Anti-idiotypic antibody molecules and uses thereof

Abstract

Antibody molecules that specifically bind to anti-APRIL antibodies are disclosed, which can be used in assays to detect anti-APRIL antibodies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 307,538, filed February 7, 2022. The contents of the above-mentioned application are incorporated herein by reference in their entirety. [Background technology]

[0002] IgA nephropathy is one of the most prevalent chronic glomerular diseases worldwide. Conservative epidemiological estimates place the global incidence at approximately 5-50 cases / million children and 10-40 cases / million adults. The disease has a geographical prevalence, with higher prevalence in Asia and both Americas, and areas of Japan and China with particularly high disease burden. There are an estimated 350,000 biopsy-confirmed cases of IgA nephropathy in Japan. In the United States, the estimate is approximately 100,000 cases, making IgA nephropathy the most frequently diagnosed primary glomerular disease in adults. Although a relatively slowly progressive disease, IgA nephropathy leads to end stage renal disease (ESRD), i.e., renal failure, in 20-50% of patients within a 20-30 year period. These numbers are likely to be grossly underreported, given the need for disease confirmation by renal biopsy, a protocol variably performed in different clinical settings. The disease has a complex pathogenesis with genetic, epidemiological, and potentially environmental components to disease etiology, pathogenesis, and progression. Similarly, it has a range of clinical manifestations, ranging from asymptomatic to end-stage renal failure (ESRD). IgA nephropathy is caused by the deposition of IgA, typically in the form of immune complexes, in the mesangium of the kidney.

[0003] Antibody drugs are being developed for the treatment of IgA nephropathy. There is a need to develop new agents and assays to evaluate antibody drugs in preclinical and clinical samples. Summary of the Invention

[0004] The present disclosure provides, at least in part, an antibody molecule that binds to an anti-APRIL antibody, such as sibeprenlimab, and comprises one or more functional and structural properties disclosed herein. In an embodiment, the antibody molecule is an anti-idiotype (anti-ID) antibody molecule. In an embodiment, the antibody molecule binds to an anti-APRIL antibody and / or reduces (e.g., inhibits, blocks, or neutralizes) one or more biological activities of an anti-APRIL antibody. In an embodiment, the antibody molecule comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) described herein. In an embodiment, the antibody molecule comprises one or more heavy chain CDRs (HCDRs) of the VH and / or one or more light chain CDRs (LCDRs) of the VL described herein. Nucleic acid molecules encoding the antibody molecules, vectors, cells, compositions, kits, and methods for making and using the antibody molecules are also provided. The antibody molecules disclosed herein are suitable for use in detecting and measuring anti-APRIL antibodies (e.g., sibeprenlimab) in samples and subjects.

[0005] Thus, in one aspect, the present disclosure provides antibody molecules (e.g., anti-idiotypic antibody molecules), e.g., antibody molecules having the following characteristics: (a) has a high affinity for an anti-APRIL antibody (e.g., sibeprenlimab), e.g., a cytotoxicity of about 1 μg / mL or less, e.g., about 900 ng / mL or less, 800 ng / mL or less, 700 ng / mL or less, 600 ng / mL or less, 500 ng / mL or less, 400 ng / mL or less, 300 ng / mL or less, 200 ng / mL or less, 100 ng / mL or less, 90 ng / mL or less, 80 ng / mL or less, 70 ng / mL or less, 60 ng / mL or less, 50 ng / mL or less, / mL or less, 40ng / mL or less, 30ng / mL or less, 20ng / mL or less, 10ng / mL or less, 5ng / mL or less, 2ng / mL or less, 1ng / mL or less, for example, between 1ng / mL and 1μg / mL, for example, between 1ng / mL and 1μg / mL, between 1ng / mL and 500ng / mL, between 1ng / mL and 200ng / mL, between 1ng / mL and 100ng / mL, between 1ng / mL and 50ng / mL, between 1ng / mL and 20ng / mL, between 1ng / mL and 10ng / mL, 1ng between 2ng / mL and 5ng / mL, between 2ng / mL and 1μg / mL, between 5ng / mL and 1μg / mL, between 10ng / mL and 1μg / mL, between 20ng / mL and 1μg / mL, between 50ng / mL and 1μg / mL, between 100ng / mL and 1μg / mL, between 200ng / mL and 1μg / mL, between 500ng / mL and 1μg / mL, between 2ng / mL and 500ng / mL, between 5ng / mL and 200ng / mL, between 10ng / mL and 100ng / mL, between 15ng / mL and 50ng / 50ng / mL, for example, about 1ng / mL, 2ng / mL, 5ng / mL, 10ng / mL, 15ng / mL, 20ng / mL, 25ng / mL, 30ng / mL, 40ng / mL, 50ng / mL, 60ng / mL, 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL, 110ng / mL, 120ng / mL, 130ng / mL, 140ng / mL, or 150ng / mL. 50 ), b) specifically binds to an epitope that is the same as, similar to, or overlaps with the epitope recognized by the idiotype of an anti-APRIL antibody (e.g., sibeprenlimab), e.g., a monoclonal antibody described herein (e.g., mAb 1H4); c) reducing (e.g., inhibiting, blocking, or neutralizing) one or more biological activities of an anti-APRIL antibody (e.g., sibeprenlimab) in vitro, ex vivo, or in vivo; d) exhibit the same or similar binding affinity or specificity, or both, as a monoclonal antibody described herein (e.g., mAb 1H4); e) exhibits the same or similar binding affinity or specificity, or both, as an antibody molecule that contains a heavy chain variable region and / or a light chain variable region described herein, e.g., a heavy chain variable region and / or a light chain variable region of a monoclonal antibody described herein (e.g., mAb 1H4); f) exhibits the same or similar binding affinity or specificity, or both, as an antibody molecule that contains one or more (e.g., two or three) heavy chain CDRs and / or one or more (e.g., two or three) light chain CDRs described herein, e.g., one or more (e.g., two or three) heavy chain CDRs and / or one or more (e.g., two or three) light chain CDRs of a monoclonal antibody described herein (e.g., mAb 1H4); g) exhibit the same or similar binding affinity or specificity, or both, as an antibody molecule comprising an amino acid sequence described herein; h) exhibit the same or similar binding affinity or specificity, or both, as an antibody molecule comprising an amino acid sequence encoded by a nucleotide sequence described herein; i) inhibits, e.g., competitively inhibits, the binding of a second antibody molecule to an anti-APRIL antibody (e.g., sibeprenlimab), where the second antibody molecule is a monoclonal antibody described herein (e.g., mAb 1H4); j) competes with a second antibody molecule for binding to an anti-APRIL antibody (e.g., cybeprenlimab), where the second antibody molecule is a monoclonal antibody described herein (e.g., mAb 1H4); k) has one or more biological properties of a monoclonal antibody described herein (e.g., mAb 1H4); l) has one or more structural characteristics of a monoclonal antibody described herein (e.g., mAb 1H4); or m) has one or more pharmacokinetic properties of a monoclonal antibody described herein (e.g., mAb 1H4); In some embodiments, the antibody molecule described herein has one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all) of the following:

[0006] In one aspect, the disclosure provides an isolated antibody molecule capable of binding to an anti-APRIL antibody, e.g., cybeprenlimab, comprising a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of SEQ ID NO: 1, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NO: 2. In one embodiment, the HCDR1 amino acid sequence comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having no more than 1, 2, or 3 amino acid differences therefrom. In one embodiment, the HCDR2 amino acid sequence comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having no more than 1, 2, or 3 amino acid differences therefrom. In one embodiment, the HCDR3 amino acid sequence comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having no more than 1, 2, or 3 amino acid differences therefrom. In one embodiment, the LCDR1 amino acid sequence comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having no more than 1, 2, or 3 amino acid differences therefrom. In one embodiment, the LCDR2 amino acid sequence comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having no more than 1, 2 or 3 amino acid differences therefrom. In one embodiment, the LCDR3 amino acid sequence comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having no more than 1, 2 or 3 amino acid differences therefrom.

[0007] In one embodiment, the VH comprises the amino acid sequence of SEQ ID NO:1, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, or differs therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues. In one embodiment, the VH comprises the amino acid sequence of SEQ ID NO:1.

[0008] In one embodiment, the VL comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues thereto. In one embodiment, the VL comprises the amino acid sequence of SEQ ID NO:2.

[0009] In one embodiment, the VH comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues therefrom, and the VL comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues therefrom. In one embodiment, the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO:2.

[0010] In one embodiment, an antibody molecule comprises two VHs and two VLs, e.g., two VHs and two VLs described herein.

[0011] In some embodiments, the antibody molecule comprises an antigen-binding fragment, hi some embodiments, the antigen-binding fragment comprises a Fab, F(ab')2, Fv, scFv, or sc(Fv)2.

[0012] In some embodiments, the antibody molecule comprises a heavy chain constant region of an IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody molecule comprises a light chain constant region of a kappa or lambda light chain. In some embodiments, the antibody molecule comprises a heavy chain constant region of an IgG1, IgG2, IgG3, or IgG4 and a light chain constant region of a kappa or lambda light chain. In some embodiments, the antibody molecule comprises a heavy chain constant region of an IgG1 (e.g., an IgG1 heavy chain constant region described herein) and a light chain constant region of a kappa light chain (e.g., a kappa light chain constant region described herein). In some embodiments, the antibody molecule comprises a heavy chain constant region of an IgG2 (e.g., an IgG2 heavy chain constant region described herein) and a light chain constant region of a kappa light chain (e.g., a kappa light chain constant region described herein).

[0013] In certain embodiments, the antibody molecule comprises an Fc region.

[0014] In some embodiments, the antibody molecule comprises a heavy chain comprising amino acids 18 to 458 of SEQ ID NO: 3. In some embodiments, the antibody molecule comprises a heavy chain comprising amino acids 18 to 464 of SEQ ID NO: 4. In some embodiments, the antibody molecule comprises a light chain comprising amino acids 18 to 235 of SEQ ID NO: 5.

[0015] In one embodiment, the antibody molecule comprises a heavy chain comprising amino acids 18 to 458 of SEQ ID NO: 3 and a light chain comprising amino acids 18 to 235 of SEQ ID NO: 5. In one embodiment, the antibody molecule comprises a heavy chain comprising amino acids 18 to 464 of SEQ ID NO: 4 and a light chain comprising amino acids 18 to 235 of SEQ ID NO: 5.

[0016] In some embodiments, the antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 5.

[0017] In one embodiment, the antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 5. In one embodiment, the antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 5.

[0018] In certain embodiments, the antibody molecule is mAb 1H4, 1A5 or 6A3. In certain embodiments, the antibody molecule is mAb 1H4.

[0019] In some embodiments, the antibody molecule is a murine antibody molecule. In some embodiments, the antibody molecule is a humanized antibody molecule. In some embodiments, the antibody molecule is a monoclonal antibody molecule. In some embodiments, the antibody molecule is a synthetic antibody molecule. In some embodiments, the antibody molecule is a purified antibody molecule, e.g., purified from hybridoma supernatant, transient transfection of cells (e.g., Expi293 cells), or a stable expression cell line (e.g., CHO cells). In some embodiments, the antibody molecule is produced, e.g., from a hybridoma, at a concentration of 0.2 mg / mL to 5 mg / mL, e.g., 0.5 mg / mL to 1.5 mg / mL. In some embodiments, the antibody molecule is a ruthenylated antibody molecule.

[0020] In some embodiments, the antibody molecule is an anti-idiotypic (anti-ID) antibody molecule. In some embodiments, the antibody molecule binds to one or more (e.g., 2, 3, 4, 5 or 6) CDRs of an anti-APRIL antibody. In some embodiments, the antibody molecule binds to the VH, VL, or both of an anti-APRIL antibody. In some embodiments, the antibody molecule does not bind, or does not substantially bind, to the Fc region of an anti-APRIL antibody.

[0021] In an embodiment, the antibody molecule binds to the antibody-APRIL antibody, e.g., in a binding ELISA assay described herein. In an embodiment, the antibody molecule binds to the antibody-APRIL antibody, e.g., in a bridging ELISA assay described herein. In certain embodiments, the antibody molecule binds to an anti-APRIL antibody with an EC50 of less than 500 ng / mL, e.g., 400 ng / mL, 300 ng / mL, 200 ng / mL, 150 ng / mL, 120 ng / mL, 100 ng / mL, 90 ng / mL, 80 ng / mL, 70 ng / mL, 60 ng / mL, 50 ng / mL, 40 ng / mL, 35 ng / mL, 30 ng / mL, 25 ng / mL, 20 ng / mL, 15 ng / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL, 1 ng / mL, or less than 0.1 ng / mL, e.g., between 1 ng / mL and 150 ng / mL, between 2 ng / mL and 100 ng / mL, between 5 ng / mL and 50 ng / mL, or between 10 ng / mL and 25 ng / mL, as determined, for example, by ELISA.

[0022] In certain embodiments, the antibody molecule reduces (e.g., neutralizes, inhibits, or blocks) binding of an anti-APRIL antibody to APRIL. In certain embodiments, the antibody molecule binds to an anti-APRIL antibody that is not bound to APRIL. In certain embodiments, the antibody molecule does not bind or substantially does not bind to an anti-APRIL antibody that is bound to APRIL. In certain embodiments, the antibody molecule binds to an anti-APRIL antibody that is not bound to APRIL and does not bind or substantially does not bind to an anti-APRIL antibody that is bound to APRIL. In certain embodiments, the antibody molecule binds to an anti-APRIL antibody that is not bound to APRIL and to an anti-APRIL antibody that is bound to APRIL.

[0023] In some embodiments, the antibody molecule bridges two anti-APRIL antibodies, e.g., binds two anti-APRIL antibodies simultaneously, hi some embodiments, the antibody molecule competes with the anti-APRIL antibody molecule for binding to APRIL.

[0024] In some embodiments, the anti-APRIL antibody binds to human APRIL. In some embodiments, the anti-APRIL antibody comprises one or more (e.g., 2, 3, 4, 5, or 6) CDRs of sibeprenlimab. In some embodiments, the anti-APRIL antibody comprises the VH, VL, or both of sibeprenlimab. In some embodiments, the anti-APRIL antibody is sibeprenlimab.

[0025] In one aspect, the disclosure provides a method of detecting an anti-APRIL antibody, the method comprising contacting an antibody molecule described herein with a sample and determining the formation of a complex between the antibody molecule and the sample (e.g., between the antibody molecule and an anti-APRIL antibody in the sample), thereby detecting the anti-APRIL antibody.

[0026] In certain embodiments, the method further comprises contacting the antibody molecule with a reference sample and determining formation of a complex between the antibody molecule and the reference sample.

[0027] In some embodiments, the anti-APRIL antibodies are detected in an ELISA assay (e.g., a pharmacokinetic ELISA). In some embodiments, the ELISA is a binding ELISA, e.g., as described herein. In some embodiments, the ELISA is a bridging ELISA, e.g., as described herein. In some embodiments, the anti-APRIL antibodies are detected in an anti-drug assay. In some embodiments, the anti-APRIL antibodies are detected in a toxicity assay. In some embodiments, the anti-APRIL antibodies are detected in a GxP assay.

[0028] In one aspect, the disclosure provides a method of evaluating a sample, the method comprising contacting an antibody molecule described herein with the sample and determining formation of a complex between the antibody molecule and the sample (e.g., between the antibody molecule and an anti-APRIL antibody in the sample), thereby evaluating the sample.

[0029] In certain embodiments, the method further comprises contacting the antibody molecule with a reference sample and determining formation of a complex between the antibody molecule and the reference sample.

[0030] In an embodiment, the sample is evaluated in an ELISA assay (e.g., a pharmacokinetic ELISA). In an embodiment, the ELISA is a binding ELISA, e.g., as described herein. In an embodiment, the ELISA is a bridging ELISA, e.g., as described herein. In an embodiment, the sample is evaluated in an anti-drug assay. In an embodiment, the sample is evaluated in a toxicity assay. In an embodiment, anti-APRIL antibodies are detected in a GxP assay.

[0031] In one aspect, the disclosure provides a method of detecting an anti-APRIL antibody, the method comprising contacting an antibody molecule described herein with a subject (e.g., administering an antibody molecule described herein to a subject) and determining formation of a complex between the antibody molecule and the subject (e.g., between the antibody molecule and an anti-APRIL antibody in the subject), thereby detecting the anti-APRIL antibody.

[0032] In certain aspects, the disclosure provides methods of evaluating a subject, the method comprising contacting the subject with an antibody molecule described herein (e.g., administering an antibody molecule described herein to the subject) and determining formation of a complex between the antibody molecule and the subject (e.g., between the antibody molecule and an anti-APRIL antibody in the subject), thereby evaluating the subject.

[0033] In certain aspects, the disclosure provides antibody molecules that bind to the same or overlapping epitopes recognized by the antibody molecules described herein.

[0034] In certain aspects, the disclosure provides compositions (e.g., pharmaceutical compositions) comprising an antibody molecule described herein and a pharma- ceutically acceptable carrier, excipient, or stabilizer. In certain embodiments, the compositions are suitable for detecting anti-APRIL antibodies in vitro, ex vivo, or in vivo.

[0035] In certain aspects, the present disclosure provides a kit comprising an antibody molecule described herein and instructions for using the antibody molecule.

[0036] In one aspect, the disclosure provides nucleic acids (e.g., isolated nucleic acids) encoding one or more (e.g., two, three, four, five, or six) CDRs, or the VH, VL, or both, of an antibody molecule described herein.

[0037] In some embodiments, the nucleic acid is humanized, hi some embodiments, the nucleic acid is codon optimized.

[0038] In one embodiment, the nucleic acid comprises nucleotides 52 to 1374 of SEQ ID NO:8, or a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or differing by no more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides therefrom.

[0039] In one embodiment, the nucleic acid comprises nucleotides 52 to 1392 of SEQ ID NO:9, or a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or differing by no more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides therefrom.

[0040] In one embodiment, the nucleic acid comprises nucleotides 52 to 705 of SEQ ID NO:10, or a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or differing by no more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides therefrom.

[0041] In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 10.

[0042] In some aspects, the disclosure provides a vector (e.g., an expression vector) comprising a nucleic acid described herein. In some embodiments, the expression vector comprises a promoter operably linked to the nucleic acid.

[0043] In one aspect, the disclosure provides a cell (e.g., a host cell) comprising a nucleic acid described herein, or a vector described herein.

[0044] In certain aspects, the disclosure provides a method of producing an antibody molecule comprising culturing a host cell described herein under conditions suitable for gene expression. In certain embodiments, the method further comprises isolating or purifying the antibody molecule.

[0045] The present disclosure contemplates all combinations of any one or more of the foregoing aspects and / or embodiments, as well as combinations with any one or more of the embodiments described in the detailed description and examples.

[0046] Other features, objects, and advantages of the compositions and methods herein will become apparent from the specification and drawings, and from the claims. [Brief description of the drawings]

[0047] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1A] Characterization of binding (FIG. 1A) and cross-linking (FIG. 1B) ELISAs is shown. [Figure 1B] Characterization of binding (FIG. 1A) and cross-linking (FIG. 1B) ELISAs is shown. [Figure 2A] Characterization of anti-ID candidates (small scale purification). Results of sibeprenlimab anti-ID binding ELISA (Figure 2A), sibeprenlimab anti-ID bridging ELISA (Figure 2B), and 2419 chimeric IgG1 binding ELISA (Figure 2C) are shown. [Figure 2B] Characterization of anti-ID candidates (small scale purification). Results of sibeprenlimab anti-ID binding ELISA (Figure 2A), sibeprenlimab anti-ID bridging ELISA (Figure 2B), and 2419 chimeric IgG1 binding ELISA (Figure 2C) are shown. [Figure 2C] Characterization of anti-ID candidates (small scale purification). Results of sibeprenlimab anti-ID binding ELISA (Figure 2A), sibeprenlimab anti-ID bridging ELISA (Figure 2B), and 2419 chimeric IgG1 binding ELISA (Figure 2C) are shown. [Figure 3A] Shown are the results of a bridging ELISA (small scale purification) in the presence of 1% human serum. Subclones 1A5 (FIG. 3A), 1H4 (FIG. 3B), and 6A3 (FIG. 3C) were tested. [Figure 3B] Shown are the results of a bridging ELISA (small scale purification) in the presence of 1% human serum. Subclones 1A5 (FIG. 3A), 1H4 (FIG. 3B), and 6A3 (FIG. 3C) were tested. [Figure 3C] Shown are the results of a bridging ELISA (small scale purification) in the presence of 1% human serum. Subclones 1A5 (FIG. 3A), 1H4 (FIG. 3B), and 6A3 (FIG. 3C) were tested. [Figure 4A] 4 shows characterization of 1H4 by binding ELISA (freeze / thaw stability). Results of a comparison of 1H4 binding ELISA lots (FIG. 4A), a stability binding ELISA of 1H4 lot #2 (FIG. 4B), and a stability binding ELISA of 1H4 lot #3 (FIG. 4C) are shown. [Figure 4B] 4 shows characterization of 1H4 by binding ELISA (freeze / thaw stability). Results of a comparison of 1H4 binding ELISA lots (FIG. 4A), a stability binding ELISA of 1H4 lot #2 (FIG. 4B), and a stability binding ELISA of 1H4 lot #3 (FIG. 4C) are shown. [Figure 4C] 4 shows characterization of 1H4 by binding ELISA (freeze / thaw stability). Results of a comparison of 1H4 binding ELISA lots (FIG. 4A), a stability binding ELISA of 1H4 lot #2 (FIG. 4B), and a stability binding ELISA of 1H4 lot #3 (FIG. 4C) are shown. [Figure 5A]Characterization of 1H4 by bridging ELISA (freeze / thaw stability and in the presence of 1% and 20% human serum) is shown. Results of a comparison of 1H4 bridging ELISA lots (FIG. 5A), a stability bridging ELISA of 1H4 lot #2 (FIG. 5B), and a stability bridging ELISA of 1H4 lot #3 (FIG. 5C) are shown. [Figure 5B] Characterization of 1H4 by bridging ELISA (freeze / thaw stability and in the presence of 1% and 20% human serum) is shown. Results of a comparison of 1H4 bridging ELISA lots (FIG. 5A), a stability bridging ELISA of 1H4 lot #2 (FIG. 5B), and a stability bridging ELISA of 1H4 lot #3 (FIG. 5C) are shown. [Figure 5C] Characterization of 1H4 by bridging ELISA (freeze / thaw stability and in the presence of 1% and 20% human serum) is shown. Results of a comparison of 1H4 bridging ELISA lots (FIG. 5A), a stability bridging ELISA of 1H4 lot #2 (FIG. 5B), and a stability bridging ELISA of 1H4 lot #3 (FIG. 5C) are shown. [Figure 6A] A comparison of the binding (FIG. 6A) and cross-linking (FIG. 6B) results between hybridoma 1H4 and recombinant 1H4 is shown. [Figure 6B] A comparison of the binding (FIG. 6A) and cross-linking (FIG. 6B) results between hybridoma 1H4 and recombinant 1H4 is shown. [Figure 7] 1 shows the results of sibeprenlimab anti-idiotype (1H4) specific ELISA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Disclosed herein are antibody molecules that bind to anti-APRIL molecules, such as sibeprenlimab, with high affinity and specificity. The generation of anti-idiotypic antibody (anti-ID) reagents that specifically recognize therapeutic monoclonal antibody (mAb) drugs (e.g., sibeprenlimab) is important to create highly sensitive antibody drug development assays (e.g., pharmacokinetic ELISA, anti-drug assays, and toxicity) for examining preclinical and clinical samples. The reagents described herein were generated using mouse immunization and hybridoma technology in combination with extensive screening for cell lines that produce mAbs specific to the variable region of sibeprenlimab. Appropriate characteristics of anti-ID mAbs include, for example, specificity for the variable region / CDR of the drug (sibeprenlimab) rather than the Fc region, negligible cross-reactivity to other human or animal immunoglobulins, creation of a "bridge" between two molecules of sibeprenlimab for use in anti-drug assay development, and easy and reproducible production. Anti-ID mAbs can be purified, for example, from either 1) hybridoma supernatants, 2) transient transfections of Expi293 cells, or 3) stably expressing CHO cell lines, whichever provides the most reproducible and high-yielding reagent lots.

[0049] definition As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

[0050] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0051] "About" and "approximately" generally refer to an acceptable degree of error for the quantity measured, given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% (e.g., within 4%, 3%, 2%, or 1%) of a given value or range of values.

[0052] The compositions and methods disclosed herein encompass polypeptides and nucleic acids having a designated sequence or a sequence substantially identical or similar thereto, for example, a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or greater to the designated sequence.

[0053] In the context of amino acid sequences, the term "substantially identical" is used herein to refer to a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are a) identical to, or b) conservative substitutions of, aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences may have a common structural domain and / or a common functional activity, e.g., an amino acid sequence that contains a common structural domain that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.

[0054] In the context of nucleotide sequences, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or a minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having a common functional activity or encode a common structural polypeptide domain or a common functional polypeptide activity, e.g., a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.

[0055] The term "functional variant" refers to a polypeptide that has substantially the same amino acid sequence as a naturally occurring sequence, or is encoded by substantially the same nucleotide sequence, and that can have one or more activities of the naturally occurring sequence.

[0056] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

[0057] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). In a typical embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

[0058] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0059] The comparison of sequences and the determination of the percent identity between two sequences can be accomplished using mathematical algorithms. In some embodiments, the percent identity between two amino acid or nucleotide sequences is determined using Clustal Omega (Sievers et al. Mol Syst Biol. 2011; 7:539). In some embodiments, the percent identity between two amino acid or nucleotide sequences is determined using Kalign2 (Lassmann et al. Nucleic Acids Res. 2009; 37(3):858-65, Lassmann and Sonnhammer BMC Bioinformatics. 2005; 6:298). In some embodiments, the percent identity between two amino acid or nucleotide sequences is determined using MAFFT (Katoh and Standley Mol Biol Evol. 2013; 30(4):772-80). In some embodiments, the percent identity between two amino acid or nucleotide sequences is determined using MUSCLE (Edgar Nucleic Acids Res. 2004; 32(5): 1792-7; Edgar BMC Bioinformatics. 2004; 5: 113). In some embodiments, the percent identity between two amino acid or nucleotide sequences is determined using MView (Brown et al. Bioinformatics. 1998; 14(4): 380-1). Other methods for determining the percent identity between two sequences are also described, for example, in Li et al. Nucleic Acids Res. 2015; 43(W1): W580-4; McWilliam et al. Nucleic Acids Res. 2013; 41(Web Server issue): W597-600.

[0060] In one embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J Mol Biol. 1970; 48(3): 443-53) algorithm as incorporated into the GAP program in the GCG software package (available at www.gcg.com) using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6. In one embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.com) using a NWSgapdna.CMP matrix, and a gap weight of 40, 50, 60, 70 or 80, and a length weight of 1, 2, 3, 4, 5 or 6. One suitable set of parameters (which should be used unless otherwise specified) is a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0061] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers and Miller (Comput Appl Biosci. 1988;4(1):11-7) as incorporated into the ALIGN program (version 2.0), using a weight residue table of PAM120, a gap length penalty of 12, and a gap penalty of 4.

[0062] The nucleic acid and protein sequences described herein can be used as "query sequences" to perform searches against public databases to identify, for example, other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. 1990; J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acids described herein. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 1997;25:3389-3402. When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0063] As used herein, the term "hybridize under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and non-aqueous methods are described in this reference, either of which can be used. Specific hybridization conditions referred to herein are as follows: 1) 6x sodium chloride / sodium citrate (sodium chloride / sodium citrate). 1) low stringency hybridization conditions at about 45° C. in 6×SSC, followed by two washes at at least 50° C. in 0.2×SSC, 0.1% SDS (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions at about 45° C. in 6×SSC, followed by one or more washes at 60° C. in 0.2×SSC, 0.1% SDS; 3) high stringency hybridization conditions at about 45° C. in 6×SSC, followed by one or more washes at 65° C. in 0.2×SSC, 0.1% SDS; and preferably 4) very high stringency hybridization conditions at 0.5 M sodium phosphate, 7% SDS, 65° C., followed by one or more washes at 0.2×SSC, 1% SDS, 65° C. Very high stringency conditions 4) are the preferred conditions and are the conditions that should be used unless otherwise specified.

[0064] It is understood that the molecules described herein may have additional conservative or non-essential amino acid substitutions which do not have a substantial effect on their function.

[0065] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that contain both amino and acid functional groups and that can be included in a polymer of naturally occurring amino acids. Exemplary amino acids include the naturally occurring amino acids; their analogs, derivatives and congeners; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes both D or L optical isomers and peptidomimetics.

[0066] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0067] The terms "polypeptide", "peptide" and "protein" (when single chain) are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified by, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Polypeptides can be isolated from natural sources, can be produced by recombinant techniques from eukaryotic or prokaryotic hosts, or can be the product of synthetic procedures.

[0068] The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may be single-stranded or double-stranded, and if single stranded, may be the coding strand or the non-coding (antisense) strand. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. A nucleic acid may be a recombinant polynucleotide or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin, that is not naturally occurring or that is linked to another polynucleotide in a non-natural configuration.

[0069] The term "isolated" as used herein refers to a material that has been removed from its original or natural environment (e.g., the natural environment, if it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide that has been separated by human intervention from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide may be part of a vector and / or such a polynucleotide or polypeptide may be part of a composition, but such a vector or composition is still isolated in that it is not part of the environment in which it is found in nature.

[0070] As used herein, the term "treating" a disorder, e.g., an APRIL-associated disorder, means that a subject (e.g., a human) having a disorder, e.g., an APRIL-associated disorder, and / or experiencing symptoms of the disorder, e.g., an APRIL-associated disorder, will, in an embodiment, experience less severe symptoms and / or recover more quickly when the antibody molecule is administered than when the antibody molecule is not administered. In an embodiment, when an APRIL-associated disorder is treated, the treated subject may have lower levels of APRIL compared to a comparable untreated subject. For example, a diagnostic assay using immunofluorescence or electron microscopy detects APRIL in a subject's biological sample after administering an antibody molecule described herein for effective treatment of an inflammatory disorder. Other assays, e.g., urinalysis, blood tests, ultrasound, x-ray, or cystoscopy, can also be used to monitor treatment in a patient or to detect the presence, e.g., decreased presence (or absence), of symptoms of a disorder, e.g., an APRIL-associated disorder, after treatment of the disorder in a subject. The treatment may, for example, partially or completely, alleviate, ameliorate, reduce, inhibit, or reduce the severity and / or incidence of one or more of the effects or symptoms, characteristics, and / or causes of a disorder, for example, an APRIL-associated disorder, and may optionally delay its onset. In an embodiment, the treatment is of a subject who does not show a specific symptom of a disorder, for example, an APRIL-associated disorder, and / or a subject who shows only early symptoms of a disorder, for example, an APRIL-associated disorder. In an embodiment, the treatment is of a subject who shows one or more established symptoms of a disorder, for example, an APRIL-associated disorder. In an embodiment, the treatment is of a subject who has been diagnosed as suffering from a disorder, for example, an APRIL-associated disorder. In an embodiment, the disorder is an APRIL-associated disorder described herein.

[0071] As used herein, the term "preventing" a disorder, e.g., an APRIL-associated disorder, means that when a subject (e.g., a human) is administered the antibody molecule, the subject is less likely to have the disorder, e.g., an APRIL-associated disorder. In some embodiments, the subject is at risk of developing a disorder, e.g., an APRIL-associated disorder. In some embodiments, the disorder is an APRIL-associated disorder described herein.

[0072] Various aspects of the compositions and methods herein are described in further detail below. Additional definitions are set forth throughout the specification.

[0073] antibody molecule In certain aspects, the disclosure provides antibody molecules that bind to anti-APRIL antibodies (eg, anti-idiotypic antibody molecules).

[0074] As used herein, the term "antibody molecule" refers to a protein that includes at least one immunoglobulin variable domain sequence, e.g., an immunoglobulin chain or fragment thereof. The term "antibody molecule" includes, for example, full length antibodies and antigen-binding fragments of antibodies.

[0075] For example, an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH) and a light (L) chain variable domain sequence (abbreviated herein as VL). In another example, an antibody molecule can include two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen binding sites, e.g., Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single chain antibodies (e.g., scFv or sc(Fv)2), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be generated by modification of whole antibodies or synthesized de novo using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. The antibodies and antibody fragments may be derived from any class of antibody, including but not limited to IgG, IgA, IgM, IgD, and IgE, and any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). The antibody molecule may be monoclonal or polyclonal. In certain embodiments, the antibody molecule is a whole IgG antibody. The antibody molecule may also be a human antibody, a humanized antibody, a CDR-grafted antibody, or an in vitro generated antibody. The antibody molecule may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, IgG4, or a chimera of two or more isotypes. The antibody molecule may also have a light chain selected from, for example, kappa or lambda. The term "immunoglobulin" (Ig) is used interchangeably with the term "antibody" herein. In certain embodiments, the antibody molecule is a multispecific antibody molecule (e.g., a bispecific antibody molecule).

[0076] Examples of antigen-binding fragments include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a diabody (dAb) fragment consisting of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv) (see, e.g., Bird et al. (1988) Science 242:423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); and (viii) a single domain antibody. These antibody fragments can be obtained using any suitable method, including a number of conventional techniques known to those with skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies.

[0077] The term "antibody" includes intact molecules and functional fragments thereof. The constant region of an antibody can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, glycosylation of the antibody, the number of cysteine ​​residues, effector cell function, or complement function).

[0078] In some embodiments, the antibody molecule is a single chain antibody. Single chain antibodies (scFv) may be engineered (see, e.g., Colcher, D. et al. (1999) Ann NY Acad Sci 880:263-80, and Reiter, Y. (1996) Clin Cancer Res 2:245-52). Single chain antibodies can be dimerized or multimerized to generate multivalent antibodies with specificities for different epitopes of the same target protein.

[0079] In an embodiment, the antibody molecule is a single domain antibody. Single domain antibodies may include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. The single domain antibody may be any of the single domain antibodies in the art, or any future single domain antibody. Single domain antibodies may be derived from any species, including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. In an embodiment, the single domain antibody is a naturally occurring single domain antibody known as a heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed, for example, in WO 94 / 04678. For clarity of explanation, this variable domain derived from a heavy chain antibody naturally devoid of light chains is known herein as a VHH or nanobody to distinguish it from the traditional VH of four-chain immunoglobulins. Such VHH molecules may be derived from antibodies raised in Camelidae species, such as camel, llama, dromedary, alpaca and guanaco. Other non-Camelidae species may also produce heavy chain antibodies naturally devoid of light chains, and such VHHs are also within the scope of the present invention.

[0080] The VH and VL regions can be subdivided into regions of hypervariability called "complementarity determining regions" (CDRs) interspersed with more conserved regions called "framework regions" (FR or FW). The terms "complementarity determining region" and "CDR" as used herein refer to sequences of amino acids within an antibody variable region that confer antigen specificity and binding affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. As used herein, the terms "framework", "FW" and "FR" are used interchangeably.

[0081] The extent of the framework regions and CDRs have been precisely defined by several methods (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 ("Kabat" numbering scheme), Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917 ("Chothia" numbering scheme), and the AbM definitions used by Oxford Molecular's AbM antibody modeling software). See generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). As used herein, CDRs defined according to the "Chothia" numbering scheme may also be referred to as "hypervariable loops". Under all definitions, each VH and VL typically contains three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0082] For example, in Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) may be numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues in the light chain variable domain (VL) may be numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia, the CDR amino acids in the VH may be numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in the VL may be numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining both the Kabat and Chothia CDR definitions, the CDRs can consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.

[0083] Generally, unless specifically indicated, the antibody molecules described herein may comprise any combination of one or more Kabat CDRs and / or Chothia hypervariable loops described herein.

[0084] As used herein, an "immunoglobulin variable domain sequence" refers to an amino acid sequence capable of forming the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two or more N-terminal or C-terminal amino acids, or may include other modifications compatible with forming a protein structure.

[0085] The term "antigen-binding region" refers to a portion of an antibody molecule that contains determinants that form an interface that binds to an antigen or epitope thereof. For proteins (or protein mimetics), the antigen-binding region typically includes one or more loops (at least, e.g., of four amino acids or amino acid mimetics) that form an interface that binds to the antigen. Typically, the antigen-binding region of an antibody molecule includes at least one or two CDRs and / or hypervariable loops, or more typically, at least three, four, five or six CDRs and / or hypervariable loops.

[0086] The terms "compete" or "cross-compete" are used interchangeably herein and refer to the ability of a first antibody molecule to interfere with the binding of a second antibody molecule to a target. The interference with binding can be direct or indirect (e.g., via allosteric modulation of the antibody molecule or the target). The extent to which an antibody molecule can interfere with the binding of another antibody molecule to a target, and therefore whether it can be said to compete, can be determined using a competitive binding assay, such as a FACS assay, an ELISA, or a BIACORE assay. In an embodiment, the competitive binding assay is a quantitative competitive assay. In certain embodiments, a first antibody molecule is said to compete with a second antibody molecule for binding to a target if binding of the first antibody molecule to the target is reduced by 10% or more, e.g., 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more in a competitive binding assay (e.g., a competitive assay described herein).

[0087] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be made by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

[0088] A "virtually human" protein is one that does not elicit a neutralizing antibody response, such as a human anti-murine antibody (HAMA) response. HAMA can be problematic in some situations, for example, when an antibody molecule is administered repeatedly, for example, in the treatment of a chronic or recurrent disease state. HAMA responses can potentially render repeated antibody administration ineffective due to increased clearance of the antibody from serum and the possibility of allergic reactions (see, e.g., Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990); LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

[0089] The antibody molecule may be a polyclonal or monoclonal antibody. In certain embodiments, the antibody may be recombinantly produced, for example by any suitable phage display or combinatorial method.

[0090] A variety of phage display and combinatorial methods for generating antibodies are known in the art (see, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. WO 92 / 18619; Dower et al. WO 91 / 17271; Winter et al. WO 92 / 20791; Markland et al. WO 92 / 15679; Breitling et al. WO 93 / 01288; McCafferty et al. WO 92 / 01047; Garrard et al. WO 92 / 09690; Ladner et al. WO 90 / 02809; Fuchs et al. (1991) Bio / Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85, Huse et al. (1989) Science 246:1275-1281, Griffths et al. (1993) EMBO J 12:725-734, Hawkins et al. (1992) J Mol Biol 226:889-896, Clackson et al. (1991) Nature 352:624-628, Gram et al. (1992) PNAS 89:3576-3580, Garrad et al. (1991) Bio / Technology 9:1373-1377, Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137, and Barbas et al. (1991) PNAS 88:7978-7982, the entire contents of which are incorporated herein by reference.

[0091] In some embodiments, the antibody molecule is a fully human antibody (e.g., an antibody made in a mouse genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody, e.g., a rodent (e.g., mouse or rat), goat, primate (e.g., monkey), or camel antibody. In some embodiments, the non-human antibody is a rodent antibody (e.g., a mouse or rat antibody). Methods for generating rodent antibodies are known in the art.

[0092] Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Spleen cells from these transgenic mice immunized with an antigen of interest are used to generate hybridomas secreting human mAbs with specific affinity for epitopes derived from human proteins (see, e.g., Wood et al. WO 91 / 00906; Kucherlapati et al. PCT WO 91 / 10741; Lonberg et al. WO 92 / 03918; Kay et al. WO 92 / 03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21; Morrison, S. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

[0093] The antibody may be one in which the variable region or a part thereof, such as the CDR, is made in a non-human organism, such as a rat or a mouse. Chimeric antibodies, CDR-grafted antibodies, and humanized antibodies are within the scope of the present invention. Antibodies made in a non-human organism, such as a rat or a mouse, and then modified, for example, in the variable framework or constant region, to reduce antigenicity in humans, are within the scope of the present invention.

[0094] Chimeric antibodies can be produced by any suitable recombinant DNA technique. Some are known in the art (Robinson et al., WO 1987 / 002671; Akira, et al., EP 184,187; Taniguchi, M., EP 171,496; Morrison et al., EP 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., EP 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (see, e.g., Nishimura et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0095] A humanized antibody or a CDR-grafted antibody has at least one or two, but typically all three, recipient CDRs (of the immunoglobulin heavy and / or light chain) replaced with donor CDRs. The antibody may have at least a portion of a non-human CDR replaced, or only a portion of the CDR replaced with a non-human CDR. It is only necessary to replace as many CDRs as are required for the binding of the humanized antibody to lipopolysaccharide. In one embodiment, the donor is a rodent antibody, such as a rat or mouse antibody, and the recipient is a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDRs is called the "donor" and the immunoglobulin providing the framework is called the "acceptor". In one embodiment, the donor immunoglobulin is non-human (e.g., rodent). The acceptor framework is typically a naturally occurring (e.g., human) framework or consensus framework, or a sequence that is about 85% or more identical thereto, such as 90%, 95%, 99% or more identical thereto.

[0096] As used herein, the term "consensus sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either may be included in the consensus sequence. "Consensus framework" refers to the framework region in the consensus immunoglobulin sequence.

[0097] Antibodies can be humanized by any suitable method, several such methods are known in the art (see, e.g., Morrison, SL, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; and Queen et al. U.S. Pat. Nos. 5,585,089, 5,693,761, and 5,693,762, the entire contents of which are incorporated herein by reference).

[0098] Humanized or CDR-grafted antibodies can be produced by CDR grafting or CDR replacement, where one, two or all CDRs of an immunoglobulin chain can be replaced. See, for example, U.S. Patent No. 5,225,539, Jones et al. 1986 Nature 321:552-525, Verhoeyan et al. 1988 Science 239:1534, Beidler et al. 1988 J.Immunol.141:4053-4060, Winter U.S. Patent No. 5,225,539, the contents of all of which are expressly incorporated herein by reference. Winter describes a CDR grafting method that can be used to prepare humanized antibodies (UK Patent Application No. 2188638(A) filed March 26, 1987, Winter U.S. Patent No. 5,225,539), the contents of which are expressly incorporated herein by reference.

[0099] Humanized antibodies in which specific amino acids are substituted, deleted or added are also provided. Criteria for selecting donor-derived amino acids are described, for example, in U.S. Patent No. 5,585,089, e.g., columns 12-16 of U.S. Patent No. 5,585,089, the contents of which are incorporated herein by reference. Other techniques for humanizing antibodies are described in Padlan et al., European Patent No. 519596(A1), published December 23, 1992.

[0100] In certain embodiments, the antibody molecule has a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE heavy chain constant regions, in particular, for example, IgG1, IgG2, IgG3 and IgG4 (e.g., human) heavy chain constant regions. In another embodiment, the antibody molecule has a light chain constant region selected from, for example, kappa or lambda (e.g., human) light chain constant regions. The constant region can be altered, e.g., mutated, to modify the properties of the antibody molecule (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, and / or complement function). In certain embodiments, the antibody molecule has effector function and can fix complement. In another embodiment, the antibody molecule does not recruit effector cells or fix complement. In certain embodiments, the antibody molecule has a reduced or no ability to bind Fc receptors. For example, it may be an isotype or subtype, fragment or other variant that does not support binding to an Fc receptor, eg, has a mutated or deleted Fc receptor binding region.

[0101] In some embodiments, the constant region of the antibody molecule is altered. Methods for altering antibody constant regions are known in the art. Antibody molecules with altered function, for example, altered affinity for effector ligands such as FcR on cells or the C1 component of complement, can be generated by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, for example, European Patent No. 388,151(A1), U.S. Patent Nos. 5,624,821 and 5,648,260, the entire contents of which are incorporated herein by reference). Amino acid mutations that stabilize antibody structure, such as S228P (S241P in EU nomenclature, Kabat nomenclature) in human IgG4, are also contemplated. Similar types of alterations can be described that would reduce or eliminate these functions when applied to mouse or other species of immunoglobulins.

[0102] In some embodiments, the amino acids in the antibody molecule are only standard amino acids. In some embodiments, the antibody molecule includes naturally occurring amino acids; their analogs, derivatives and congeners; amino acid analogs with variant side chains; and / or all stereoisomers of any of the foregoing. The antibody molecule may include D- or L-optical isomers of amino acids and peptidomimetics.

[0103] In some embodiments, an antibody molecule comprises a monoclonal antibody (e.g., a full-length antibody having an immunoglobulin Fc region). In some embodiments, an antibody molecule comprises a full-length antibody or a full-length immunoglobulin chain. In some embodiments, an antibody molecule comprises an antigen-binding or functional fragment of a full-length antibody or a full-length immunoglobulin chain.

[0104] In certain embodiments, the antibody molecule is a monospecific antibody molecule, e.g., it binds to a single epitope. For example, a monospecific antibody molecule can have multiple immunoglobulin variable region sequences that each bind to the same epitope.

[0105] In an embodiment, the antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable region sequences, a first immunoglobulin variable region sequence of the plurality having binding specificity for a first epitope and a second immunoglobulin variable region sequence of the plurality having binding specificity for a second epitope. In an embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap. In an embodiment, the first and second epitopes do not overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In an embodiment, the multispecific antibody molecule comprises a third, fourth, or fifth immunoglobulin variable domain. In an embodiment, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0106] In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is typically characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody molecule comprises a heavy chain variable region sequence and a light chain variable region sequence having binding specificity for a first epitope and a heavy chain variable region sequence and a light chain variable region sequence having binding specificity for a second epitope. In certain embodiments, a bispecific antibody molecule comprises a half antibody that has binding specificity for a first epitope and a half antibody that has binding specificity for a second epitope. In certain embodiments, a bispecific antibody molecule comprises a half antibody, or fragment thereof, that has binding specificity for a first epitope and a half antibody, or fragment thereof, that has binding specificity for a second epitope. In certain embodiments, a bispecific antibody molecule comprises an scFv, or fragment thereof, that has binding specificity for a first epitope and an scFv, or fragment thereof, that has binding specificity for a second epitope.

[0107] Protocols for making bispecific or heterodimeric antibody molecules are known in the art and include, for example, the "knobs-in-holes" approach as described in U.S. Pat. No. 5,731,168; electrostatic steering Fc pairing as described in, for example, WO 09 / 089004, WO 06 / 106905, and WO 2010 / 129304; Strand Exchange Engineered Domains as described in, for example, WO 07 / 110205; Fab arm exchange, e.g., as described in WO 08 / 119353, WO 2011 / 131746, and WO 2013 / 060867; bispecific antibody conjugates by antibody crosslinking, e.g., using heterobifunctional reagents with amine-reactive groups and sulfhydryl-reactive groups to create bispecific structures, e.g., as described in U.S. Pat. No. 4,433,059; bispecific antibody determinants created by recombining half-antibodies (heavy-light chain pairs or Fabs) from different antibodies through cycles of reduction and oxidation of the disulfide bond between the two heavy chains, e.g., as described in U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., via sulfhydryl-reactive groups, e.g., as described in U.S. Pat. No. 5,273,743. three cross-linked Fab' fragments; biosynthetic binding proteins, e.g. as described in US Pat. No. 5,534,254, e.g. pairs of scFv cross-linked via their C-terminal tails, preferably via disulfide or amine reactive chemical cross-linking; bifunctional antibodies, e.g. Fab fragments with different binding specificities dimerized via leucine zippers (e.g. c-fos and c-jun) replacing the constant domains, e.g. as described in US Pat. No. 5,582,996; bispecific and oligospecific mono- and oligovalent receptors, e.g. the VH-CH1 regions of two antibodies (two Fab fragments) linked via a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody, typically with associated light chains, e.g. as described in US Pat. No. 5,591,828;Bispecific DNA-antibody conjugates, e.g., cross-linking of antibodies or Fab fragments via a double-stranded piece of DNA, as described, for example, in U.S. Pat. No. 5,635,602; bispecific fusion proteins, e.g., expression constructs containing two scFvs with a hydrophilic helical peptide linker between them and a complete constant region, as described, for example, in U.S. Pat. No. 5,637,481; multivalent and multispecific binding proteins, e.g., dimers of polypeptides having a first domain with a binding region of an Ig heavy chain variable region and a second domain with a binding region of an Ig light chain variable region, commonly referred to as diabodies (also higher order structures creating bispecific, trispecific or tetraspecific molecules are disclosed); minibodies in which linked VL and VH chains are further connected by peptide spacers to antibody hinge and CH3 regions, which can dimerize to form bispecific / multivalent molecules, as described, for example, in U.S. Pat. No. 5,837,821. diconstructs; VH and VL domains linked by short peptide linkers (e.g., 5 or 10 amino acids) in either orientation or no linker at all, which can form dimers to form bispecific diabodies; trimers and tetramers, e.g., as described in U.S. Pat. No. 5,844,094; consecutive VH domains (or VL domains in family members) connected by peptide linkages at C-terminal crosslinking groups and further associated with VL domains to form a series of FVs (or scFvs), e.g., as described in U.S. Pat. No. 5,864,019; and single-chain binding polypeptides having both VH and VL domains linked via peptide linkers, which are combined into multivalent structures through non-covalent or chemical crosslinking, e.g., using both scFv or diabody-type formats to form homo-, hetero-, tri-, and tetravalent structures, e.g., as described in U.S. Pat. No. 5,869,620. The contents of the above-referenced applications are incorporated herein by reference in their entirety;

[0108] Further methods for making multispecific or bispecific antibody molecules are described in, e.g., U.S. Patent Nos. 5,910,573, 5,932,448, 5,959,083, 5,989,830, 6,005,079, 6,239,259, 6,294,353, 6,333,396, 6,476,198, 6,511,663, 6,670,453, 6,743,896, 6,809,185, 6,833,441, 7,129,330, 7,183,076, 7,521,056, 7,527,787, 7,534,866, 7,542,872, 7,552,882, 7,562,892, 7,572,892, 7,582,892, 7,623,198, 6,742,198, 6,511,663, 6,670,453, 6,742,892, 7,572,872, 7,582,892, 7,534,866, 7,542,872, 7,552,882, 7,562,892, 7,572,892, 7,582,892, 7,592,90 ... No. 7612181, U.S. Patent Application Publication Nos. 2002 / 004587, 2002 / 076406, 2002 / 103345, 2003 / 207346, 2003 / 211078, 2004 / 219643, 2004 / 220388, 2004 / 242847, 2005 / 003403, 2005 / 004352, 2005 / 069552, 2005 / 079170, 2005 / 100543, 2005 / 136049, 2005 / 136051, 2005 / 163782, 2005 / 266425, 2006 / 083747, 2006 / 120960, 2006 / 20 No. 4493, No. 2006 / 263367, No. 2007 / 004909, No. 2007 / 087381, No. 2007 / 1281 No. 50, No. 2007 / 141049, No. 2007 / 154901, No. 2007 / 274985, No. 2008 / 050370 No. 2008 / 069820, No. 2008 / 152645, No. 2008 / 171855, No. 2008 / 241884, No. 2008 / 254512, No. 2008 / 260738, No. 2009 / 130106, No. 2009 / 148905, No. 2009 / 155275, No. 2009 / 162359, No. 2009 / 162360, No. 2009 / 175851, No. 2009 / 175867, No. 2009 / 232811, No. 2009 / 234105, No. 2009 / 263392, No. 2009 / 274649, European Patent No. 346087, International Publication No. 00 / 06605, International Publication No. 02 / 072635, International Publication No. 04 / 081051,Nos. 06 / 020258, 2007 / 044887, 2007 / 095338(A2), 2007 / 137760(A2), 2008 / 119353, 2009 / 021754, 2009 / 068630, 91 / 03493, 93 / 23537, 94 / 09131, 94 / 12625, 95 / 09917, 96 / 37621, 99 / 64460. The contents of the above-referenced applications are incorporated herein by reference in their entirety.

[0109] The polypeptides of the antibody molecules described herein may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. Antibody molecules may also be modified by, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Polypeptides may be isolated from natural sources, may be produced by recombinant techniques from eukaryotic or prokaryotic hosts, or may be the product of synthetic procedures.

[0110] The antibody molecules described herein may be used alone in unconjugated form or may be conjugated to a substance, e.g., a toxin or moiety (e.g., a therapeutic agent; a compound that emits radiation; a molecule of plant, fungal, or bacterial origin; or a biological protein (e.g., a protein toxin) or particle (e.g., via a viral coat protein, e.g., a recombinant viral particle). For example, the antibody molecule may be conjugated to a radioactive isotope, such as an alpha, beta, or gamma emitter, or beta and gamma emitters.

[0111] An antibody molecule may be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a "derivatized" antibody molecule is an antibody molecule that has been modified. Methods of derivatization include, but are not limited to, the addition of fluorescent moieties, radionucleotides, toxins, enzymes, or affinity ligands, such as biotin. Thus, antibody molecules are intended to include derivatized and otherwise modified forms of the antibodies described herein, such as immunoadhesin molecules. For example, an antibody molecule may be functionally linked (by chemical coupling, genetic fusion, non-covalent binding, or other methods) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a toxin, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of an antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0112] Several types of derivatized antibody molecules are produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include crosslinkers that are heterobifunctional, having two distinctly different reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional crosslinkers (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0113] Useful detection agents with which the anti-dengue antibody molecule can be derivatized (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, fluorescent metal atoms such as europium (Eu) and other anthanides, and radioactive materials (described below). Exemplary fluorescent detection agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5 dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. Antibodies can also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, acetylcholinesterase, glucose oxidase, and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detection agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that is detectable. Antibody molecules may also be derivatized with prosthetic groups (e.g., streptavidin / biotin and avidin / biotin). For example, an antibody can be derivatized with biotin and detected through indirect measurement of avidin or streptavidin binding. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin, an example of a luminescent material is luminol, and examples of bioluminescent materials include luciferase, luciferin, and aequorin.

[0114] Labeled antibody molecules can be used diagnostically and / or experimentally in a number of situations, for example: (i) to isolate a given antigen by standard techniques, such as affinity chromatography or immunoprecipitation; (ii) to detect a given antigen (e.g., in a cell lysate or cell supernatant) to assess protein abundance and expression patterns; (iii) to monitor protein levels in tissues as part of a clinical trial procedure, for example, to determine the efficacy of a given treatment regimen.

[0115] The antibody molecules described herein may be conjugated to another molecular entity, typically a label or a therapeutic (e.g., an antimicrobial (e.g., antibacterial or bactericidal), immunomodulatory, immunostimulatory, cytotoxic, or cytostatic) agent or moiety. Radioisotopes can be used for diagnostic or therapeutic applications. Radioisotopes that can be attached to antibody molecules include, but are not limited to, alpha, beta, or gamma emitters, or beta and gamma emitters. Such radioisotopes include iodine ( 131 I or 125 I), yttrium ( 90 Y), lutetium ( 177 Lu), Actinium ( 225 Ac), praseodymium, astatine ( 211 At), Rhenium ( 186 Re), Bismuth ( 212 Bi or 213 Bi), Indium ( 111 In), Technetium ( 99 mTc), phosphorus ( 32 P), Rhodium ( 188 Rh), Sulfur ( 35 S), carbon ( 14 C), tritium ( 3 H), Chromium ( 51 Cr), Chlorine ( 36 Cl), Cobalt ( 57 Co or 58 Co), Iron ( 59 Fe), Selenium ( 75 Se), or Gallium ( 67Radioisotopes useful as therapeutic agents include, but are not limited to, yttrium ( 90 Y), lutetium ( 177 Lu), Actinium ( 225 Ac), praseodymium, astatine ( 211 At), Rhenium ( 186 Re), Bismuth ( 212 Bi or 213 Bi), and rhodium ( 188 For example, radioisotopes useful as labels for use in diagnostics include iodine ( 131 I or 125 I), Indium ( 111 In), Technetium ( 99 mTc), phosphorus ( 32 P), carbon ( 14 C), and tritium ( 3 H), or one or more of the therapeutic isotopes listed above.

[0116] The present disclosure provides radiolabeled antibody molecules and methods for labeling the same. In certain embodiments, a method for labeling an antibody molecule is disclosed. The method includes contacting the antibody molecule with a chelating agent, thereby producing a conjugated antibody. The conjugated antibody is a chelating agent that is chelating with a radioisotope, e.g., 111 indium, 90 Yttrium, and 177 It is radioactively labeled with lutetium, thereby producing a labeled antibody molecule.

[0117] In certain embodiments, the antibody molecule is conjugated to a therapeutic agent.Therapeutically active radioisotopes are disclosed herein. Examples of other therapeutic agents include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, such as maytansinol (see, e.g., U.S. Pat. No. 5,208,020), CC-1065 (see, e.g., U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545), and analogs or homologs thereof. Therapeutic agents include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II). These include, but are not limited to, cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0118] In certain embodiments, the antibody molecule (e.g., a monospecific, bispecific or multispecific antibody molecule) is covalently linked, e.g., fused, to another partner, e.g., a protein, e.g., as a fusion molecule (e.g., a fusion protein).

[0119] As used herein, "fusion protein" and "fusion polypeptide" refer to a polypeptide having at least two moieties covalently linked to each other, where each of the moieties is a polypeptide. In certain embodiments, each of the moieties is a polypeptide having a different property. The property can be a biological property, e.g., activity in vitro or in vivo. The property can also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. The two moieties can be linked directly by a single peptide bond or through a linker (e.g., a peptide linker), but are in reading frame with each other.

[0120] In one aspect, the disclosure features a method for providing an antibody molecule that specifically binds to a target protein. The method may include providing a protein that includes at least a portion of a target protein that is homologous to the corresponding portion of the target protein (e.g., at least 70, 75, 80, 85, 87, 90, 92, 94, 95, 96, 97, 98% identical) but differs by at least one amino acid (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids), obtaining an antibody molecule that specifically binds to the target protein, and evaluating the effectiveness of the antibody molecule in modulating the activity of the target protein. The method may further include contacting the antibody molecule or derivative with, for example, a sample from a subject. The method may further include administering the antibody molecule or derivative to a subject, for example, a human.

[0121] In another aspect, the disclosure provides a method of making an antibody molecule disclosed herein. The method may include providing an antigen or a fragment thereof, obtaining an antibody molecule that specifically binds to the antigen, and evaluating the effectiveness of the antibody molecule in modulating the activity of the antigen and / or an organism expressing the antigen. The method may further include contacting the antibody molecule or derivative with, for example, a sample from a subject. The method may further include administering the antibody molecule or derivative to a subject, for example, a human.

[0122] The present disclosure provides nucleic acids encoding the antibody molecules described herein, as well as vectors and host cells containing the nucleic acids. Nucleic acids include, but are not limited to, RNA, genomic DNA, and cDNA.

[0123] The amino acid and nucleotide sequences of exemplary antibody molecules are set forth below.

[0124] Amino acid sequence of 1H4_VH (CDR sequences are underlined)

[0125] [Table 1]

[0126] [Table 2]

[0127] Amino acid sequence of 1H4_Vk (CDR sequences are underlined)

[0128] [Table 3]

[0129] [Table 4]

[0130] Amino acid sequence of 1H4_VH_IgG1 (Osteo Leader: underlined; 1H4_VH: standard; linker: underlined and italic; mouse IgG1 constant region: italic; CDR sequence: bold and underlined)

[0131] [Table 5]

[0132] Amino acid sequence of 1H4_VH_IgG2 (Osteo Leader: underlined; 1H4_VH: standard; linker: underlined and italic; mouse IgG2 constant region: italic; CDR sequence: bold and underlined)

[0133] [Table 6]

[0134] Amino acid sequence of 1H4_Vk (Osteo Leader: underlined; 1H4_VH: standard; kappa constant region: italics; CDR sequences: bold and underlined)

[0135] [Table 7]

[0136] Nucleotide sequence of 1H4_VH (CDR-encoding sequences are bold and underlined)

[0137] [Table 8]

[0138] [Table 9]

[0139] Nucleotide sequence of 1H4_Vk (CDR-encoding sequences are bold and underlined)

[0140] [Table 10]

[0141] [Table 11]

[0142] Nucleotide sequence of 1H4_VH_IgG1 (Osteo Leader: underlined; 1H4_VH: standard; linker: underlined and italicized; mouse IgG1 constant region: italicized; CDR coding sequence: bold and underlined)

[0143] [Table 12]

[0144] Nucleotide sequence of 1H4_VH_IgG2 (Osteo Leader: underlined; 1H4_VH: standard; linker: underlined and italicized; mouse IgG2 constant region: italicized; CDR coding sequence: bold and underlined)

[0145] [Table 13]

[0146] Nucleotide sequence of 1H4_Vk (Osteo Leader: underlined; 1H4_VH: standard; kappa constant region: italics; CDR coding sequences: bold and underlined)

[0147] [Table 14]

[0148] In some embodiments, the antibody molecule comprises one, two or three CDRs of the VH region of a monoclonal antibody described herein (e.g., any of monoclonal antibodies 1H4, 1A5, 6A3, 1B8, 1E5, 6C1, 3F4, 6C12, 2F5 or 6H5), using the Kabat or Chothia definitions of CDRs. In some embodiments, the antibody molecule comprises one, two or three CDRs of the VL region of a monoclonal antibody described herein (e.g., any of monoclonal antibodies 1H4, 1A5, 6A3, 1B8, 1E5, 6C1, 3F4, 6C12, 2F5 or 6H5), using the Kabat or Chothia definitions of CDRs. In one embodiment, the antibody molecule comprises one or more (e.g., two or three) CDRs of the VH region and / or one or more (e.g., two or three) CDRs of the VL region of a monoclonal antibody described herein (e.g., any of monoclonal antibodies 1H4, 1A5, 6A3, 1B8, 1E5, 6C1, 3F4, 6C12, 2F5, or 6H5), using the Kabat or Chothia definitions of CDRs.

[0149] In some embodiments, an antibody molecule comprises one, two, or three VH CDRs as described herein. In some embodiments, an antibody molecule comprises one, two, or three VL CDRs as described herein. In some embodiments, an antibody molecule comprises one or more (e.g., two or three) VH CDRs and / or one or more (e.g., two or three) VL CDRs as described herein.

[0150] In certain embodiments, an antibody molecule comprises one, two, or three VH CDRs comprising the amino acid sequences of SEQ ID NOs: 11, 12, and / or 13, or amino acid sequences that have no more than one, two, or three amino acid differences therefrom. In certain embodiments, an antibody molecule comprises a set of three VH CDRs comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 13, or amino acid sequences that have no more than one, two, or three amino acid differences therefrom. In certain embodiments, an antibody molecule comprises one, two, or three VH CDRs comprising the amino acid sequences of SEQ ID NOs: 11, 12, and / or 13. In certain embodiments, an antibody molecule comprises a set of three VH CDRs comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 13.

[0151] In certain embodiments, an antibody molecule comprises one, two, or three VL CDRs comprising the amino acid sequences of SEQ ID NOs: 14, 15, and / or 16, or amino acid sequences that have no more than one, two, or three amino acid differences therefrom. In certain embodiments, an antibody molecule comprises a set of three VL CDRs comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 16, or amino acid sequences that have no more than one, two, or three amino acid differences therefrom. In certain embodiments, an antibody molecule comprises one, two, or three VL CDRs comprising the amino acid sequences of SEQ ID NOs: 14, 15, and / or 16. In certain embodiments, an antibody molecule comprises a set of three VL CDRs comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 16.

[0152] In certain embodiments, an antibody molecule comprises one, two, or three VH CDRs comprising an amino acid sequence encoded by SEQ ID NO: 17, 18, and / or 19, or a nucleic acid sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences therefrom. In certain embodiments, an antibody molecule comprises a set of three VH CDRs comprising CDR sequences encoded by SEQ ID NO: 17, 18, and 19, or a nucleic acid sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences therefrom. In certain embodiments, an antibody molecule comprises one, two, or three VH CDRs comprising an amino acid sequence encoded by SEQ ID NO: 17, 18, and / or 19. In certain embodiments, an antibody molecule comprises a set of three VH CDRs comprising CDR sequences encoded by SEQ ID NO: 17, 18, and 19.

[0153] In some embodiments, an antibody molecule comprises one, two, or three VL CDRs comprising an amino acid sequence encoded by SEQ ID NO: 20, 21, and / or 22, or a nucleic acid sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences therefrom. In some embodiments, an antibody molecule comprises a set of three VL CDRs comprising CDR sequences encoded by SEQ ID NO: 20, 21, and 22, or a nucleic acid sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences therefrom. In some embodiments, an antibody molecule comprises one, two, or three VL CDRs comprising an amino acid sequence encoded by SEQ ID NO: 20, 21, and / or 22. In some embodiments, an antibody molecule comprises a set of three VL CDRs comprising CDR sequences encoded by SEQ ID NO: 20, 21, and 22.

[0154] In some embodiments, the antibody molecule comprises one, two, three or four frameworks of the VH region of a monoclonal antibody described herein (e.g., any of monoclonal antibodies 1H4, 1A5, 6A3, 1B8, 1E5, 6C1, 3F4, 6C12, 2F5 or 6H5). In some embodiments, the antibody molecule comprises one, two, three or four frameworks of the VL region of a monoclonal antibody described herein (e.g., any of monoclonal antibodies 1H4, 1A5, 6A3, 1B8, 1E5, 6C1, 3F4, 6C12, 2F5 or 6H5). In certain embodiments, the antibody molecule comprises one or more (e.g., two, three or four) frameworks of the VH region and / or one or more (e.g., two, three or four) frameworks of the VL region of a monoclonal antibody described herein (e.g., any of monoclonal antibodies 1H4, 1A5, 6A3, 1B8, 1E5, 6C1, 3F4, 6C12, 2F5, or 6H5).

[0155] In some embodiments, the antibody molecule comprises a heavy chain variable region of a monoclonal antibody described herein (e.g., any of monoclonal antibodies 1H4, 1A5, 6A3, 1B8, 1E5, 6C1, 3F4, 6C12, 2F5, or 6H5). In some embodiments, the antibody molecule comprises a light chain variable region of a monoclonal antibody described herein (e.g., any of monoclonal antibodies 1H4, 1A5, 6A3, 1B8, 1E5, 6C1, 3F4, 6C12, 2F5, or 6H5). In some embodiments, the antibody molecule comprises a heavy chain variable region and a light chain variable region of a monoclonal antibody described herein (e.g., any of monoclonal antibodies 1H4, 1A5, 6A3, 1B8, 1E5, 6C1, 3F4, 6C12, 2F5, or 6H5).

[0156] In some embodiments, an antibody molecule comprises a heavy chain variable region having an amino acid sequence described herein, or a substantially identical amino acid sequence thereof. In some embodiments, an antibody molecule comprises a light chain variable region having an amino acid sequence described herein, or a substantially identical amino acid sequence thereof. In some embodiments, an antibody molecule comprises a heavy chain variable region having an amino acid sequence described herein (or a substantially identical amino acid sequence thereof) and a light chain variable region having an amino acid sequence described herein.

[0157] In some embodiments, an antibody molecule comprises a heavy chain having an amino acid sequence described herein, or a substantially identical amino acid sequence thereof. In some embodiments, an antibody molecule comprises a light chain having an amino acid sequence described herein, or a substantially identical amino acid sequence thereof. In some embodiments, an antibody molecule comprises a heavy chain having an amino acid sequence described herein (or a substantially identical amino acid sequence thereof) and a light chain having an amino acid sequence described herein.

[0158] In some embodiments, an antibody molecule comprises a heavy chain variable region encoded by a nucleotide sequence described herein, or a substantially identical nucleotide sequence thereof. In some embodiments, an antibody molecule comprises a light chain variable region encoded by a nucleotide sequence described herein, or a substantially identical nucleotide sequence thereof. In some embodiments, an antibody molecule comprises a heavy chain variable region encoded by a nucleotide sequence described herein (or a substantially identical nucleotide sequence thereof) and a light chain variable region encoded by a nucleotide sequence described herein (or a substantially identical nucleotide sequence thereof).

[0159] In some embodiments, the antibody molecule further comprises a heavy chain constant region. In some embodiments, the heavy chain constant region is an IgG1 constant region or a functional portion thereof. In another embodiment, the heavy chain constant region is an IgG2 constant region or a functional portion thereof. In some embodiments, the antibody molecule further comprises a light chain constant region. In some embodiments, the antibody molecule further comprises a heavy chain constant region. In some embodiments, the heavy chain constant region is an IgG3 constant region or a functional portion thereof. In some embodiments, the antibody molecule further comprises a heavy chain constant region. In some embodiments, the heavy chain constant region is an IgG4 constant region or a functional portion thereof. In some embodiments, the antibody molecule further comprises a heavy chain constant region and a light chain constant region. In some embodiments, the antibody molecule comprises the heavy chain constant region, light chain constant region, and heavy and light chain variable regions of a monoclonal antibody described herein (e.g., any of monoclonal antibodies 1H4, 1A5, 6A3, 1B8, 1E5, 6C1, 3F4, 6C12, 2F5, or 6H5). In one embodiment, the antibody molecule comprises a heavy chain constant region, a light chain constant region, and a variable region comprising one, two, three, four, five, or six CDRs of a monoclonal antibody described herein (e.g., any of monoclonal antibodies 1H4, 1A5, 6A3, 1B8, 1E5, 6C1, 3F4, 6C12, 2F5, or 6H5).

[0160] In some embodiments, the antibody molecule further comprises a linker, e.g., a linker described herein. In some embodiments, the antibody molecule further comprises a leader, e.g., a leader described herein.

[0161] APRIL APRIL (A Proliferation Inducing Ligand), also known as CD256, TNF-and APOL-related Leukocyte Expressed Ligand 2 (TALL-2), or TNF-related Death Ligand 1 (TRDL-1), is a TNF family cytokine encoded by the Tumor Necrosis Factor Ligand Superfamily Member 13 (TNFSF13) gene (also known as APRIL, TALL2, or ZTNF2). APRIL plays a role in several biological processes, such as signal transduction, regulation of cell proliferation, and IgA class switching (Hahne et al. (1998) J. Exp. Med. 188:1185-1190 (1998); Castigli et al. Proc. Natl. Acad. Sci. USA 101:3903-3908 (2004)).

[0162] APRIL is functionally and structurally related to BAFF (B Cell Activating Factor F13B), also known as BLyS (B lymphocyte stimulator). Both cytokines are involved in regulating important aspects of innate and adaptive immune functions. Both APRIL and BAFF bind to lymphocyte receptors TACI (transmembrane activator and CAML interactor) and BCMA (B cell maturation antigen). APRIL and BAFF appear to heterologously interact with each other through protein-protein interactions. Although both APRIL and BAFF share biochemical (receptor binding), immunological, and even some structural overlap (e.g., related to the three-dimensional topology of their respective receptor binding domains), the two cytokines are nevertheless structurally and functionally distinct. APRIL binds to biologically relevant heparan sulfate (present in the extracellular matrix of cells as heparan sulfate proteoglycans), whereas BAFF does not. This interaction serves an important biological function in promoting the oligomerization state of APRIL in concert with its localized interaction with TACI, which also requires HSPGS for full activity. More specifically, unlike BAFF, which acts as a potent activator of B cells, including both proliferation and differentiation, APRIL appears to function in regulating B cell phenotype, as it is associated with, for example, IgA production and differentiation / survival of IgA-positive plasma cells. Thus, targeted disruption of APRIL-receptor signaling is expected to have less disruptive effects on B cell homeostasis and overall immune function, compared to other immune-related therapeutics that target BAFF (e.g., belimumab), or anti-CD20 therapies that primarily target pre-B and early B cells (e.g., rituximab).APRIL has also been shown to be expressed at high levels in other bone marrow-related cells and lymphoid tissues, as well as in hematological cancers (e.g., myeloma, chronic lymphocytic leukemia (CLL)) and solid tumors (e.g., colon, thyroid, and breast).

[0163] Exemplary amino acid and nucleotide sequences of human APRIL are described, for example, in Hahne et al. J. Exp. Med. 188:1185-1190 (1998), Shu et al. J. Leukoc. Biol. 65:680-683 (1999), Kelly et al. Cancer Res. 60:1021-1027 (2000), and Pradette-Balade et al. EMBO J. 21:5711-5720 (2002). Other variants and alternative sequences of human APRIL are described, for example, in The MGC Project Team, Genome Res. 14:2121-2127 (2004), Ota et al. Nat. Genet. 36:40-45 (2004), and Kelly et al. Cancer Res. 60:1021-1027 (2000). Exemplary amino acid and nucleotide sequences of human and mouse APRIL are also described, for example, in WO 2017 / 091683, the contents of which are incorporated by reference in their entirety.

[0164] Animal models The antibody molecules described herein can be evaluated in vivo, for example, using various animal models. For example, animal models can be used to test the pharmacokinetic and / or pharmacodynamic properties of the antibody molecules described herein.

[0165] Exemplary animal models of IgA nephropathy that can be used to evaluate the antibody molecules described herein include the ddY mouse model of spontaneous IgA nephritis (Imai et al. Kidney Int. 1985;27(5):756-761); mouse models utilizing inactive proteins or common viral pathogens as provoking antigens (Emancipator et al. Curr. Protoc. Immunol. 2001 May;Chapter 15:Unit 15.11), rat models with non-infectious protein antigens (Emancipator et al. Curr. Protoc. Immunol. 2001 May;Chapter 15:Unit 15.11); a chronic mouse model of IgA immune complex-associated nephropathy (Montinaro et al. Nephrol. Dial. Transplant. 1995;10(11):2035-2042); the Gne M712T mouse as a model of human glomerulopathy (Kakani et al. al. Am. J. Pathol. 2012; 180(4): 1431-1440); a mouse IgA nephropathy model using MBP-20 peptide fusion protein (Zhang et al. Anat. Rec. (Hoboken). 2010; 293(10): 1729-1737); and a mouse model of IgA immune complex nephritis (Rifai et al. J Exp Med. 1979; 150(5): 1161-1173). Other animal models of IgA nephropathy are described, for example, in Tomino et al. J. Nephrol. 2008; 21(4): 463-467, Endo Ren. Fail. 1997; 19(3): 347-371, and Rifai Kidney Int. 1987; 31(1): 1-7.

[0166] Exemplary animal models of other disorders described herein are also known in the art. Exemplary animal types that can be used to evaluate the antibody molecules described herein include, but are not limited to, mice, rats, rabbits, guinea pigs, and monkeys.

[0167] composition The antibody molecule described herein can be included in a composition. In some embodiments, the composition is a pharmaceutical composition, for example, including a pharma- ceutically acceptable carrier. The composition described herein can be used in an assay for evaluating anti-APRIL antibody molecules in a sample or subject.

[0168] As used herein, a "pharmaceutical acceptable carrier" includes any physiologically compatible solvent, dispersion medium, isotonicity agent, absorption delaying agent, and the like. The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion). In some embodiments, less than about 5%, e.g., less than about 4%, 3%, 2%, or 1%, of the antibody molecules in the pharmaceutical composition are present as aggregates. In other embodiments, at least about 95%, e.g., at least about 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.8% or more of the antibody molecules in the pharmaceutical composition are present as monomers. In some embodiments, the level of aggregates or monomers is determined by chromatography, e.g., high performance size exclusion chromatography (HP-SEC).

[0169] The compositions described herein may be in various forms. Forms include liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, liposomes, and suppositories. The suitable form depends on the intended mode of administration and therapeutic application. Exemplary suitable compositions are in the form of injection or infusion solutions. One suitable mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody molecule is administered by intravenous infusion or injection. In some embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0170] The phrases "parenteral administration" and "administered parenterally", as used herein, mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0171] Therapeutic compositions should typically be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high antibody concentrations. Sterile injectable solutions may be prepared by incorporating the required amount of active compound (i.e., antibody or antibody moiety) in a suitable solvent with one or a combination of ingredients listed above, as required, followed by filtration sterilization. In general, dispersions may be prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying to obtain a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof. The proper fluidity of the solution may be maintained, for example, by the use of coating agents, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The time of absorption of the injectable composition may be prolonged by including in the composition an agent that delays absorption, such as monostearate salts and gelatin.

[0172] The antibody molecules described herein can be administered by a variety of methods, several of which are known in the art, and for many therapeutic, prophylactic, or diagnostic applications, a suitable route / mode of administration is intravenous injection or infusion. For example, antibody molecules can be administered by intravenous infusion at a rate of about 1-100 mg / m at a rate of less than 10 mg / min, preferably 5 mg / min or less. 2 , preferably about 5 to 50 mg / m 2 , about 7~25mg / m 2 , more preferably about 10 mg / m 2can be administered until a dose of 100 mg / kg is reached. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. In some embodiments, the active compound can be prepared with a carrier that protects the compound from rapid release, such as a sustained release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0173] In certain embodiments, the antibody molecule may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The antibody molecule (and other ingredients, if desired) may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the antibody molecule may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer the antibody molecule by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material that prevents its inactivation. Therapeutic, prophylactic, or diagnostic compositions may also be administered using medical devices, several of which are known in the art.

[0174] Dosage regimens are adjusted to provide the desired response (e.g., therapeutic, prophylactic, or diagnostic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in unit dosage form. Unit dosage form, as used herein, refers to physically discrete units appropriate for the subject to be treated as a unitary dosage, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage form are determined and directly influenced by (a) the unique characteristics of the antibody molecule and the particular therapeutic, prophylactic, or diagnostic effect to be achieved, and (b) the limitations inherent in the technology of compounding such antibody molecules to treat the susceptibility of an individual.

[0175] Exemplary non-limiting ranges for a therapeutically, prophylactically, or diagnostically effective amount of an antibody molecule are about 0.1-50 mg / kg of subject body weight, e.g., about 0.1-30 mg / kg, e.g., about 1-30, 1-15, 1-10, 1-5, 5-10, or 1-3 mg / kg, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 mg / kg. The antibody molecule can be administered by intravenous infusion at a rate of less than 10 mg / min, e.g., 5 mg / min or less, at a rate of about 1-100 mg / m 2 , for example, about 5 to 50 mg / m 2 , about 7~25mg / m 2 , for example, about 10 mg / m 2 It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated. It should be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and that dosage ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions.

[0176] The pharmaceutical compositions herein may include a "therapeutically effective amount," a "prophylactically effective amount," or a "diagnostically effective amount" of an antibody molecule described herein.

[0177] A "therapeutically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve a desired therapeutic result. A therapeutically effective amount of an antibody molecule may vary depending on factors such as the condition, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody molecule are outweighed by the therapeutically beneficial effects. A "therapeutically effective dosage" typically inhibits a measurable parameter by at least about 20%, e.g., at least about 40%, at least about 60%, or at least about 80%, compared to an untreated subject. The measurable parameter may be, for example, hematuria, colored urine, foamy urine, pain, swelling of the hands and feet (edema), or high blood pressure. The ability of an antibody molecule to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in treating or preventing a disorder. Alternatively, this property of a composition can be evaluated by examining the ability of the antibody molecule to inhibit a measurable parameter in an in vitro or ex vivo assay.

[0178] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0179] A "diagnostically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired diagnostic result. Typically, a diagnostically effective amount is an amount that allows a disorder to be diagnosed in vitro, ex vivo, or in vivo.

[0180] kit The antibody molecules described herein may be comprised in a kit.

[0181] The kit may include one or more other components, including instructions for use; other reagents, such as a label, a therapeutic agent, or an agent useful for chelating or otherwise binding the antibody molecule to a label or therapeutic agent, or a radiation protection composition; devices or other materials for preparing the antibody molecule for administration; a pharma- ceutically acceptable carrier; and devices or other materials for administration to a subject.

[0182] nucleic acid The present disclosure provides a nucleic acid comprising a nucleotide sequence (e.g., a nucleotide sequence described herein) encoding an antibody molecule described herein, or a functional fragment thereof (e.g., a VH, a VL, or both).

[0183] For example, the disclosure features a first nucleic acid and a second nucleic acid encoding, respectively, a heavy chain variable region and a light chain variable region of an antibody molecule described herein or a portion of an antibody molecule described herein. The nucleic acids can include a nucleotide sequence that encodes an amino acid sequence described herein, or a nucleotide sequence substantially identical thereto (e.g., a nucleotide sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto, or that differs therefrom by no more than 3, 6, 15, 30, or 45 nucleotides).

[0184] In certain embodiments, the nucleic acid comprises a nucleotide sequence encoding at least one, two or three CDRs from a heavy chain variable region having an amino acid sequence described herein (e.g., a CDR comprising the amino acid sequence of SEQ ID NO: 11, 12 and / or 13), or a CDR from a heavy chain variable region having an amino acid sequence substantially identical to an amino acid sequence described herein (e.g., an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto and / or has one or more substitutions, such as conserved substitutions). In certain embodiments, the nucleic acid comprises a nucleotide sequence encoding at least one, two or three CDRs from a light chain variable region having an amino acid sequence described herein (e.g., a CDR comprising the amino acid sequence of SEQ ID NO: 14, 15 and / or 16), or a CDR from a light chain variable region having an amino acid sequence substantially identical to an amino acid sequence described herein (e.g., an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto and / or has one or more substitutions, such as conserved substitutions). In certain embodiments, the nucleic acid comprises a nucleotide sequence encoding at least one, two, or three CDRs from a heavy chain variable region having an amino acid sequence described herein (e.g., a CDR comprising the amino acid sequence of SEQ ID NO: 11, 12, and / or 13), or a CDR substantially identical to an amino acid sequence described herein (e.g., an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto, and / or has one or more substitutions, such as conserved substitutions), and a nucleotide sequence encoding at least one, two, or three CDRs from a light chain variable region having an amino acid sequence described herein (e.g., a CDR comprising the amino acid sequence of SEQ ID NO: 14, 15, and / or 16), or a CDR substantially identical to an amino acid sequence described herein (e.g., an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto, and / or has one or more substitutions, such as conserved substitutions).

[0185] In some embodiments, the nucleic acid comprises a nucleotide sequence described herein, or a nucleotide sequence substantially homologous thereto (e.g., a nucleotide sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto and / or capable of hybridizing under stringency conditions described herein). In some embodiments, the nucleic acid comprises a portion (e.g., a functional portion) of a nucleotide sequence described herein, or a sequence substantially homologous thereto (e.g., a nucleotide sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto and / or capable of hybridizing under stringency conditions described herein). The portion may, for example, encode a variable region (e.g., VH or VL), one, two, or three or more CDRs, or one, two, three, or four or more framework regions.

[0186] The nucleic acids described herein comprise deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may be single-stranded or double-stranded, and if single-stranded, may be the coding strand or the non-coding (antisense) strand. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. A nucleic acid may be a recombinant polynucleotide, a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin, that is not naturally occurring or that is linked to another polynucleotide in a non-natural configuration.

[0187] The nucleic acids described herein can be contained in vectors and host cells. The nucleic acids can be present in a single vector or in separate vectors, and can be present in the same host cell or in separate host cells, as described in more detail below.

[0188] vector The disclosure provides vectors (eg, cloning and expression vectors) that contain the nucleic acids described herein.

[0189] In one embodiment, the vector comprises a nucleic acid comprising a nucleotide sequence encoding an antibody molecule as described herein. In another embodiment, the vector comprises a nucleic acid comprising a nucleotide sequence as described herein. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).

[0190] A number of vector systems may be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV or MOMLV) or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, Eastern equine encephalitis virus and flaviviruses.

[0191] Furthermore, cells that have stably integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers can provide, for example, prototropy to auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or can be introduced into the same cell by co-transformation. Additional elements may be required for optimal synthesis of mRNA. These elements can include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0192] Once the vector is prepared for expression, it can be transfected or introduced into a suitable host cell. To achieve this, various techniques can be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are grown in culture and screened for the appropriate activity.

[0193] Methods and conditions for culturing the resulting transfected cells and for recovering the produced antibody molecules are known to those skilled in the art and, based on the present description, may be varied or optimized depending on the particular expression vector and mammalian host cell used.

[0194] cell The disclosure provides a cell (eg, a host cell) comprising a nucleic acid described herein, or a vector described herein.

[0195] In some embodiments, the cells are genetically engineered to contain a nucleic acid described herein or a vector described herein. In some embodiments, the cells are genetically engineered using an expression cassette. The phrase "expression cassette" refers to a nucleotide sequence that can affect the expression of a gene in a host that is compatible with such sequences. Such a cassette can include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or useful for effecting expression, such as, for example, an inducible promoter, can also be used.

[0196] The cell may be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In some embodiments, the cell (e.g., a host cell) is an isolated cell.

[0197] Uses of antibody molecules The antibody molecules disclosed herein and the compositions disclosed herein have in vitro, ex vivo, and in vivo utilities.

[0198] In certain embodiments, the antibody molecules reduce (e.g., inhibit, block, or neutralize) one or more biological activities of an anti-APRIL antibody (e.g., sibeprenlimab). For example, these antibody molecules can be administered to cells in culture, in vitro or ex vivo, or to a subject, e.g., a human subject, e.g., in vivo, to reduce (e.g., inhibit, block, or neutralize) one or more biological activities of an anti-APRIL antibody. In certain embodiments, the antibody molecules compete with the anti-APRIL antibody for binding to APRIL. In certain embodiments, the antibody molecules inhibit or substantially inhibit binding of the anti-APRIL antibody to APRIL. Thus, in certain aspects, the disclosure provides a method of evaluating a subject or a sample from a subject treated with an anti-APRIL antibody, comprising contacting the sample or subject with an antibody molecule described herein such that the sample or subject is evaluated.

[0199] As used herein, the term "subject" is intended to include humans and non-human animals. In some embodiments, the subject is a human subject, e.g., a human patient with or at risk of having an APRIL-associated disorder. In some embodiments, the APRIL-associated disorder is IgA nephropathy. The term "non-human animal" includes mammals and non-mammals, e.g., non-human primates. In some embodiments, the subject is a human. The antibody molecules described herein are suitable for determining the level or activity of anti-APRIL antibodies in a subject. Subjects with an APRIL-associated disorder include, for example, subjects who have developed an APRIL-associated disorder but are (at least temporarily) asymptomatic, subjects who show symptoms of an APRIL-associated disorder, or subjects who have a disorder associated with an APRIL-associated disorder.

[0200] Diagnostic methods The present disclosure provides diagnostic methods for detecting the presence of anti-APRIL antibodies in vitro (e.g., in a sample such as a blood sample or biopsy) or in vivo (e.g., in vivo imaging in a subject). The methods include (i) contacting a sample with an antibody molecule described herein or administering an antibody molecule described herein to a subject, and (optionally) (ii) contacting a reference sample (e.g., a control sample) with the antibody molecule or administering the antibody molecule to a reference subject (e.g., a control subject), and (iii) detecting the formation of a complex between the antibody molecule and an anti-APRIL antibody in the sample or subject or the reference sample or reference subject, where a change, e.g., a statistically significant change, in the formation of the complex in the sample or subject compared to the reference sample or reference subject is indicative of the presence of an anti-APRIL antibody in the sample or subject. The antibody molecule may be directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody molecules. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials, as described above and in more detail below.

[0201] The term "sample", when referring to a sample used to detect a polypeptide or a nucleic acid encoding a polypeptide, includes, but is not limited to, a body fluid (e.g., blood, serum, or urine), a cell, a cell lysate, a protein or membrane extract of a cell, or a tissue sample (e.g., a biopsy).

[0202] The complex formation between the antibody molecule and the anti-APRIL antibody can be detected by measuring or visualizing either the antibody molecule bound to the anti-APRIL antibody or the unbound antibody molecule. Any suitable detection assay can be used, and conventional detection assays include enzyme-linked immunosorbent assays (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry. Instead of labeling the antibody molecule, the presence of the anti-APRIL antibody can be assayed in the sample by a competitive immunoassay that utilizes a standard labeled with a detectable substance and an unlabeled antibody molecule. In this assay, the sample, the labeled standard and the antibody molecule are combined and the amount of the labeled standard bound to the unlabeled binding molecule is determined. The amount of anti-APRIL antibody in the sample is inversely proportional to the amount of the labeled standard bound to the antibody molecule.

[0203] The antibody molecules described herein can be used to diagnose disorders that can be treated or prevented by anti-APRIL antibodies. The detection or diagnostic methods described herein can be used in combination with other methods described herein to treat or prevent disorders described herein.

[0204] Enumeration of embodiments 1. An antibody molecule capable of binding to an anti-APRIL antibody, comprising a heavy chain variable region (VH) comprising the HCDR1 amino acid sequence, HCDR2 amino acid sequence, and HCDR3 amino acid sequence of SEQ ID NO:1, and a light chain variable region (VL) comprising the LCDR1 amino acid sequence, LCDR2 amino acid sequence, and LCDR3 amino acid sequence of SEQ ID NO:2. 2. (a) (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having no more than 1, 2 or 3 amino acid differences therefrom; (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having no more than 1, 2 or 3 amino acid differences therefrom; and / or (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having no more than 1, 2, or 3 amino acid differences therefrom; and / or a heavy chain variable region (VH) comprising one, two or three of (b) (iv) LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having no more than 1, 2 or 3 amino acid differences therefrom; (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having no more than 1, 2 or 3 amino acid differences therefrom; and / or (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having no more than 1, 2, or 3 amino acid differences therefrom; An antibody molecule capable of binding to an anti-APRIL antibody, comprising a light chain variable region (VL) comprising one, two or three of: 3. The antibody molecule of embodiment 1 or 2, wherein the VH comprises an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or which differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids therefrom. 4. An antibody molecule according to any one of embodiments 1 to 3, wherein VH comprises the amino acid sequence of SEQ ID NO:1. 5. The antibody molecule of any of embodiments 1 to 4, wherein the VL comprises an amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or which differs therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. 6. An antibody molecule according to any one of embodiments 1 to 5, wherein the VL comprises the amino acid sequence of SEQ ID NO:2. 7. An antibody molecule according to any one of embodiments 1 to 6, comprising an antigen-binding fragment. 8. The antibody molecule of embodiment 7, wherein the antigen-binding fragment comprises a Fab, F(ab')2, Fv, scFv, or sc(Fv)2. 9. An antibody molecule according to any one of embodiments 1 to 8, comprising a heavy chain constant region. 10. The antibody molecule of embodiment 9, wherein the heavy chain constant region comprises an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. 11. The antibody molecule of any of embodiments 1 to 10, comprising a light chain constant region. 12. The antibody molecule of embodiment 11, wherein the light chain constant region comprises the light chain constant region of a kappa or lambda light chain. 13. An antibody molecule according to any one of embodiments 1 to 12, comprising an Fc region. 14. The antibody molecule of any of embodiments 1 to 13, comprising a heavy chain comprising amino acids 18 to 458 of SEQ ID NO:3 or amino acids 18 to 464 of SEQ ID NO:4. 15. The antibody molecule of any of embodiments 1 to 14, comprising a light chain comprising amino acids 18 to 235 of SEQ ID NO:5. 16. (a) a heavy chain comprising amino acids 18 to 458 of SEQ ID NO: 3 and a light chain comprising amino acids 18 to 235 of SEQ ID NO: 5, or (b) a heavy chain comprising amino acids 18 to 464 of SEQ ID NO: 4 and a light chain comprising amino acids 18 to 235 of SEQ ID NO: 5 16. The antibody molecule of any one of embodiments 1 to 15, comprising: 17. The antibody molecule of any of embodiments 1 to 16, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 or 4. 18. The antibody molecule of any of embodiments 1 to 17, comprising a light chain comprising the amino acid sequence of SEQ ID NO:5. 19. (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 5, or (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 5 19. The antibody molecule of any one of embodiments 1 to 18, comprising: 20. The antibody molecule according to any one of embodiments 1 to 19, which is mAb 1H4. 21. An antibody molecule according to any one of embodiments 1 to 20, which is a murine antibody molecule. 22. An antibody molecule according to any one of embodiments 1 to 21, which is an isolated antibody molecule. 23. An antibody molecule according to any one of embodiments 1 to 22, which is a monoclonal antibody molecule. 24. The antibody molecule of any of embodiments 1 to 23, which is an anti-idiotypic antibody molecule. 25. An antibody molecule according to any one of embodiments 1 to 24, which binds to the VH, the VL, or both, of an anti-APRIL antibody. 26. An antibody molecule according to any of embodiments 1 to 25, which binds to one or more (e.g. 2, 3, 4, 5 or 6) CDRs of an anti-APRIL antibody. 27. An antibody molecule according to any one of embodiments 1 to 26, which does not bind or does not substantially bind to the Fc region of an anti-APRIL antibody. 28. The antibody molecule according to any of embodiments 1 to 27, which binds to an anti-APRIL antibody with an EC50 of less than 500 ng / mL, e.g., 400 ng / mL, 300 ng / mL, 200 ng / mL, 150 ng / mL, 120 ng / mL, 100 ng / mL, 90 ng / mL, 80 ng / mL, 70 ng / mL, 60 ng / mL, 50 ng / mL, 40 ng / mL, 35 ng / mL, 30 ng / mL, 25 ng / mL, 20 ng / mL, 15 ng / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL, 1 ng / mL, or less than 0.1 ng / mL, e.g., 1 ng / mL to 150 ng / mL, 2 ng / mL to 100 ng / mL, 5 ng / mL to 50 ng / mL, or 10 ng / mL to 25 ng / mL, as determined, for example, by ELISA. 29. An antibody molecule according to any of embodiments 1 to 28, which reduces (eg neutralizes, inhibits or blocks) binding of an anti-APRIL antibody to APRIL. 30. An antibody molecule according to any of embodiments 1 to 29, which binds to an anti-APRIL antibody that is not bound to APRIL. 31. An antibody molecule according to any of embodiments 1 to 30, which does not bind or does not substantially bind to an anti-APRIL antibody that is bound to APRIL. 32. An antibody molecule according to any of embodiments 1 to 30, which binds to an anti-APRIL antibody that is not bound to APRIL, and does not bind or does not substantially bind to an anti-APRIL antibody that is bound to APRIL. 33. An antibody molecule according to any of embodiments 1 to 32, which binds to an anti-APRIL antibody that is not bound to APRIL, and does not bind or does not substantially bind to an anti-APRIL antibody that is bound to APRIL. 34. An antibody molecule according to any of embodiments 1 to 33, which bridges, e.g. binds simultaneously, two anti-APRIL antibodies, e.g. as determined by a bridging ELISA assay. 35. An antibody molecule according to any of embodiments 1 to 34, which competes with an anti-APRIL antibody for binding to APRIL. 36. The antibody molecule of any of embodiments 1-35, wherein the anti-APRIL antibody binds to human APRIL. 37. The antibody molecule of any of embodiments 1-36, wherein the anti-APRIL antibody is cybeprenlimab. 38. An antibody molecule that competes with an antibody molecule of any of embodiments 1 to 37 for binding to an anti-APRIL antibody. 39. An antibody molecule that binds to an epitope that is the same as or overlaps with an epitope recognized by an antibody molecule according to any one of embodiments 1 to 37. 40. A method for detecting anti-APRIL antibodies, comprising: (a) contacting an antibody molecule according to any one of embodiments 1 to 39 with a sample; (b) determining the formation of complexes between the antibody molecules and the sample (e.g., between the antibody molecules and anti-APRIL antibodies in the sample); Thereby, detecting anti-APRIL antibodies; A method comprising: 41. A method for evaluating a sample, comprising: (a) contacting an antibody molecule according to any one of embodiments 1 to 39 with a sample; (b) determining the formation of complexes between the antibody molecules and the sample (e.g., between the antibody molecules and anti-APRIL antibodies in the sample); evaluating the sample therewith; and A method comprising: 42. (c) contacting the antibody molecule with a reference sample; (d) determining the formation of a complex between the antibody molecule and the reference sample (e.g., between the antibody molecule and an anti-APRIL antibody in the reference sample); 42. The method of embodiment 40 or 41, further comprising: 43. The method of any of embodiments 40 to 42, further comprising providing the sample or reference sample prior to contacting the antibody molecule with the sample or reference sample. 44. The method according to any one of embodiments 40 to 43, which is an ELISA assay. 45. The method according to any of embodiments 40 to 44, which is an anti-drug assay, a toxicity assay, or a GxP assay. 46. ​​A method for detecting anti-APRIL antibodies, comprising: (a) administering to a subject an antibody molecule according to any one of embodiments 1 to 39; (b) determining the formation of a complex between the antibody molecule and the subject (e.g., between the antibody molecule and an anti-APRIL antibody in the subject); Thereby, detecting anti-APRIL antibodies; A method comprising: 47. A method for evaluating a subject, comprising: (a) administering to a subject an antibody molecule according to any one of embodiments 1 to 39; (b) determining the formation of a complex between the antibody molecule and the subject (e.g., between the antibody molecule and an anti-APRIL antibody in the subject); Evaluating the subject therewith; and A method comprising: 48. A composition (e.g., a pharmaceutical composition) comprising an antibody molecule according to any one of embodiments 1 to 39, optionally comprising a pharma- ceutically acceptable carrier, excipient or stabilizer. 49. A nucleic acid (e.g., an isolated nucleic acid) encoding the VH, VL, or both, of an antibody molecule according to any one of embodiments 1 to 39. 50. The nucleic acid of embodiment 49, comprising nucleotides 52 to 1374 of SEQ ID NO:8, or a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or differing therefrom by no more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides. 51. The nucleic acid of embodiment 49 or 50, comprising the nucleotide sequence of SEQ ID NO:8. 52. The nucleic acid of embodiment 49, comprising nucleotides 52 to 1392 of SEQ ID NO:9, or a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or differing therefrom by no more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides. 53. The nucleic acid of embodiment 49 or 52, comprising the nucleotide sequence of SEQ ID NO:9. 54. The nucleic acid of any of embodiments 49-53, comprising nucleotides 52-705 of SEQ ID NO: 10, or a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or differing therefrom by no more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides. 55. The nucleic acid according to any one of embodiments 49 to 54, comprising the nucleotide sequence of SEQ ID NO: 10. 56. A vector (e.g., an expression vector) comprising a nucleic acid according to any one of embodiments 49 to 55. 57. The vector of embodiment 56, comprising a promoter operably linked to the nucleic acid. 58. A cell (eg, a host cell) comprising a nucleic acid according to any of embodiments 49 to 55 or a vector according to embodiment 56 or 57. 59. A method for producing an antibody molecule, comprising culturing a cell according to embodiment 58 under conditions suitable for gene expression. 60. The method of embodiment 59, further comprising isolating or purifying the antibody molecule. 61. The method of embodiment 59 or 60, wherein the antibody molecule is produced at a concentration of 0.2 mg / mL to 5 mg / mL, for example, 0.5 mg / mL to 1.5 mg / mL. EXAMPLES

[0205] Example 1: Generation of anti-idiotype antibodies Mice were immunized with sibeprene limab, which was used as a counterscreening reagent to assess specificity in pre-subclone screening.

[0206] The first immunization was successful in eliciting a strong serological response to sibeprenlimab as shown by titer testing, and all three mice were harvested for fusion. In the titer testing, sibeprenlimab was coated onto an ELISA plate, and antisera from each of the three mice were titrated on the plate with normal mouse serum as a control. All three mice showed immunization to sibeprenlimab compared to normal mouse serum, and were recommended and used for hybridoma generation.

[0207] Lymph nodes from three mice yielded eight 96-well plates of hybridomas after fusion. Hybridomas were plated based on cell density to maximize monoclonality. Primary screening of the eight fusion plates assessed sibeprenlimab binding by enzyme-linked immunosorbent assay (ELISA) and counterscreened for reactivity to human IgG2. Clones positive to sibeprenlimab (and negative to human IgG2) were ranked, selected, and transferred to a new single 96-well plate consisting of 75 clone wells and 20 blank wells, plus one well containing an antiserum positive control. Clones were grown in new 96-well plates for pre-subclonal screening, and supernatants were used for secondary screening for binding to sibeprenlimab, human IgG2, human IgG, 20% pooled human serum, 20% pooled cynomolgus serum, and 0.8% BSA. Of the 75 clones, 68 clones showed reactivity to sibeprenlimab and little or no reactivity to human IgG2 counterscreen, human IgG, 20% pooled human serum, 20% pooled cynomolgus serum, and 0.8% BSA. Seven clones that showed binding to human IgG2 and / or human Ig were excluded from all further analysis. Most clones showed a maximum signal at 450 nM with an OD value of ≥ 1.5. The 68 parental clones were frozen and the supernatants were sent for tertiary screening.

[0208] Clonal selection All 68 hybridoma clones were titrated on an anti-mouse IgG Fv biosensor chip on an Octet in the Octect assay, normalized to 1 μg / mL in 1×PBS, and stored at 4° C. for use in tertiary screening assays. The 68 candidate anti-ID hybridomas were screened for binding to a panel of mAbs to determine anti-ID specificity. mAbs from the panel were coated onto ELISA plates and incubated overnight at 4° C. before each specificity ELISA. Based on the results of the specificity screening, 24 hybridomas showed high reactivity to sibeprenlimab, mAb 2419 (chimeric hIgG1), and mAb 2419-043 (IgG2, similar to sibeprenlimab), and low / no reactivity to the control mAb. mAb 2419 is a mouse IgG1 version of humanized sibeprenlimab with conserved CDR loops but with changes in the framework regions and a different constant region. Binding to mAb 2419 is consistent with an anti-ID targeting the CDR regions of sibeprenlimab.

[0209] The top 24 hybridomas were further characterized in binding ("PK-like") and bridging ELISA (Figures 1A-1B). For the bridging ELISA assay, sibeprenlimab was biotinylated. In this assay, the biotinylated version of sibeprenlimab was detectable by both goat anti-human IgG and streptavidin-HRP secondary antibodies, whereas non-biotinylated sibeprenlimab was only detected by goat anti-human IgG, as expected.

[0210] Based on the data from the binding and bridging ELISA, all 24 selected clones showed strong binding to sibeprenlimab with various EC50 values. Of the 24 clones, 7 were identified as "bridgers" to various degrees. The list of clones to pursue for further characterization was based on the ability of mouse mAbs to form bridges in the bridging ELISA. Hybridoma clones bound well to sibeprenlimab in the indirect binding ELISA, but only a subset performed well in the bridging ELISA. All clones were also tested to determine the mAb isotype. Clones with IgG2 isotype and favorable binding / bridging profile were initially selected for subcloning because of the ease and reproducibility of purification on Protein A columns in the scale-up of anti-idiotype production. Table 1 summarizes the 10 clones selected for subcloning and the results for this initial experimental panel.

[0211] [Table 15]

[0212] Characterization of final subclones Ten subcloned hybridomas (two parents each) were prepared as (1) frozen hybridomas, (2) hybridoma supernatants, and (3) live cells in 6-well plates. Subclone supernatants were titrated on an anti-mouse IgG Fv biosensor chip on an Octet, normalized to 1 μg / mL in 1× PBS, and stored at 4°C for use in later experiments. Live hybridoma cells in 6-well plates were passaged using standard hybridoma techniques. Hybridoma cells were frozen within the first few weeks of passaging. Hybridomas were maintained at 65-85% plate confluency or a concentration of 0.25-1.0×106 cells / mL. General passaging was performed with a scaling of 1:5 or 1:10 every 48-72 hours.

[0213] Hybridoma supernatants were characterized using a panel of tertiary screening assays to determine the final subclones for purification and further characterization. Experiments to characterize the 20 subclones included sibeprenlimab binding ELISA, bridging ELISA, and rapid ELISA mAb isotyping. After completion of this round of experiments, several subclones at 1 μg / mL had reduced affinity for sibeprenlimab in the binding ELISA and were removed from selection. Based on the results from the bridging ELISA, 5 of the 20 subclones were still strong "bridgers" and 7 of the 10 selected clones were strong "bridgers" prior to subcloning. When the isotypes of the subclones were tested, no IgG2a / b isotypes were observed (3 of the 10 parents were identified as IgG2 prior to subcloning). The change in the properties of the subclones meant that there were no more IgG2a / b hybridomas for a simple Protein A purification strategy. Therefore, we focused our efforts on (1) purifying the hybridomas using protein G and low IgG FBS, and (2) sequencing the variable regions of the lead hybridomas and cloning them into mouse IgG2 vectors. Table 2 summarizes the characterization of all 20 subclones. 1A5.F4, 1H4.F4, and 6A3.E6 were selected for large-scale hybridoma scale-up.

[0214] [Table 16]

[0215] Sequences and recombinant expression 1A5.F4, 1A5.F6, 1H4.F4, 6A3.E6, 6A3.F8, and 6C12.F12 were selected and cultured in 6-well plates and proceeded to sequencing and cloning into mouse IgG2a platform vectors. Four unique productive HC and LC sequences were identified. Binding and bridging ELISAs were performed with supernatants from all four transfections. Transfections of 1A5 and both 1H4 (HC_VH1 and HC_VH4_5_10) did not retain sibeprenlimab binding or bridging. Some activity was retained for 6C12. Therefore, 6C12 was purified using a Protein A column and retested for activity. Compared to 6C12 hybridoma supernatant, recombinant purified 6C12 had lower bridging activity and slightly lower binding activity, which was not ideal for the final anti-idiotypic antibody. Attempts to recombinantly produce the anti-idiotype were re- undertaken with the final candidate, 1H4.

[0216] Three subclones, 1A5.F4, 1H4.F4, and 6A3.E6, were selected based on activity in an established experimental panel for large-scale hybridoma generation and purification, followed by qualification.

[0217] Hybridoma Scaling Up in CELLine Bioreactor Flasks 1A5.F4, 1H4.F4, and 6A3.E6 were scaled up to five T-150 flasks in low IgG medium. Once confluent, one of the T-150 flasks of each subclone was used to seed a CeLLine 1000 bioreactor flask and incubated at 37°C and 5% CO2 for 7 days followed by harvest 1. The other four T-150 flasks were incubated at 37°C and 5% CO2 for 7 days, pooled, harvested, purified on a 1 mL protein G column, dialyzed in 1x PBS, filter sterilized, and quantified. In Figures 2A-2C, small-scale purified lots of 1A4, 1H4, and 6A3 were characterized by binding ELISA (Figure 2A), bridging ELISA (Figure 2B), and specificity ELISA (Figure 2C) against 2419 before selecting anti-IDs for purification from their respective CeLLine 1000 bioreactor flasks. While production in bioreactor flasks can take up to 4 weeks, this parallel workflow of small-scale purification characterization during incubation / production in CeLLine 1000 bioreactor flasks allowed for more rapid large-scale production of selected anti-IDs.

[0218] The binding data in Figures 2A-2C suggest that all three anti-ID candidates bind similarly to sibeprenlimab. 1H4 appears to be the strongest "bridger" and only 1H4 and 6A3 bind to 2419. 2419 has the same CDR regions as sibeprenlimab. In Figures 3A-3C, the bridging ELISA was also performed in the presence of 1% human serum (during the assay) to ensure there was no cross-reactivity. Additionally, SDS PAGER was run for 1A5, 1H4, and 6A3 to ensure there was no mixture of heavy and light chains. Reduced and non-reduced samples were run on 4-12% Bis-Tris gels and bands were visualized with Simple Blue Strain. The profile of 1A5 (reduced) differs slightly from that of 1H4 and 6A3.

[0219] Generation of GXP assays Based on the characterization data, 1H4 was selected as the anti-ID mAb to generate for the GXP assay. IgG1 hybridomas generated in CeLLine 1000 bioreactor flasks were purified on a 1 mL protein G column. Three hybridoma production lots of 1H4 were qualified for the GXP assay by performing one and three freeze / thaw cycles and evaluating by binding ELISA (Figures 4A-4C), bridging ELISA (in the presence of 1% and 20% human serum to ensure no cross-reactivity) (Figures 5A-5C), reducing and non-reducing SDS-PAGE (visualized with 4-12% Bis-Tris gels and Simple Blue Stain), and mAb isotyping ELISA to confirm the IgG1 κ isotype.

[0220] These lots of 1H4 were determined to behave similarly in the binding / bridging ELISA: 1H4 in the presence of 1% and 20% human serum and 1 and 3 freeze / thaw cycles did not affect SDS-PAGE reducing and non-reducing profiles and performance in the binding / bridging ELISA.

[0221] Additional lots of hybridoma-produced 1H4 were similarly characterized and compared to previous lots using binding and bridging ELISA and SDS-PAGE (reduced and non-reduced, 4-12% Bis-Tris gels, visualized with Simple Blue stain). Additional lots of 1H4 showed equivalent binding and bridging activity as previous lots. Furthermore, SDS-PAGE reducing and non-reducing profiles were comparable.

[0222] Sequence and recombinant expression of 1H4 1H4 was for ADA assay development. The sequence of the 1H4.F4 anti-idiotype was identified for recombinant expression in expi293 cells.

[0223] To compare the activity of 1H4 in an IgG1 backbone, both were carried forward. The 1H4_VH sequence and the IgG1 constant region were cloned into a vector. The 1H4_Vk sequence was also cloned into a vector. The nucleotide sequence was humanized and codon optimized. Colonies were sequenced and plasmids were maxiprepped using standard laboratory methods. Two 200 mL expi293 transfections were performed with 1H4(IgG1) and 1H4(IgG2). IgG1 was purified on a 1 mL Protein G column and IgG2 was purified on a 1 mL Protein A column. It is noteworthy that the IgG1 recombinant was expressed at a lower level compared to the IgG2 recombinant.

[0224] In Figures 6A-6B, hybridoma-produced 1H4 was compared to recombinantly produced and purified 1H4 (IgG1 and IgG2 backbones). The three lots appeared comparable in the binding ELISA, but cross-linking results indicate that the 1H4 hybridoma was the best "cross-linker" and that there was some variability between the recombinant lots. Additionally, hybridoma-produced 1H4 was compared to recombinantly produced and purified 1H4 (IgG1 and IgG2 backbones) in SDS-PAGE (4-12% Bis-Tris gels and visualized with Simple Blue Stain). Hybridoma-produced 1H4 and recombinantly produced 1H4 had comparable reducing and non-reducing SDS-PAGE profiles.

[0225] Comparison of binding between "free" and "whole" sibeprenlimab To further characterize the sibeprenlimab anti-idiotypic antibody 1H4, 1H4 was evaluated for binding specificity, whether it recognized "free drug" (i.e., sibeprenlimab unbound to APRIL) or "total drug" (i.e., sibeprenlimab bound or unbound to APRIL). 12 ng / mL sibeprenlimab and titrated 1H4 were preincubated overnight at 4°C and added to APRIL precoated plates (0.5 μg / mL). Similarly, 12 ng / mL sibeprenlimab and titrated APRIL were preincubated overnight at 4°C and added to 1H4 precoated plates (1.0 μg / mL). In FIG. 7, 1H4 was found to be neutral (i.e., blocks the binding of sibeprenlimab to APRIL) and recognized only the "free drug" and not the "total drug", i.e., did not recognize sibeprenlimab in the context of binding to APRIL.

[0226] summary Anti-idiotypic (anti-ID) monoclonal antibodies were generated that recognize sibeprenlimab. 1A5, 1H4, and 6A3 were characterized, and 1H4 was determined to be suitable for use in preclinical and clinical antibody drug development assays. Production of 1H4 was performed using a hybridoma cell line in a CeLLine 1000 bioreactor flask. 1H4 was then sequenced, recombinantly expressed, produced in both IgG1 and IgG2 VH backbones, and evaluated in a panel of anti-ID characterization assays.

[0227] Incorporation by Reference All publications, patents, and accession numbers mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0228] Equivalent Although specific embodiments of the present invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the present invention will become apparent to those skilled in the art upon review of this specification and the following claims. The full scope of the present invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

[0229] Incorporation by Reference All publications, patents, and accession numbers mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0230] Equivalent Although specific embodiments of the present invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the present invention will become apparent to those skilled in the art upon review of this specification and the following claims. The full scope of the present invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

1. (a) (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having no more than 1, 2, or 3 amino acid differences therefrom; (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having no more than 1, 2, or 3 amino acid differences therefrom; and / or (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having no more than 1, 2, or 3 amino acid differences therefrom; and / or a heavy chain variable region (VH) comprising one, two or three of: (b) (iv) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having no more than 1, 2, or 3 amino acid differences therefrom; (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having no more than 1, 2, or 3 amino acid differences therefrom; and / or (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having no more than 1, 2, or 3 amino acid differences therefrom. An antibody molecule capable of binding to an anti-APRIL antibody, comprising a light chain variable region (VL) comprising one, two or three of:

2. The antibody molecule of claim 1, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO:11, the HCDR2 comprises the amino acid sequence of SEQ ID NO:12, the HCDR3 comprises the amino acid sequence of SEQ ID NO:13, the LCDR1 comprises the amino acid sequence of SEQ ID NO:14, the LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:

16.

3. The antibody molecule of claim 1, wherein the VH comprises an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids therefrom.

4. The antibody molecule of any one of claims 1 to 3, wherein the VH comprises the amino acid sequence of SEQ ID NO:

1.

5. 5. The antibody molecule of any one of claims 1 to 4, wherein the VL comprises an amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids therefrom.

6. The antibody molecule of any one of claims 1 to 5, wherein the VL comprises the amino acid sequence of SEQ ID NO:

2.

7. An antibody molecule according to any one of claims 1 to 6, comprising an antigen-binding fragment.

8. The antibody molecule of claim 7 , wherein the antigen-binding fragment comprises Fab, F(ab′)2, Fv, scFv, or sc(Fv)2.

9. An antibody molecule according to any one of claims 1 to 8, comprising a heavy chain constant region.

10. The antibody molecule of claim 9, wherein the heavy chain constant region comprises an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

11. An antibody molecule according to any one of claims 1 to 10, comprising a light chain constant region.

12. The antibody molecule of claim 11 , wherein the light chain constant region comprises a kappa or lambda light chain light chain constant region.

13. An antibody molecule according to any one of claims 1 to 12, comprising an Fc region.

14. 14. The antibody molecule of any one of claims 1 to 13, comprising a heavy chain comprising amino acids 18 to 458 of SEQ ID NO:3 or amino acids 18 to 464 of SEQ ID NO:

4.

15. 15. The antibody molecule of any one of claims 1 to 14, comprising a light chain comprising amino acids 18 to 235 of SEQ ID NO:

5.

16. (a) a heavy chain comprising amino acids 18 to 458 of SEQ ID NO:3 and a light chain comprising amino acids 18 to 235 of SEQ ID NO:5; or (b) a heavy chain comprising amino acids 18 to 464 of SEQ ID NO:4 and a light chain comprising amino acids 18 to 235 of SEQ ID NO:

5. The antibody molecule of any one of claims 1 to 15, comprising:

17. 17. The antibody molecule of any one of claims 1 to 16, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 or 4.

18. 18. The antibody molecule of any one of claims 1 to 17, comprising a light chain comprising the amino acid sequence of SEQ ID NO:

5.

19. (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:3 and a light chain comprising the amino acid sequence of SEQ ID NO:5; or (b) a heavy chain comprising the amino acid sequence of SEQ ID NO:4 and a light chain comprising the amino acid sequence of SEQ ID NO:

5. The antibody molecule of any one of claims 1 to 18, comprising:

20. The antibody molecule of any one of claims 1 to 19, which is mAb 1H4.

21. The antibody molecule of any one of claims 1 to 20, which is a mouse antibody molecule.

22. The antibody molecule of any one of claims 1 to 21, which is an isolated antibody molecule.

23. An antibody molecule according to any one of claims 1 to 22 which is a monoclonal antibody molecule.

24. An antibody molecule according to any one of claims 1 to 23, which is an anti-idiotypic antibody molecule.

25. The antibody molecule of any one of claims 1 to 24, which binds to the VH, VL, or both of the anti-APRIL antibody.

26. 26. The antibody molecule of any one of claims 1 to 25, which binds to one or more (e.g., 2, 3, 4, 5 or 6) CDRs of the anti-APRIL antibody.

27. The antibody molecule of any one of claims 1 to 26, which does not bind or does not substantially bind to the Fc region of the anti-APRIL antibody.

28. The anti-APRIL antibody has a serum concentration of less than 500 ng / mL, e.g., 400 ng / mL, 300 ng / mL, 200 ng / mL, 150 ng / mL, 120 ng / mL, 100 ng / mL, 90 ng / mL, 80 ng / mL, 70 ng / mL, 60 ng / mL, 50 ng / mL, 40 ng / mL, 35 ng / mL, 30 ng / mL, 25 ng / mL, or ...350 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 350 ng / mL, 300 ng / mL, 250 ng / mL, or less than 500 ng / mL, e.g., 400 ng / mL, 300 ng / mL, 350 ng / mL, 300 ng / mL, 350 ng / mL, 300 ng / mL, 350 ng / mL, 300 ng / mL, 350 ng / mL, 300 ng / mL, 28. The antibody molecule of any one of claims 1 to 27, which binds with an EC50 of less than 1 ng / mL, 20 ng / mL, 15 ng / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL, 1 ng / mL, or 0.1 ng / mL, for example between 1 ng / mL and 150 ng / mL, between 2 ng / mL and 100 ng / mL, between 5 ng / mL and 50 ng / mL, or between 10 ng / mL and 25 ng / mL.

29. The antibody molecule of any one of claims 1 to 28, which reduces (e.g. neutralizes, inhibits or blocks) binding of the anti-APRIL antibody to APRIL.

30. The antibody molecule of any one of claims 1 to 29, which binds to an anti-APRIL antibody that does not bind to APRIL.

31. The antibody molecule of any one of claims 1 to 30, which does not bind or does not substantially bind to an anti-APRIL antibody that binds to APRIL.

32. An antibody molecule according to any one of claims 1 to 30, which binds to an anti-APRIL antibody that is not bound to APRIL, and does not bind or does not substantially bind to an anti-APRIL antibody that is bound to APRIL.

33. An antibody molecule according to any one of claims 1 to 32, which binds to an anti-APRIL antibody that is not bound to APRIL and does not bind or does not substantially bind to an anti-APRIL antibody that is bound to APRIL.

34. 34. The antibody molecule of any one of claims 1 to 33, which bridges, e.g. simultaneously binds, two anti-APRIL antibodies, e.g. as determined by a bridge ELISA assay.

35. The antibody molecule of any one of claims 1 to 34, which competes with said anti-APRIL antibody for binding to APRIL.

36. The antibody molecule of any one of claims 1 to 35, wherein the anti-APRIL antibody binds to human APRIL.

37. The antibody molecule of any one of claims 1 to 36, wherein the anti-APRIL antibody is sibeprenlimab.

38. An antibody molecule that competes with the antibody molecule of any one of claims 1 to 37 for binding to an anti-APRIL antibody.

39. An antibody molecule that binds to an epitope that is the same as or overlaps with an epitope recognized by an antibody molecule according to any one of claims 1 to 37.

40. A method for detecting anti-APRIL antibodies, comprising: (a) contacting an antibody molecule according to any one of claims 1 to 39 with a sample; (b) determining the formation of a complex between the antibody molecule and the sample (e.g., between the antibody molecule and the anti-APRIL antibody in the sample); thereby detecting the anti-APRIL antibody; A method comprising:

41. 1. A method for evaluating a sample, comprising: (a) contacting the sample with an antibody molecule according to any one of claims 1 to 39; (b) determining the formation of a complex between the antibody molecule and the sample (e.g., between the antibody molecule and the anti-APRIL antibody in the sample); evaluating said sample therewith; and A method comprising:

42. (c) contacting the antibody molecule with a reference sample; (d) determining the formation of a complex between the antibody molecule and the reference sample (e.g., between the antibody molecule and the anti-APRIL antibody in the reference sample); 42. The method of claim 40 or 41, further comprising:

43. The method of any one of claims 40 to 42, further comprising providing a sample or a reference sample prior to contacting said antibody molecule with said sample or reference sample.

44. The method of any one of claims 40 to 43, which is an ELISA assay.

45. The method of any one of claims 40 to 44, which is an anti-drug assay, a toxicity assay, or a GxP assay.

46. A method for detecting anti-APRIL antibodies, comprising: (a) administering to a subject an antibody molecule according to any one of claims 1 to 39; (b) determining the formation of a complex between the antibody molecule and the subject (e.g., between the antibody molecule and the anti-APRIL antibody in the subject); thereby detecting the anti-APRIL antibody; A method comprising:

47. 1. A method for evaluating a subject, comprising: (a) administering to said subject an antibody molecule according to any one of claims 1 to 39; (b) determining the formation of a complex between the antibody molecule and the subject (e.g., between the antibody molecule and an anti-APRIL antibody in the subject); evaluating said subject therewith; A method comprising:

48. A composition (e.g. a pharmaceutical composition) comprising an antibody molecule according to any one of claims 1 to 39, optionally comprising a pharma- ceutically acceptable carrier, excipient or stabilizer.

49. A nucleic acid (eg, an isolated nucleic acid) encoding the VH, VL, or both, of an antibody molecule according to any one of claims 1 to 39.

50. 50. The nucleic acid of claim 49, comprising nucleotides 52 to 1374 of SEQ ID NO:8, or a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or differing by no more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides thereto.

51. 51. The nucleic acid of claim 49 or 50, comprising the nucleotide sequence of SEQ ID NO:

8.

52. 50. The nucleic acid of claim 49, comprising nucleotides 52 to 1392 of SEQ ID NO:9, or a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or differing by no more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides therefrom.

53. 53. The nucleic acid of claim 49 or 52, comprising the nucleotide sequence of SEQ ID NO:

9.

54. 54. The nucleic acid of any one of claims 49 to 53, comprising nucleotides 52 to 705 of SEQ ID NO: 10, or a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or differing by no more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides thereto.

55. 55. The nucleic acid according to any one of claims 49 to 54, comprising the nucleotide sequence of SEQ ID NO:

10.

56. A vector (eg, an expression vector) comprising the nucleic acid of any one of claims 49 to 55.

57. 57. The vector of claim 56, comprising a promoter operably linked to the nucleic acid.

58. A cell (eg a host cell) comprising a nucleic acid according to any one of claims 49 to 55 or a vector according to claim 56 or 57.

59. 60. A method for producing an antibody molecule comprising culturing a cell according to claim 58 under conditions suitable for gene expression.

60. 60. The method of claim 59, further comprising isolating or purifying the antibody molecule.

61. 61. The method of claim 59 or 60, wherein the antibody molecule is produced at a concentration of 0.2 mg / mL to 5 mg / mL, such as 0.5 mg / mL to 1.5 mg / mL.

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