appropriate and / or equivalent dose by looking at the dose of an approved composition for treating and preventing cancer.

[0158] In one embodiment, a pharmaceutical composition is provided comprising a therapeutically effective amount of recombinant protein and a pharmaceutically acceptable excipient, including an embodiment provided herein.

[0159] The therapeutically effective dose provided herein refers to an amount effective for achieving the intended purpose. The actual amount effective for a specific application will vary depending particularly on the condition being treated. When administered to treat a disease in the above method, the pharmaceutical composition described herein will contain an amount of recombinant protein (e.g., bispecific antibody) effective for achieving the desired result, e.g., modulating the activity of a target molecule (e.g., BAFF-R), activating effector cells (e.g., T cells), or reducing, eliminating, or delaying the progression of disease symptoms (e.g., cancer, autoimmune disease). Determining the therapeutically effective dose of the recombinant protein (e.g., bispecific antibody) provided herein is within the capacity of a person skilled in the art, particularly in light of the detailed disclosure herein.

[0160] IV. Treatment Methods

[0161] In one embodiment, a method for treating cancer in a subject requiring treatment is provided, comprising administering a therapeutically effective amount of a recombinant protein (e.g., a bispecific antibody) including an embodiment thereof described herein to the subject, thereby treating the cancer in said subject. In an embodiment, said cancer is lymphoma, leukemia, or multiple myeloma. In an embodiment, said cancer is lymphoma. In an embodiment, said lymphoma is mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, or Burkitt lymphoma. In an embodiment, said lymphoma is mantle cell lymphoma. In an embodiment, said lymphoma is follicular lymphoma. In an embodiment, said lymphoma is diffuse large B-cell lymphoma. In an embodiment, said lymphoma is marginal zone lymphoma. In an embodiment, said lymphoma is Burkitt lymphoma.

[0162] In an embodiment, the cancer is leukemia. In an embodiment, the leukemia is lymphoblastic leukemia, chronic lymphoblastic leukemia, or hairy cell leukemia. In an embodiment, the leukemia is lymphoblastic leukemia. In an embodiment, the leukemia is chronic lymphoblastic leukemia. In an embodiment, the leukemia is hairy cell leukemia.

[0163] In an embodiment, the cancer is myeloma. In an embodiment, the myeloma is multiple myeloma.

[0164] In an embodiment, the method further comprises administering a second therapeutic agent to a subject. In an embodiment, the therapeutic agent is a chimeric monoclonal antibody capable of binding to the CD20 antigen. In an embodiment, the therapeutic agent is rituximab. The term "rituximab" refers, in the ordinary sense, to a monoclonal antibody against protein CD20 identified by ATCC code L01XC02.

[0165] In another aspect, a method for treating an autoimmune disease in a subject requiring treatment is provided. The method comprises administering a therapeutically effective amount of a recombinant protein (e.g., a bispecific antibody) including an embodiment provided herein to the subject, thereby treating the autoimmune disease in said subject. In an embodiment, said autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, Sjögren's syndrome, or autoimmune hemolytic anemia. In an embodiment, said autoimmune disease is rheumatoid arthritis. In an embodiment, said autoimmune disease is systemic lupus erythematosus. In an embodiment, said autoimmune disease is multiple sclerosis. In an embodiment, said autoimmune disease is glomerulonephritis. In an embodiment, said autoimmune disease is Sjögren's syndrome. In an embodiment, said autoimmune disease is autoimmune hemolytic anemia. In an embodiment, said method further comprises administering a second therapeutic agent to the subject.

[0166] The therapeutically effective dose provided herein means an amount effective for achieving the intended purpose. The actual amount effective for a specific application will vary depending particularly on the condition being treated. When administered to treat a disease in the above method, the pharmaceutical composition described herein will contain an amount of recombinant protein (BAFF-R bispecific antibody), including embodiments provided herein, which is effective in achieving the desired result, for example, modulating the activity of a target molecule (e.g., BAFF-R), activating effector cells, or reducing, eliminating, or delaying the progression of disease symptoms (e.g., cancer, autoimmune disease). Determining the therapeutically effective dose of the recombinant protein (BAFF-R bispecific antibody) provided herein is within the capacity of a person skilled in the art, particularly in light of the detailed disclosure herein.

[0167] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citrates, or acetates with a pH of typically 5.0 to 8.0, optionally 6.0 to 7.0; salts such as sodium chloride, potassium chloride, etc., that create isotonicity; antioxidants; preservatives; low molecular weight polypeptides; proteins; hydrophilic polymers such as polysorbate 80; amino acids such as glycine; carbohydrates; chelating agents; sugars; and other standard ingredients known to those skilled in the art (Remington: The Science and Practice of Pharmacy, 22nd ed., Loyd V. Allen et al., editors, Pharmaceutical Press (2012)). mAb may be present at a concentration of 0.1 to 100 mg / mL, e.g. 1 to 10 mg / mL or 10 to 50 mg / mL, e.g. 5, 10, 20, 30, 40, 50 or 60 mg / mL.

[0168] Pharmaceutical compositions (including embodiments thereof) comprising a recombinant protein (BAFF-R bispecific antibody), including embodiments provided herein, may be administered by various methods known in the art. The route and / or mode of administration depends on the desired result. Optionally, administration may be performed intravenously, intramuscularly, intraperitoneally, or subcutaneously, or administered near the target site. Pharmaceutically acceptable excipients may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0169] Pharmaceutical compositions of recombinant proteins (BAFF-R bispecific antibodies) including embodiments provided herein may be prepared according to methods widely known in the art and routinely practiced. For example, see Remington: The Science and Practice of Pharmacy, 22nd ed., Loyd V. Allen et al., editors, Pharmaceutical Press (2012); and Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably prepared under GMP conditions. Typically, a therapeutically effective amount or effective dose of recombinant proteins (BAFF-R bispecific antibodies) including embodiments provided herein is used in pharmaceutical compositions. Recombinant proteins (BAFF-R bispecific antibodies) including embodiments provided herein may be formulated into pharmaceutically acceptable dose forms by conventional methods known to those skilled in the art. The dose regimen is adjusted to provide a desired optimal response (e.g., a therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. It may be advantageous to formulate a recombinant protein (BAFF-R bispecific antibody) including embodiments provided herein in combination with other therapies or agents. It may be advantageous to formulate the parenteral composition in dose units for ease of administration and uniformity of dose.The dosage unit form used herein refers to a physically distinct unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined quantity of recombinant protein (BAFF-R bispecific antibody), including embodiments provided herein, calculated to produce a desired therapeutic effect in relation to the necessary pharmaceutical excipients.

[0170] The actual dose level of the active ingredient in a pharmaceutical composition may vary to obtain an amount of the active ingredient effective in achieving a desired therapeutic response for a specific patient, composition, and mode of administration without toxicity to the patient. The selected dose level depends on various pharmacodynamic factors, including the activity of the specific composition used, the route of administration, the time of administration, the elimination rate of the specific antibody in use, the duration of treatment, other drugs, compounds, and / or substances used in combination with the specific composition employed, age, sex, body weight, medical condition, the general health and medical history of the patient under treatment, and similar factors.

[0171] Physicians or veterinarians may start with a dose of the recombinant protein (BAFF-R bispecific antibody) provided herein, including embodiments used in pharmaceutical compositions, at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. Generally, the effective dose of the composition depends on many different factors, including the specific disease or condition to be treated, the means of administration, the target site, the patient's physiological condition, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. It is necessary to titrate the therapeutic dose to optimize safety and efficacy. In the case of administration using the bispecific antibody, the dose is in the range of about 0.0001 to 100 mg / kg of host body weight, more generally 0.01 to 5 mg / kg. For example, the dose may be 1 mg / kg body weight or 10 mg / kg body weight or within the range of 1 to 10 mg / kg. Exemplary therapeutic regimens include administration once every 2 or 3 weeks, once every 1 month, or once every 3 to 6 months.

[0172] The recombinant protein (BAFF-R bispecific antibody) provided herein may be administered multiple times, including embodiments thereof. The interval between single doses may be weekly, monthly, or annually. The interval may also be irregular as indicated by measuring the patient's blood level of the bispecific antibody. In some methods, the dose is adjusted to achieve a plasma antibody concentration of 1 to 1000 μg / mL, and in some methods, 25 to 300 μg / mL.

[0173] Alternatively, the recombinant protein (BAFF-R bispecific antibody) provided herein may be administered as a sustained-release formulation, including embodiments thereof, in which case less frequent administration is required. The dose and frequency of administration depend on the half-life of the recombinant protein (BAFF-R bispecific antibody) provided herein, including embodiments thereof, in the patient. The dose and frequency of administration may depend on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dose is administered over a long period at relatively infrequent intervals. Some patients continue to receive treatment for the remainder of their lives. In therapeutic applications, a relatively high dose is sometimes required at relatively short intervals until disease progression is reduced or terminated, preferably until the patient shows partial or complete improvement in disease symptoms. Subsequently, prophylactic administration may be given to the patient.

[0174] Examples

[0175] Bispecific T cell engagers (BiTEs) are a class of bispecific monoclonal antibodies that can be used as targeted immunotherapies. Unlike traditional monoclonal antibodies, BiTE antibodies consist of two distinct variable fragments arranged as two single-chain variable fragments (scFvs). One scFv targets and engages T cells via the CD3 molecule, while the other targets cell surface antigens of tumor cells. Targeting both molecules not only links T cells to tumor cells but also activates them through CD3 signaling. Subsequently, the activated T cells will exert cytotoxic activity against the tumor cells.

[0176] BAFF-R BiTE (bispecific antibody) was constructed from scFvs derived from the heavy and light chains of a previously developed humanized anti-BAFF-R monoclonal antibody, as disclosed in the aforementioned international patent application PCT / US2017 / 036178. Preliminary testing of BAFF-R BiTE demonstrated potent specific binding to BAFF-R and human T cells. BAFF-R BiTE In vitro BAFF-R expressing lymphomas and leukemias can induce potent T cell responses. Healthy donor B cells were also exposed to cytotoxicity in the presence of BAFF-R BiTE and T cell effectors due to normal BAFF-R expression.

[0177] The BAFF-R BiTE bispecific antibody was generated by the knob-hole technology. To facilitate heterodimer formation, a hole-in-knob structure was applied to the bispecific antibody. For BAFF-R BiTE (H90Bi), the gel was developed under reducing and non-reducing conditions. The control group included the control / unrelated BiTE (control Bi, 4-4Bi) and the original humanized BAFF-R antibody (H90 Ab, also referred to herein as C90).

[0178] The binding specificity of the bispecific antibody was measured using FACS histograms of wild-type mouse fibroblast L cells, engineered BAFF-R expressing L cells (B2D L cells), and T cells isolated from healthy donors bound to the BAFF-R / CD3 bispecific antibody (H90 BiTE), as shown in Figure 2. Antibody concentrations ranged from 0.05 μg to 5 μg. An unrelated BiTE antibody was used as a control. An anti-human IgG antibody was used as the secondary antibody. BAFF-R BiTE binding was further evaluated using FACS plots and was confirmed to bind independently to BAFF-R and T cells, as shown in Figures 8a through 8f.

[0179] The specific cytotoxicity of the bispecific antibody was measured against BAFF-R-expressing L cells. The plot shows the percentage of specific cytotoxicity calculated from the chromium release assay (Fig. 3). The BAFF-R / CD3 bispecific antibody (H90 BiTE) was incubated for 4 hours with chromium-labeled target wild-type or BAFF-R-expressing L cells along with effector CD8 T cells isolated from a healthy single donor at an effector-to-target (E:T) ratio of 10:1. Controls included control / unrelated BiTE (control Bi, 4-4Bi) and the original humanized BAFF-R antibody (H90 Ab) or CD8 T cells alone (no antibody).

[0180] The cytotoxicity of bispecific antibodies against malignant B cell lines was measured. The plot shows the percentage of specific cytotoxicity calculated from the chromium release assay against various malignant B cell lines as indicated (Fig. 4). The BAFF-R / CD3 bispecific antibody (H90 BiTE) was incubated with effector CD8 T cells isolated from a healthy single donor for 4 hours with the chromium-labeled target cell lines as indicated, at an effector-to-target (E:T) ratio of 10:1. The controls included control / unrelated BiTE (control Bi, 4-4Bi) and the original humanized BAFF-R antibody (H90 Ab) or CD8 T cells alone (no antibody).

[0181] The dose-dependent cytotoxicity of the bispecific antibodies was measured. The plot shows the percentage of specific cytotoxicity calculated by the chromium release assay for JeKo-1 mantle cell lymphoma and normal isolated B cells (Fig. 5). Various antibody concentrations of BAFF-R / CD3 bispecific antibodies (H90 BiTE) were incubated with chromium-labeled target JeKo-1 or normal B cells isolated from a healthy single donor at an effector-to-target (E:T) ratio of 10:1 for 4 hours. Controls included control / unrelated BiTE (control Bi, 4-4Bi) and the original humanized BAFF-R antibody (H90 Ab) or CD8 T cells alone (no antibody).

[0182] An exemplary treatment schedule using a Z-138 MCL tumor mouse model treated with BAFF-R BiTE is illustrated in the schematic diagram of Fig. 6. Mice were treated to evaluate the efficacy of BiTE in reducing tumor growth. The effects of administration of BAFF-R BiTE, control T cells, and PBS on tumor growth in these animals are shown in Fig. 7.

[0183] Unofficial ranking list

[0184] Humanized C90 / H90 CDR L1: ESVDNYGIS (Sequence No. 1)

[0185] Humanized C90 / H90 CDR L2: AAS (Sequence No. 2)

[0186] Humanized C90 / H90 CDR L3: QQSKEVPWT (Sequence No. 3)

[0187] Humanized C90 / H90 CDR H1: GDSITSGY (Sequence No. 4)

[0188] Humanized C90 / H90 CDR H2: ISYSGST (Sequence No. 5)

[0189] Humanization C90 / H90 CDR H3: ASPNYPFYAMDY (Sequence No. 6)

[0190] Humanized C90-1 light chain variable domain protein sequence METDTLLLWVLLLWVPGSTGEIVLTQSPATLSLSPGERATLSCRASESVDNYGISFLNWFQQKPGQAPRLLIYAASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSKEVPWTFGGGTKVEIKRTV (Sequence No. 7)

[0191] Humanized C90-2 light chain variable domain protein sequence METDTLLLWVLLLWVPGSTGDIVLTQSPATLSLSPGERATLSCRASESVDNYGISFMNWFQQKPGQAPRLLIYAASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSKEVPWTFGGGTKVEIKRTV (Sequence No. 8)

[0192] Humanized C90-3 light chain variable domain protein sequence METDTLLLWVLLLWVPGSTGDIVMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSKEVPWTFGQGTKVEIKRTV (Sequence No. 9)

[0193] Humanized C90-1 heavy chain variable domain protein sequence MDPKGSLSWRILLFLSLAFELSYGQVQLQESGPGLVKPSQTLSLTCTVSGDSITSGYWNWIRQHPGKGLEYIGYISYSGSTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCASPNYPFYAMDYWGQGTLVTVSS (Sequence No. 10)

[0194] Humanized C90-2 heavy chain variable domain protein sequence MDPKGSLSWRILLFLSLAFELSYGEVQLQESGPGLVKPSQTLSLTCTVSGDSITSGYWNWIRQHPGKGLEYIGYISYSGSTYYNPSLKSRVTISRDTSKNQYSLKLSSVTAADTAVYYCASPNYPFYAMDYWGQGTLVTVSS (Sequence No. 11)

[0195] Humanized C90-3 heavy chain variable domain protein sequence MDPKGSLSWRILLFLSLAFELSYGEVQLQESGPGLVKPSETLSLTCSVSGDSITSGYWNWIRQPPGKGLEYIGYISYSGSTYYNPSLKSRVTISRDTSKNQYSLRLSSVTAADTALYYCASPNYPFYAMDYWGQGTRVTVSS (Sequence No. 12)

[0196] Humanization C90-1 FR L1: METDTLLLWVLLLWVPGSTGEIVLTQSPATLSLSPGERATLSCRAS (Sequence No. 13)

[0197] Humanization C90-1 FR L2: FLNWFQQKPGQAPRLLIY (Sequence No. 14)

[0198] Humanization C90-1 FR L3: NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (Sequence No. 15)

[0199] Humanization C90-1 FR L4: FGGGTKVEIKRTV (Sequence No. 16)

[0200] Humanization C90-2 FR L1: METDTLLLWVLLLWVPGSTGDIVLTQSPATLSLSPGERATLSCRAS (Sequence No. 17)

[0201] Humanization C90-2 FR L2: FMNWFQQKPGQAPRLLIY (Sequence No. 18)

[0202] Humanization C90-2 FR L3: NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (Sequence No. 19)

[0203] Humanization C90-2 FR L4: FGGGTKVEIKRTV (Sequence No. 20)

[0204] Humanization C90-3 FR L1: METDTLLLWVLLLWVPGSTGDIVMTQSPSSLSASVGDRVTITCRAS (Sequence No. 21)

[0205] Humanization C90-3 FR L2: FMNWFQQKPGKAPKLLIY (Sequence No. 22)

[0206] Humanization C90-3 FR L3: NLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (Sequence No. 23)

[0207] Humanization C90-3 FR L4: FGQGTKVEIKRTV (Sequence No. 24)

[0208] Humanization C90-1 FR H1: MDPKGSLSWRILLFLSLAFELSYGQVQLQESGPGLVKPSQTLSLTCTVS (Sequence No. 25)

[0209] Humanized C90-1 FR H2: WNWIRQHPGKGLEYIGY (Sequence No. 26)

[0210] Humanization C90-1 FR H3: YYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYC (Sequence No. 27)

[0211] Humanized C90-1 FR H4: WGQGTLVTVSS (Sequence No. 28)

[0212] Humanization C90-2 FR H1: MDPKGSLSWRILLFLSLAFELSYGEVQLQESGPGLVKPSQTLSLTCTVS (Sequence No. 29)

[0213] Humanized C90-2 FR H2: WNWIRQHPGKGLEYIGY (Sequence No. 30)

[0214] Humanization C90-2 FR H3: YYNPSLKSRVTISRDTSKNQYSLKLSSVTAADTAVYYC (Sequence No. 31)

[0215] Humanization C90-2 FR H4: WGQGTLVTVSS (Sequence No. 32)

[0216] Humanization C90-3 FR H1: MDPKGSLSWRILLFLSLAFELSYGEVQLQESGPGLVKPSETLSLTCSVS (Sequence No. 33)

[0217] Humanized C90-3 FR H2: WNWIRQPPGKGLEYIGY (Sequence No. 34)

[0218] Humanization C90-3 FR H3: YYNPSLKSRVTISRDTSKNQYSLRLSSVTAADTALYYC (Sequence No. 35)

[0219] Humanized C90-3 FR H4: WGQGTRVTVSS (Sequence No. 36)

[0220] Humanized C90 or C55 Linker: GGGSGGGSGGGS (Sequence No. 37)

[0221] Humanization C55 CDR L1: QDISNY (Sequence No. 38)

[0222] Humanized C55 CDR L2: YTS (Sequence No. 39)

[0223] Humanized C55 CDR L3: HQFSELPWT (Sequence No. 40)

[0224] Humanized C55 CDR H1: GFSLSTSGMG (Sequence No. 41)

[0225] Humanized C55 CDR H2: IWWDDDK (Sequence No. 42)

[0226] Humanized C55 CDR H3: ARSFGYGLDY (Sequence No. 43)

[0227] Humanized C55-1 light chain variable domain protein sequence METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSSLHTGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCHQFSELPWTFGGGTKVEIKRTV (Sequence No. 44)

[0228] Humanized C55-2 light chain variable domain protein sequence METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSSLHTGVPSRFSGSGSGTDYTLTISSLQPEDIATYYCHQFSELPWTFGGGTKVEIKRTV (Sequence No. 45)

[0229] Humanized C55-3 light chain variable domain protein sequence METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKTPKLLIYYTSSLHTGVPSRFSGSGSGTDYTLTISSLQPEDIATYYCHQFSELPWTFGGGTKVEIKRTV (Sequence No. 46)

[0230] Humanized C55-1 heavy chain variable domain protein sequence MDPKGSLSWRILLFLSLAFELSYGQVTLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARSFGYGLDYWGQGTLVTVSS (Sequence No. 47)

[0231] Humanized C55-2 heavy chain variable domain protein sequence MDPKGSLSWRILLFLSLAFELSYGQVTLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYNSSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARSFGYGLDYWGQGTLVTVSS (Sequence No. 48)

[0232] Humanized C55-3 heavy chain variable domain protein sequence MDPKGSLSWRILLFLSLAFELSYGQVTLKESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYNTSLKSRLTITKDTSKNQVVLKMTNMDPVDTATYYCARSFGYGLDYWGQGTLVTVSS (Sequence No. 49)

[0233] Humanization C55-1 FR L1: METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCQAS (Sequence No. 50)

[0234] Humanization C55-1 FR L2: LNWYQQKPGKAPKLLIY (Sequence No. 51)

[0235] Humanization C55-1 FR L3: SLHTGVPSRFSGSGSGTDYTFTISSLQPEDIATYYC (Sequence No. 52)

[0236] Humanization C55-1 FR L4: FGGGTKVEIKRTV (Sequence No. 53)

[0237] Humanization C55-2 FR L1: METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCSAS (Sequence No. 54)

[0238] Humanization C55-2 FR L2: LNWYQQKPGKAPKLLIY (Sequence No. 55)

[0239] Humanization C55-2 FR L3: SLHTGVPSRFSGSGSGTDYTLTISSLQPEDIATYYC (Sequence No. 56)

[0240] Humanization C55-2 FR L4: FGGGTKVEIKRTV (Sequence No. 57)

[0241] Humanization C55-3 FR L1: METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCQAS (Sequence No. 58)

[0242] Humanization C55-3 FR L2: LNWYQQKPGKTPKLLIY (Sequence No. 59)

[0243] Humanization C55-3 FR L3: SLHTGVPSRFSGSGSGTDYTLTISSLQPEDIATYYC (Sequence No. 60)

[0244] Humanization C55-3 FR L4: FGGGTKVEIKRTV (Sequence No. 61)

[0245] Humanization C55-1 FR H1: MDPKGSLSWRILLFLSLAFELSYGQVTLKESGPTLVKPTQTLTLTCTFS (Sequence No. 62)

[0246] Humanization C55-1 FR H2: VGWIRQPPGKALEWLAH (Sequence No. 63)

[0247] Humanization C55-1 FR H3: YYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYC (Sequence No. 64)

[0248] Humanization C55-1 FR H4: WGQGTLVTVSS (Sequence No. 65)

[0249] Humanization C55-2 FR H1: MDPKGSLSWRILLFLSLAFELSYGQVTLKESGPTLVKPTQTLTLTCTFS (Sequence No. 66)

[0250] Humanization C55-2 FR H2: VGWIRQPPGKALEWLAH (Sequence No. 67)

[0251] Humanization C55-2 FR H3: YYNSSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYC (Sequence No. 68)

[0252] Humanized C55-2 FR H4: WGQGTLVTVSS (Sequence No. 69)

[0253] Humanization C55-3 FR H1: MDPKGSLSWRILLFLSLAFELSYGQVTLKESGPALVKPTQTLTLTCTFS (Sequence No. 70)

[0254] Humanization C55-3 FR H2: VGWIRQPPGKALEWLAH (Sequence No. 71)

[0255] Humanization C55-3 FR H3: YYNTSLKSRLTITKDTSKNQVVLKMTNMDPVDTATYYC (Sequence No. 72)

[0256] Humanized C55-3 FR H4: WGQGTLVTVSS (Sequence No. 73)

[0257] Anti-CD3 scFv 1 CDR L1: TGAVTSGNY (Sequence No. 74)

[0258] Anti-CD3 scFv 1 CDR L2: GTK (Sequence No. 75)

[0259] Anti-CD3 scFv 1 CDR L3: VLWYSNRWV (Sequence No. 76)

[0260] Anti-CD3 scFv 1 CDR H1: GFTFNKYA (Sequence No. 77)

[0261] Anti-CD3 scFv 1 CDR H2: IRSKYNNYAT (Sequence No. 78)

[0262] Anti-CD3 scFv 1 CDR H3: VRHGNFGNSYISYWAY (Sequence No. 79)

[0263] Anti-CD3 scFv 1 protein sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL (Sequence No. 80)

[0264] Anti-CD3 scFv 2 CDR L1: SSVSY (Sequence No. 81)

[0265] Anti-CD3 scFv 2 CDR L2: DTS (Sequence No. 82)

[0266] Anti-CD3 scFv 2 CDR L3: QQWSSNPFT (Sequence No. 83)

[0267] Anti-CD3 scFv 2 CDR H1: GYTFTRYT (Sequence No. 84)

[0268] Anti-CD3 scFv 2 CDR H2: INPSRGYT (Sequence No. 85)

[0269] Anti-CD3 scFv 2 CDR H3: ARYYDDHYCLDY (Sequence No. 86)

[0270] Anti-CD3 scFv 2 protein sequence QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSG GGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 87)

[0271] Implementation example

[0272] Embodiment 1. A recombinant protein comprising the following:

[0273] (i) a first antibody region capable of binding to an effector cell ligand; and

[0274] (ii) As a second antibody region,

[0275] (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 1, CDR L2 presented in SEQ ID NO. 2, and CDR L3 presented in SEQ ID NO. 3; and

[0276] (b) A second antibody region comprising a heavy chain variable domain comprising CDR H1 as presented in SEQ ID NO. 4, CDR H2 as presented in SEQ ID NO. 5, and CDR H3 as presented in SEQ ID NO. 6.

[0277] Embodiment 2. The recombinant protein of Embodiment 1, wherein the light chain variable domain comprises the sequence of SEQ ID NO. 7.

[0278] Embodiment 3. The recombinant protein in Embodiment 1 or 2, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO. 10.

[0279] Embodiment 4. In any one of Embodiments 1 to 3, the recombinant protein wherein the light chain variable domain comprises FR L1 presented in SEQ ID NO. 13, FR L2 presented in SEQ ID NO. 14, FR L3 presented in SEQ ID NO. 15, and FR L4 presented in SEQ ID NO. 16.

[0280] Embodiment 5. In any one of Embodiments 1 to 4, the recombinant protein wherein the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 25, FR H2 presented in SEQ ID NO. 26, FR H3 presented in SEQ ID NO. 27, and FR H4 presented in SEQ ID NO. 28.

[0281] Embodiment 6. The recombinant protein of Embodiment 1, wherein the light chain variable domain comprises the sequence of SEQ ID NO. 8.

[0282] Embodiment 7. The recombinant protein in Embodiment 1 or 6, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO. 11.

[0283] Embodiment 8. In any one of Embodiments 1, 6, or 7, the recombinant protein wherein the light chain variable domain comprises FR L1 presented in SEQ ID NO. 17, FR L2 presented in SEQ ID NO. 18, FR L3 presented in SEQ ID NO. 19, and FR L4 presented in SEQ ID NO. 20.

[0284] Embodiment 9. A recombinant protein in any one of Embodiments 1 or 6 to 8, wherein the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 29, FR H2 presented in SEQ ID NO. 30, FR H3 presented in SEQ ID NO. 31, and FR H4 presented in SEQ ID NO. 32.

[0285] Embodiment 10. The recombinant protein of Embodiment 1, wherein the light chain variable domain comprises the sequence of SEQ ID NO. 9.

[0286] Embodiment 11. The recombinant protein in Embodiment 1 or 10, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO. 12.

[0287] Embodiment 12. In any one of Embodiments 1, 10, or 11, the recombinant protein wherein the light chain variable domain comprises FR L1 presented in SEQ ID NO. 21, FR L2 presented in SEQ ID NO. 22, FR L3 presented in SEQ ID NO. 23, and FR L4 presented in SEQ ID NO. 24.

[0288] Embodiment 13. A recombinant protein in any one of Embodiments 1 or 10 to 12, wherein the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 33, FR H2 presented in SEQ ID NO. 34, FR H3 presented in SEQ ID NO. 35, and FR H4 presented in SEQ ID NO. 36.

[0289] Example 14. Recombinant protein comprising the following:

[0290] (i) a first antibody region capable of binding to an effector cell ligand; and

[0291] (ii) As a second antibody region,

[0292] (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 38, CDR L2 presented in SEQ ID NO. 39, and CDR L3 presented in SEQ ID NO. 40; and

[0293] (b) A second antibody region comprising a heavy chain variable domain comprising CDR H1 as presented in SEQ ID NO. 41, CDR H2 as presented in SEQ ID NO. 42, and CDR H3 as presented in SEQ ID NO. 44.

[0294] Embodiment 15. The recombinant protein of Embodiment 14, wherein the light chain variable domain comprises the sequence of SEQ ID NO. 44.

[0295] Embodiment 16. The recombinant protein in Embodiment 14 or 15, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO. 47.

[0296] Embodiment 17. In any one of Embodiments 14 to 16, the recombinant protein wherein the light chain variable domain comprises FR L1 presented in SEQ ID NO. 50, FR L2 presented in SEQ ID NO. 51, FR L3 presented in SEQ ID NO. 52, and FR L4 presented in SEQ ID NO. 53.

[0297] Embodiment 18. In any one of Embodiments 14 to 17, the recombinant protein wherein the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 62, FR H2 presented in SEQ ID NO. 63, FR H3 presented in SEQ ID NO. 64, and FR H4 presented in SEQ ID NO. 65.

[0298] Embodiment 19. The recombinant protein of Embodiment 14, wherein the light chain variable domain comprises the sequence of SEQ ID NO. 45.

[0299] Embodiment 20. The recombinant protein in Embodiment 14 or 19, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO. 48.

[0300] Embodiment 21. In any one of Embodiments 14, 19, or 20, the recombinant protein wherein the light chain variable domain comprises FR L1 presented in SEQ ID NO. 54, FR L2 presented in SEQ ID NO. 55, FR L3 presented in SEQ ID NO. 56, and FR L4 presented in SEQ ID NO. 57.

[0301] Embodiment 22. A recombinant protein in any one of Embodiments 14 or 19 to 21, wherein the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 66, FR H2 presented in SEQ ID NO. 67, FR H3 presented in SEQ ID NO. 68, and FR H4 presented in SEQ ID NO. 69.

[0302] Embodiment 23. The recombinant protein of Embodiment 14, wherein the light chain variable domain comprises the sequence of SEQ ID NO. 46.

[0303] Embodiment 24. The recombinant protein in Embodiment 14 or 23, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO. 49.

[0304] Embodiment 25. In any one of Embodiments 14, 23, or 24, the recombinant protein wherein the light chain variable domain comprises FR L1 presented in SEQ ID NO. 58, FR L2 presented in SEQ ID NO. 59, FR L3 presented in SEQ ID NO. 60, and FR L4 presented in SEQ ID NO. 61.

[0305] Embodiment 26. A recombinant protein in any one of Embodiments 14 or 23 to 25, wherein the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 70, FR H2 presented in SEQ ID NO. 71, FR H3 presented in SEQ ID NO. 72, and FR H4 presented in SEQ ID NO. 73.

[0306] Embodiment 27. A recombinant protein in any one of Embodiments 1 to 26, wherein the first antibody region is a first Fab' fragment or the second antibody region is a second Fab' fragment.

[0307] Embodiment 28. A recombinant protein in any one of Embodiments 1 to 26, wherein the first antibody region is a first single-chain variable fragment (scFv) or the second antibody region is a second single-chain variable fragment (scFv).

[0308] Embodiment 29. In any one of Embodiments 1 to 28, the recombinant protein, wherein the second antibody region is capable of binding to the BAFF-R protein with an equilibrium dissociation constant (KD) of less than about 5 nM.

[0309] Embodiment 30. In any one of Embodiments 1 to 29, the recombinant protein, wherein the second antibody region is capable of binding to the BAFF-R protein with an equilibrium dissociation constant (KD) of less than about 4 nM.

[0310] Embodiment 31. In any one of Embodiments 1 to 30, the first antibody region is

[0311] (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 74, CDR L2 presented in SEQ ID NO. 75, and CDR L3 presented in SEQ ID NO. 76; and

[0312] (b) A recombinant protein comprising a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 77, CDR H2 presented in SEQ ID NO. 78, and CDR H3 presented in SEQ ID NO. 79.

[0313] Embodiment 32. In any one of Embodiments 1 to 30, the first antibody region is

[0314] (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 81, CDR L2 presented in SEQ ID NO. 82, and CDR L3 presented in SEQ ID NO. 83; and

[0315] (b) A recombinant protein comprising a heavy chain variable domain including CDR H1 presented in SEQ ID NO. 84, CDR H2 presented in SEQ ID NO. 85, and CDR H3 presented in SEQ ID NO. 86.

[0316] Embodiment 33. In any one of Embodiments 1 to 32, the recombinant protein, wherein the first antibody region is a first single-chain variable fragment (scFv).

[0317] Embodiment 34. The recombinant protein of Embodiment 33, wherein the first scFv comprises the sequence of SEQ ID NO. 80 or SEQ ID NO. 87.

[0318] Embodiment 35. In any one of Embodiments 1 to 34, the recombinant protein wherein the first effector cell ligand is a CD3 protein.

[0319] Embodiment 36. In any one of Embodiments 1 to 35, the second antibody region is a recombinant protein that binds to the BAFF-R protein.

[0320] Embodiment 37. In Embodiment 36, the BAFF-R protein is a recombinant protein, wherein the BAFF-R protein is a human BAFF-R protein.

[0321] Embodiment 38. In Embodiment 36, the BAFF-R protein is a recombinant protein that forms part of a cell.

[0322] Embodiment 39. In Embodiment 38, the BAFF-R protein is a recombinant protein expressed on the surface of the cell.

[0323] Embodiment 40. In Embodiment 39, the cell is a lymphoid cell, and the recombinant protein.

[0324] Embodiment 41. In Embodiment 39, the cell is a B cell, and the recombinant protein.

[0325] Embodiment 42. In Embodiment 39, the cell is a cancer cell, and the recombinant protein.

[0326] Embodiment 43. In Embodiment 42, the cell is a lymphoma cell, recombinant protein.

[0327] Embodiment 44. A pharmaceutical composition comprising a therapeutically effective amount of any one of the recombinant proteins of Embodiments 1 to 43 and a pharmaceutically acceptable excipient.

[0328] Embodiment 45. A method for treating cancer in a subject requiring treatment, comprising administering a therapeutically effective amount of any one of Embodiments 1 to 43 recombinant protein to the subject, thereby treating the cancer of the subject.

[0329] Embodiment 46. Method according to Embodiment 45, wherein the cancer is lymphoma, leukemia, or myeloma.

[0330] Embodiment 47. A method for treating an autoimmune disease in a subject requiring treatment, comprising administering a therapeutically effective amount of a recombinant protein of any one of Embodiments 1 to 43 to the subject, thereby treating the autoimmune disease of the subject.

Claims

Claim 1 A recombinant protein comprising: (i) a first antibody region capable of binding to an effector cell ligand; and (ii) a second antibody region comprising: (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 1, CDR L2 presented in SEQ ID NO. 2, and CDR L3 presented in SEQ ID NO. 3; and (b) a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 4, CDR H2 presented in SEQ ID NO. 5, and CDR H3 presented in SEQ ID NO. 6; and a second antibody region comprising: Claim 2 In claim 1, the light chain variable domain comprises the sequence of SEQ ID NO. 7, a recombinant protein. Claim 3 A recombinant protein according to claim 1 or 2, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO.

10. Claim 4 In claim 1, the light chain variable domain comprises FR L1 presented in SEQ ID NO. 13, FR L2 presented in SEQ ID NO. 14, FR L3 presented in SEQ ID NO. 15, and FR L4 presented in SEQ ID NO. 16, a recombinant protein. Claim 5 In claim 1, the recombinant protein comprising the heavy chain variable domain FR H1 presented in SEQ ID NO. 25, FR H2 presented in SEQ ID NO. 26, FR H3 presented in SEQ ID NO. 27, and FR H4 presented in SEQ ID NO.

28. Claim 6 In claim 1, the light chain variable domain comprises the sequence of SEQ ID NO. 8, a recombinant protein. Claim 7 A recombinant protein according to claim 1 or 6, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO.

11. Claim 8 In claim 1, the light chain variable domain comprises FR L1 presented in SEQ ID NO. 17, FR L2 presented in SEQ ID NO. 18, FR L3 presented in SEQ ID NO. 19, and FR L4 presented in SEQ ID NO. 20, a recombinant protein Claim 9 In claim 1, the recombinant protein comprising the heavy chain variable domain FR H1 presented in SEQ ID NO. 29, FR H2 presented in SEQ ID NO. 30, FR H3 presented in SEQ ID NO. 31, and FR H4 presented in SEQ ID NO.

32. Claim 10 In claim 1, the recombinant protein wherein the light chain variable domain comprises the sequence of SEQ ID NO.

9. Claim 11 A recombinant protein according to claim 1 or 10, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO.

12. Claim 12 In claim 1, the light chain variable domain comprises FR L1 presented in SEQ ID NO. 21, FR L2 presented in SEQ ID NO. 22, FR L3 presented in SEQ ID NO. 23, and FR L4 presented in SEQ ID NO. 24, a recombinant protein. Claim 13 In claim 1, the recombinant protein comprising the heavy chain variable domain FR H1 presented in SEQ ID NO. 33, FR H2 presented in SEQ ID NO. 34, FR H3 presented in SEQ ID NO. 35, and FR H4 presented in SEQ ID NO.

36. Claim 14 A recombinant protein comprising: (i) a first antibody region capable of binding to an effector cell ligand; and (ii) a second antibody region comprising: (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 38, CDR L2 presented in SEQ ID NO. 39, and CDR L3 presented in SEQ ID NO. 40; and (b) a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 41, CDR H2 presented in SEQ ID NO. 42, and CDR H3 presented in SEQ ID NO. 43; and a second antibody region comprising: Claim 15 In claim 14, the light chain variable domain comprises the sequence of SEQ ID NO. 44, a recombinant protein. Claim 16 A recombinant protein according to claim 14 or 15, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO.

47. Claim 17 In claim 14, the light chain variable domain comprises FR L1 presented in SEQ ID NO. 50, FR L2 presented in SEQ ID NO. 51, FR L3 presented in SEQ ID NO. 52, and FR L4 presented in SEQ ID NO. 53, a recombinant protein. Claim 18 In claim 14, the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 62, FR H2 presented in SEQ ID NO. 63, FR H3 presented in SEQ ID NO. 64, and FR H4 presented in SEQ ID NO. 65, a recombinant protein. Claim 19 In claim 14, the light chain variable domain comprises the sequence of SEQ ID NO. 45, a recombinant protein. Claim 20 A recombinant protein according to claim 14 or 19, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO.

48. Claim 21 In claim 14, the light chain variable domain comprises FR L1 presented in SEQ ID NO. 54, FR L2 presented in SEQ ID NO. 55, FR L3 presented in SEQ ID NO. 56, and FR L4 presented in SEQ ID NO. 57, a recombinant protein. Claim 22 In claim 14, the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 66, FR H2 presented in SEQ ID NO. 67, FR H3 presented in SEQ ID NO. 68, and FR H4 presented in SEQ ID NO. 69, a recombinant protein. Claim 23 In claim 14, the light chain variable domain comprises the sequence of SEQ ID NO. 46, a recombinant protein. Claim 24 A recombinant protein according to claim 14 or 23, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO.

49. Claim 25 In claim 14, the light chain variable domain comprises FR L1 presented in SEQ ID NO. 58, FR L2 presented in SEQ ID NO. 59, FR L3 presented in SEQ ID NO. 60, and FR L4 presented in SEQ ID NO. 61, a recombinant protein. Claim 26 In claim 14, the heavy chain variable domain comprises FR H1 presented in SEQ ID NO. 70, FR H2 presented in SEQ ID NO. 71, FR H3 presented in SEQ ID NO. 72, and FR H4 presented in SEQ ID NO. 73, a recombinant protein. Claim 27 A recombinant protein according to claim 1, wherein the first antibody region is a first Fab' fragment or the second antibody region is a second Fab' fragment. Claim 28 A recombinant protein according to claim 1, wherein the first antibody region is a first single-chain variable fragment (scFv) or the second antibody region is a second single-chain variable fragment (scFv). Claim 29 In claim 1, the second antibody region is a recombinant protein capable of binding to the BAFF-R protein with an equilibrium dissociation constant (KD) of less than about 5 nM. Claim 30 In claim 1, the second antibody region is a recombinant protein capable of binding to the BAFF-R protein with an equilibrium dissociation constant (KD) of less than about 4 nM. Claim 31 A recombinant protein according to claim 1, wherein the first antibody region comprises: (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 74, CDR L2 presented in SEQ ID NO. 75, and CDR L3 presented in SEQ ID NO. 76; and (b) a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 77, CDR H2 presented in SEQ ID NO. 78, and CDR H3 presented in SEQ ID NO.

79. Claim 32 A recombinant protein according to claim 1, wherein the first antibody region comprises: (a) a light chain variable domain comprising CDR L1 presented in SEQ ID NO. 81, CDR L2 presented in SEQ ID NO. 82, and CDR L3 presented in SEQ ID NO. 83; and (b) a heavy chain variable domain comprising CDR H1 presented in SEQ ID NO. 84, CDR H2 presented in SEQ ID NO. 85, and CDR H3 presented in SEQ ID NO.

86. Claim 33 In claim 1 or 32, the first antibody region is a recombinant protein that is a first single-chain variable fragment (scFv). Claim 34 In claim 33, the first scFv is a recombinant protein comprising the sequence of SEQ ID NO. 80 or SEQ ID NO.

87. Claim 35 In claim 1, the first effector cell ligand is a recombinant protein, which is a CD3 protein. Claim 36 In claim 1, the second antibody region is a recombinant protein that binds to the BAFF-R protein. Claim 37 In paragraph 36, the above BAFF-R protein is a recombinant protein that is a human BAFF-R protein. Claim 38 In paragraph 36, the above BAFF-R protein is a recombinant protein that forms part of a cell. Claim 39 In claim 38, the BAFF-R protein is a recombinant protein expressed on the surface of the cell. Claim 40 In paragraph 39, the cell is a lymphoid cell, a recombinant protein. Claim 41 In paragraph 39, the cell is a B cell, a recombinant protein. Claim 42 In Clause 39, the cell is a cancer cell, a recombinant protein. Claim 43 In Clause 42, the cell is a recombinant protein that is a lymphoma cell. Claim 44 A pharmaceutical composition comprising a therapeutically effective amount of the recombinant protein of claim 1 and a pharmaceutically acceptable excipient. Claim 45 A method for treating cancer in a subject requiring treatment, comprising administering a therapeutically effective amount of the recombinant protein of claim 1 to said subject, thereby treating cancer in said subject. Claim 46 In paragraph 45, the above cancer is lymphoma, leukemia, or myeloma, method. Claim 47 A method for treating an autoimmune disease in a subject requiring treatment, comprising administering a therapeutically effective amount of the recombinant protein of claim 1 to said subject, thereby treating the autoimmune disease in said subject.

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