Anti-lambda myeloma antigen (LMA)-binding proteins for treating LMA-expressing cancers and autoimmune disorders

Abstract

To provide antibodies, compositions and nucleic acids that may be used in the treatment of LMA-expressing pathologies.SOLUTION: The present application is directed to anti-lambda myeloma antigen (LMA) binding proteins, wherein the binding protein is an antibody. Compositions comprising these antibodies and nucleic acids encoding these antibodies are also disclosed.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to anti-LMA binding proteins. Such binding proteins may be useful in treating disorders associated with the abnormal proliferation of plasma cells and / or their precursors. [Background technology]

[0002] Abnormal proliferation of plasma cells and / or their precursor cells is a hallmark of various human pathologies. One example is multiple myeloma (MM), a malignant tumor of bone marrow plasma cells. The disease is characterized by malignant plasma cells secreting either lambda or lambda light chain-restricted monoclonal paraproteins. Lambda restriction occurs in approximately 40% of myeloma patients, and expression of the lambda myeloma antigen (LMA) is highly restricted to malignant effector cells. Despite recent advances in treatment, multiple myeloma remains incurable. Its clinical course is characterized by initial therapeutic responses, followed by repeated relapses with eventual resistance to all forms of treatment. It is also associated with significant morbidity and disability due to both the disease itself and toxicity from available treatments.

[0003] Therefore, new approaches to treating the abnormal proliferation of plasma cells and / or their precursors are needed. Summary of the Invention [Problem to be solved by the invention]

[0004] In producing the human binding proteins of the present disclosure, the inventors identified a selection of human antibodies that bind to and kill LMA-positive cell lines. The inventors also identified human antibodies with heavy chain modifications that lead to preferential binding to LMA (i.e., cell surface antigens) over free lambda light chains (e.g., serum antigens). Such modifications may be incorporated into various binding proteins to preferentially target cells that express LMA. Such binding proteins may be particularly effective in mediating targeted killing of cells that express LMA, such as LMA-expressing cancer cells. [Means for solving the problem]

[0005] Thus, in a first aspect, the disclosure relates to an anti-lambda myeloma antigen (LMA) binding protein having an antigen-binding domain that preferentially binds LMA over free lambda light chains and a heavy chain variable region (V H ) and the light chain variable region (V L ), including V H comprises a complementarity determining region (CDR) 1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3. H comprises the amino acid sequence set forth in SEQ ID NO: 4. In another example, V L comprises a CDR1 set forth in SEQ ID NO: 6, a CDR2 set forth in SEQ ID NO: 7, and a CDR3 set forth in SEQ ID NO: 8 or SEQ ID NO: 13. L comprises the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 14. In another example, V L comprises the amino acid sequence set forth in SEQ ID NO: 9. In one example, the above-referenced binding protein binds to lambda isotypes 2 and 3. In one example, the above-referenced antibody does not bind to lambda isotype 1. In another example, the above-referenced antibody has a K D is 1×10 10 In another example, the above-referenced antibodies bind to free lambda light chains with a K measured by SPR of less than D is 5 x 10 9 In another example, the above-referenced antibodies bind to free lambda light chains with a K measured by SPR of less than D is 1×10 9 In another example, the above-referenced antibodies bind to free lambda light chains with a K measured by SPR of less than D is 5 x 10 8 In another example, the above-referenced antibodies bind to free lambda light chains with a K measured by SPR of less than D is 5 x 10 7 The free lambda light chains bind to less than 100 .mu.m.

[0006] In another example, the disclosure encompasses an anti-LMA binding protein having an antigen binding domain, wherein the antigen binding domain binds to LMA and H and V L Including, -V H comprises a CDR1 as set forth in SEQ ID NO: 31, a CDR2 as set forth in SEQ ID NO: 32, and a CDR3 as set forth in SEQ ID NO: 33, and V L comprises a CDR1 as set forth in SEQ ID NO: 36 or SEQ ID NO: 41, a CDR2 as set forth in SEQ ID NO: 37 or SEQ ID NO: 42, and a CDR3 as set forth in SEQ ID NO: 38 or SEQ ID NO: 43, or -V H comprises a CDR1 as set forth in SEQ ID NO: 61, a CDR2 as set forth in SEQ ID NO: 62, and a CDR3 as set forth in SEQ ID NO: 63, and V L comprises a CDR1 as set forth in SEQ ID NO: 66 or SEQ ID NO: 71, a CDR2 as set forth in SEQ ID NO: 67 or SEQ ID NO: 72, and a CDR3 as set forth in SEQ ID NO: 68 or SEQ ID NO: 73. H comprises the amino acid sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 64. L comprises the amino acid sequence set forth in any one of SEQ ID NOs: 39, 44, 69, or 74.

[0007] In another example, the disclosure encompasses an anti-LMA binding protein having an antigen binding domain, wherein the antigen binding domain binds to LMA and comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), -V H comprises the amino acid sequence set forth in SEQ ID NO: 5, and V L comprises the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 15, -V H comprises the amino acid sequence set forth in SEQ ID NO: 34, and V L comprises the amino acid sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 45, -V Hcomprises the amino acid sequence set forth in SEQ ID NO: 65, and V L comprises the amino acid sequence shown in SEQ ID NO:70 or SEQ ID NO:75.

[0008] In another example, the disclosure encompasses an anti-LMA binding protein having an antigen binding domain, wherein the antigen binding domain binds to LMA and comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), -V H comprises CDR1, CDR2, and CDR3 derived from the amino acid sequence set forth in SEQ ID NO: 5, and V L comprises CDR1, CDR2, and CDR3 derived from the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 15, -V H comprises CDR1, CDR2, and CDR3 derived from the amino acid sequence set forth in SEQ ID NO: 34, and V L comprises CDR1, CDR2, and CDR3 derived from the amino acid sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 45; -V H comprises CDR1, CDR2, and CDR3 derived from the amino acid sequence set forth in SEQ ID NO: 65, and V L comprises CDR1, CDR2, and CDR3 derived from the amino acid sequence set forth in SEQ ID NO: 70 or SEQ ID NO: 75; CDRs are assigned using Kabat.

[0009] In another example, the disclosure encompasses an anti-LMA binding protein having an antigen binding domain, wherein the antigen binding domain binds to LMA and comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), -V H comprises CDR1, CDR2, and CDR3 derived from the amino acid sequence set forth in SEQ ID NO: 5, and V L comprises CDR1, CDR2, and CDR3 derived from the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 15, -V Hcomprises CDR1, CDR2, and CDR3 derived from the amino acid sequence set forth in SEQ ID NO: 34, and V L comprises CDR1, CDR2, and CDR3 derived from the amino acid sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 45; -V H comprises CDR1, CDR2, and CDR3 derived from the amino acid sequence set forth in SEQ ID NO: 65, and V L comprises CDR1, CDR2, and CDR3 derived from the amino acid sequence set forth in SEQ ID NO: 70 or SEQ ID NO: 75; CDRs are assigned using IMGT.

[0010] In another example, the binding proteins disclosed herein specifically bind to LMA. In another example, the binding proteins disclosed herein are antibodies. In one example, the antibodies specifically bind to LMA. In one example, the antibodies are human antibodies. In one example, the V of the binding proteins disclosed herein H and V L is present in a single polypeptide chain. For example, the binding protein may be: (i) single-chain Fv fragments (scFv), (ii) dimeric scFv (di-scFv), (iii) trimeric scFv (tri-scFv), (iv) the constant region of an antibody, Fc, or heavy chain constant domain C H 2 and / or C H Any one of (i), (ii), or (iii) linked to 3.

[0011] In another example, the V of the binding proteins disclosed herein H and V L are present on separate polypeptide chains. For example, the binding protein may be: (i) diabodies, (ii) triabodies, (iii) tetrabodies, (iv) Fab, (v) F(ab')2, (vi) Fv, (vii) the constant region of an antibody, Fc, or heavy chain constant domain C H 2 and / or C H One of (i) to (vi) connected to 3, or (viii) Intact antibody.

[0012] In another example, the disclosure includes a nucleic acid encoding a binding protein disclosed herein. In another example, the disclosure includes a vector comprising a nucleic acid encoding a binding protein disclosed herein. In one example, such a vector can be provided to a host cell for expression of the nucleic acid. Thus, in one example, the disclosure includes an isolated or recombinant cell that expresses a binding protein defined herein.

[0013] In another example, the disclosure includes a composition comprising a pharmaceutical carrier and any of the binding proteins defined herein.

[0014] In another example, the disclosure includes a method of treating an LMA-expressing cancer in a subject, the method comprising administering to the subject an effective amount of a binding protein defined herein. In another example, the disclosure includes the use of a binding protein defined herein in the manufacture of a medicament for treating an LMA-expressing cancer. In another example, the disclosure includes a binding protein defined herein for use in treating an LMA-expressing cancer. In one example, the LMA-expressing cancer is multiple myeloma, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma (DLBCL), POEMS syndrome, or amyloidosis.

[0015] In another example, the disclosure includes a method of treating an autoimmune disorder in a subject, the method comprising administering to the subject an effective amount of a binding protein as defined herein. In another example, the disclosure includes the use of a binding protein as defined herein in the manufacture of a medicament for treating an autoimmune disorder. In another example, the disclosure includes a binding protein as defined herein for use in treating an autoimmune disorder. In one example, the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, and multiple sclerosis.

[0016] Any example herein should be construed as applying mutatis mutandis to any other example, unless specifically stated otherwise.

[0017] The present disclosure is not intended to be limited in scope by the specific examples described herein. Such specific examples are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure as described herein.

[0018] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps, or group of compositions of matter should be interpreted as encompassing one and more (i.e., one or more) of that step, composition, group of steps, or group of compositions.

[0019] The present disclosure will now be described by way of the following non-limiting examples and with reference to the accompanying drawings. [Brief explanation of the drawings]

[0020] [Figure 1]4G7 monoclonal antibody epitope of lambda free light chain. The left panel shows an alignment of the amino acid sequences of lambda free light chain 1, 2, and 3 isotypes, with lambda isotypes 2 and 3 comprising 95% of the expressed lambda light chain repertoire. Asterisks indicate sequence identity. Different amino acids are highlighted. The right panel shows identified peptides of the 3D structure of the lambda light chain dimer (MCG dimer) and MCG Ig, demonstrating that within the folded light chain protein, the two peptides form a conjugate to create a non-idiotypic conformational epitope. [Figure 2A] Affinity of human anti-LMA antibodies to purified lambda light chain. (A) The graph shows the SPR value of 4G7 for human anti-LMA antibody clones (1A11, 7F11, 10B3, 18E8, 18E11, and 18F9) (20 μg / mL) against purified lambda light chain BJP (LAM034MD, LAM134MD, MAL788MD, LAM885D, LAM893D) (20 ng / mL). Anti-κBJP monoclonal antibodies MDX-1097 and κBJP (20 ng / mL) (KAP960M) were used as negative controls. The human anti-LMA antibody clones showed similarly high selective affinity for purified lambda light chain BJP, except for 7F11, which showed lower affinity. Abbreviations for the termini of lambda light chain BJP: M: monomer; MD: mixture of monomer and dimer; D: dimer. [Figure 2B] Affinity of human anti-LMA antibodies to purified lambda light chain. (B) The graph shows the SPR values ​​of human anti-LMA antibody clones (18E8, 7F11, and 18F9) (700 ng / mL) to commercially available lambda light chain (Bethyl). Human anti-LMA antibody 7F11 binds weaker to commercially available lambda light chain (Bethyl) compared to clones 18E8 and 18F9. [Figure 3]Affinity of human anti-LMA antibodies to recombinant lambda light chain isolates derived from HEK cells. The graph shows SPR values ​​of human anti-LMA antibody candidates (1A11, 7F11, 10B3, 18E8, 18E11, and 18F9) (20 μg / mL) against supernatants from cynomolgus lambda light chains (CYNO1 and CYNO3), lambda light chain-transfected HEK cells, the LP-1 lambda light chain isolate (LP-1 ISO), and the RPMI-8226 lambda light chain isolate (RPMI-8226 ISO). The anti-κBJP monoclonal antibody MDX-1097 was used as a negative control. The human anti-LMA antibody clones, except for 7F11, showed similarly high selective affinity for cynomolgus lambda light chains and the lambda light chain isolates derived from HEK, LP-1, and RPMI-8226, whereas 7F11 showed lower affinity. [Figure 4] Affinity of human anti-LMA antibodies to supernatant human multiple myeloma cell lines. The graph shows the SPR values ​​of human anti-LMA antibody candidates (6A1, 13H3, and 4A1) (20 μg / mL) for the supernatant of human multiple myeloma cell lines (RPMI-8226 (lambda isotype 2), LP-1 (lambda isotype 1), JJN3 (kappa), and OPM-2 (lambda isotype 3)). The human anti-LMA antibody clones showed high selective affinity for the soluble lambda light chain isotype expressed by the human multiple myeloma cell lines, but not for the kappa light chain expressed by the JJN3 cell line. [Figure 5A] Binding of human anti-LMA antibodies to LMA-positive human myeloma cell lines. (A) The graph shows the relative staining (geometric mean) of LMA-positive human myeloma cell lines (RPMI-8226 (lambda isotype 2), U266 (lambda isotype 2), JJN3 (kappa), OPM-2 (lambda isotype 3)) by human anti-LMA antibody candidates (1A11, 7F11, 10B3, 18E8, 18E11, and 18F9) compared to the no primary antibody control as determined by flow cytometry analysis. The human anti-LMA antibodies showed selective staining of all human myeloma cell lines tested, but did not (or only poorly) stain the JJN3 (kappa) cell line. [Figure 5B]Binding of human anti-LMA antibodies to LMA-positive human myeloma cell lines. (B) Graph shows relative staining (geometric mean) of LMA-positive human myeloma cell lines (U266 (lambda isotype 2), OPM-2 (lambda isotype 3), and KMS-18 (lambda isotype 1)) by human anti-LMA antibody candidates (1A11, 7F11, 10B3, 18E8, 18E11, 18F9, 6A1, 4A1, and 13H3) compared to no primary antibody control as determined by flow cytometry analysis. [Figure 6] Antibody-dependent cellular cytotoxicity (ADCC) against myeloma cells. [Figure 7] Complement-mediated cytotoxicity (CDC) against myeloma cells. [Figure 8] Sequence alignment comparing 7F11 with 10B3, 18E11, and 18E8. Heavy chain CDRs underlined. Asterisks indicate sequence identity. [Figure 9] Cryosection of human tonsil (HT2449-1) stained with 10 μg / mL 10B3. 40x objective lens. [Figure 10] Cryosection of human tonsil (HT2449-1) stained with 10 μg / mL of 7F11. 40x objective lens. DETAILED DESCRIPTION OF THE INVENTION

[0021] Deciphering the sequence listing NB: Kabat numbering system used to assign CDR and framework sequences SEQ ID NO: 1-7F11 heavy chain CDR1 amino acid sequence SEQ ID NO: 2-7F11 heavy chain CDR2 amino acid sequence SEQ ID NO: 3-7F11 heavy chain CDR3 amino acid sequence SEQ ID NOs: 4-7F11 heavy chain framework amino acid sequence SEQ ID NO: 5-7F11 heavy chain full length amino acid sequence SEQ ID NO: 6-7F11 Light Chain 1-CDR1 Amino Acid Sequence SEQ ID NO: 7-7F11 light chain 1-CDR2 amino acid sequence SEQ ID NO:8-7F11 light chain 1-CDR3 amino acid sequence SEQ ID NO: 9 - 7F11 light chain 1 - framework amino acid sequence SEQ ID NO: 10 - 7F11 light chain 1 - full length amino acid sequence SEQ ID NO: 11-7F11 light chain 2-CDR1 amino acid sequence SEQ ID NO: 12-7F11 light chain 2-CDR2 amino acid sequence SEQ ID NO: 13-7F11 light chain 2-CDR3 amino acid sequence SEQ ID NO: 14 - 7F11 light chain 2 - framework amino acid sequence SEQ ID NO: 15 - 7F11 light chain 2 - full length amino acid sequence SEQ ID NO: 16-7F11 heavy chain CDR1 DNA sequence SEQ ID NO: 17-7F11 heavy chain CDR2 DNA sequence SEQ ID NO: 18-7F11 heavy chain CDR3 DNA sequence SEQ ID NO: 19-7F11 heavy chain framework DNA sequence SEQ ID NO: 20-7F11 heavy chain full length DNA sequence SEQ ID NO: 21-7F11 Light Chain 1-CDR1 DNA sequence SEQ ID NO: 22-7F11 light chain 1-CDR2 DNA sequence SEQ ID NO: 23-7F11 Light Chain 1-CDR3 DNA Sequence SEQ ID NO: 24-7F11 light chain 1 - framework DNA sequence SEQ ID NO: 25 - 7F11 light chain 1 - full length DNA sequence SEQ ID NO: 26-7F11 Light Chain 2-CDR1 DNA Sequence SEQ ID NO: 27-7F11 Light chain 2-CDR2 DNA sequence SEQ ID NO: 28-7F11 light chain 2-CDR3 DNA sequence SEQ ID NO: 29 - 7F11 light chain 2 - framework DNA sequence SEQ ID NO:30 - 7F11 light chain 2 - full length DNA sequence SEQ ID NO: 31-18E8 heavy chain CDR1 amino acid sequence SEQ ID NO: 32-18E8 heavy chain CDR2 amino acid sequence SEQ ID NO: 33-18E8 heavy chain CDR3 amino acid sequence SEQ ID NO: 34-18E8 heavy chain framework amino acid sequence SEQ ID NO: 35-18E8 heavy chain full length amino acid sequence SEQ ID NO: 36-18E8 light chain 1-CDR1 amino acid sequence SEQ ID NO: 37-18E8 light chain 1-CDR2 amino acid sequence SEQ ID NO: 38 - 18E8 light chain 1-CDR3 amino acid sequence SEQ ID NO: 39 - 18E8 light chain 1 - framework amino acid sequence SEQ ID NO: 40 - 18E8 light chain 1 - full length amino acid sequence SEQ ID NO: 41-18E8 light chain 2-CDR1 amino acid sequence SEQ ID NO: 42-18E8 light chain 2-CDR2 amino acid sequence SEQ ID NO: 43-18E8 light chain 2-CDR3 amino acid sequence SEQ ID NO: 44-18E8 light chain 2 - framework amino acid sequence SEQ ID NO: 45 - 18E8 light chain 2 - full length amino acid sequence SEQ ID NO: 46-18E8 heavy chain CDR1 DNA sequence SEQ ID NO: 47-18E8 heavy chain CDR2 DNA sequence SEQ ID NO: 48-18E8 heavy chain CDR3 DNA sequence SEQ ID NO: 49-18E8 heavy chain framework DNA sequence SEQ ID NO: 50-18E8 heavy chain full length DNA sequence SEQ ID NO: 51-18E8 light chain 1-CDR1 DNA sequence SEQ ID NO: 52-18E8 light chain 1-CDR2 DNA sequence SEQ ID NO: 53-18E8 light chain 1-CDR3 DNA sequence SEQ ID NO: 54-18E8 light chain 1 - framework DNA sequence SEQ ID NO:55-18E8 light chain 1 - full length DNA sequence SEQ ID NO: 56-18E8 light chain 2-CDR1 DNA sequence SEQ ID NO: 57-18E8 light chain 2-CDR2 DNA sequence SEQ ID NO: 58-18E8 light chain 2-CDR3 DNA sequence SEQ ID NO: 59-18E8 light chain 2 - framework DNA sequence SEQ ID NO: 60-18E8 light chain 2 - full length DNA sequence SEQ ID NO: 61-10B3 heavy chain CDR1 amino acid sequence SEQ ID NO: 62-10B3 heavy chain CDR2 amino acid sequence SEQ ID NO: 63-10B3 heavy chain CDR3 amino acid sequence SEQ ID NO: 64-10B3 heavy chain framework amino acid sequence SEQ ID NO: 65-10B3 heavy chain full length amino acid sequence SEQ ID NO: 66-10B3 Light Chain 1-CDR1 Amino Acid Sequence SEQ ID NO: 67-10B3 Light Chain 1-CDR2 Amino Acid Sequence SEQ ID NO: 68-10B3 Light chain 1-CDR3 amino acid sequence SEQ ID NO: 69-10B3 light chain 1 - framework amino acid sequence SEQ ID NO: 70 - 10B3 Light Chain 1 - Full Length Amino Acid Sequence SEQ ID NO: 71-10B3 Light Chain 2-CDR1 Amino Acid Sequence SEQ ID NO: 72-10B3 Light Chain 2-CDR2 Amino Acid Sequence SEQ ID NO: 73-10B3 Light chain 2-CDR3 amino acid sequence SEQ ID NO: 74-10B3 light chain 2 - framework amino acid sequence SEQ ID NO: 75 - 10B3 light chain 2 - full length amino acid sequence SEQ ID NO: 76-10B3 heavy chain CDR1 DNA sequence SEQ ID NO: 77-10B3 heavy chain CDR2 DNA sequence SEQ ID NO: 78-10B3 heavy chain CDR3 DNA sequence SEQ ID NO: 79-10B3 heavy chain framework DNA sequence SEQ ID NO: 80-10B3 heavy chain full length DNA sequence SEQ ID NO: 81-10B3 Light Chain 1-CDR1 DNA Sequence SEQ ID NO: 82-10B3 Light Chain 1-CDR2 DNA Sequence SEQ ID NO: 83-10B3 Light chain 1-CDR3 DNA sequence SEQ ID NO: 84-10B3 Light chain 1 - framework DNA sequence SEQ ID NO: 85-10B3 Light Chain 1 - Full Length DNA Sequence SEQ ID NO: 86-10B3 Light Chain 2-CDR1 DNA Sequence SEQ ID NO: 87-10B3 Light chain 2-CDR2 DNA sequence SEQ ID NO: 88-10B3 Light chain 2-CDR3 DNA sequence SEQ ID NO: 89-10B3 Light chain 2 - framework DNA sequence SEQ ID NO: 90-10B3 Light Chain 2 - Full Length DNA Sequence SEQ ID NO: 91-4G7 epitope (lambda isotypes 2 and 3) epitope 1 SEQ ID NO: 92-4G7 epitope (lambda isotypes 2 and 3) epitope 2 SEQ ID NO: 93-4G7 epitope (lambda isotype 1) epitope 1 SEQ ID NO: 94-4G7 epitope (lambda isotype 1) epitope 2

[0022] General Techniques and Selected Definitions Unless specifically stated otherwise, all technical and scientific terms used herein (e.g., molecular biology, biochemistry, antibodies, antibody fragments, and clinical trials) should be understood to have the same meaning as commonly understood by one of ordinary skill in the art.

[0023] "Lambda myeloma antigen" (LMA) is a cell membrane antigen found on the surface of plasma cells, such as malignant myeloma cells, and in some cases, non-malignant plasma cell precursors, such as plasmablasts. Specifically, LMA consists of free lambda light chains expressed on the cell membrane. Anti-LMA binding proteins encompassed by the present disclosure specifically recognize a conformational epitope on the lambda light chain that is only available for binding when the lambda light chain is not associated with a heavy chain. Thus, anti-LMA binding proteins encompassed by the present disclosure do not bind to intact lambda chain-containing IgG, IgM, IgE, or IgA.

[0024] As used herein, the term "binds" with respect to the interaction of a binding protein and LMA described herein means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the LMA. For example, binding proteins generally recognize and bind to a specific antigen, rather than an antigen. For example, if a binding protein binds to epitope "A," the presence of a molecule containing epitope "A" (or free, unlabeled "A") in a reaction involving labeled "A" and the binding protein will reduce the amount of labeled "A" bound to the binding protein. In one example, an LMA-binding protein disclosed herein preferentially binds to LMA (i.e., a cell surface antigen) over free lambda light chains (e.g., serum antigens). A binding protein disclosed herein that preferentially binds to LMA over free lambda light chains reacts or associates more frequently, rapidly, for a longer period of time, and / or with greater affinity with LMA than free light chains.

[0025] As used herein, the term "specifically binds" should be interpreted to mean that the binding interaction between the binding protein and LMA is dependent on the detection of LMA by the binding protein. Thus, the binding protein specifically binds to or recognizes LMA even when present in a mixture of other molecules, cells, or organisms. In one example, the binding protein reacts or associates with LMA more frequently, rapidly, for a longer period of time, and / or with a higher affinity than with alternative antigens or cells. In one example, a binding protein disclosed herein that specifically binds to LMA can also preferentially bind to or recognize LMA over free light chain. It is also understood from reading this definition that, for example, a binding protein that specifically binds to LMA may or may not specifically bind to a second antigen. As such, "specific binding" does not necessarily require exclusive or undetectable binding of another antigen. The term "specifically binds" can be used interchangeably herein with "selectively bind." Generally, references to binding herein refer to specific binding, with each term being understood to explicitly support the other. Methods for determining specific binding will be apparent to those skilled in the art. For example, a binding protein of the present disclosure is contacted with LMA or a surrogate antigen. The binding of the binding protein to LMA or the surrogate antigen is then determined, and as described above, a binding protein that binds to LMA rather than the surrogate antigen is considered to specifically bind to LMA. Similar methods can be used to identify preferential binding. In this case, the surrogate antigen would be free light chain.

[0026] The term "immunoglobulin" will be understood to include anti-LMA binding proteins that contain immunoglobulin domains. Exemplary immunoglobulins are antibodies. Additional proteins encompassed by the term "immunoglobulin" include domain antibodies, camelid antibodies, and antibodies from cartilaginous fish (i.e., immunoglobulin novel antigen receptors (IgNARs)). Generally, camelid antibodies and IgNARs are composed of V H Including V LThey lack the T-cell receptor and are often called heavy chain immunoglobulins. Other "immunoglobulins" contain a T-cell receptor.

[0027] The term "binding protein" is used in the context of this disclosure to refer to human immunoglobulin molecules that immunologically react with a specific antigen, and includes both polyclonal and monoclonal antibodies. The term "binding protein" is also used in the context of the Pierce Catalogue and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3 rd "Antibody" encompasses antigen-binding forms of antibodies, including fragments having antigen-binding ability (e.g., Fab', F(ab')2, Fab, Fv, and rIgG) as discussed in "Antibody" (Ed.), W.H. Freeman & Co., New York (1998). The term is also used to refer to recombinant single-chain Fv fragments (scFv) and their bivalent (di-scFv) and trivalent (tri-scFv) forms. The term antibody also includes diabodies, triabodies, and tetrabodies.

[0028] An "antigen-binding fragment" of an antibody contains one or more variable regions of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; and single-chain antibody molecules formed from antibody fragments. For example, the term antigen-binding fragment may be used to refer to recombinant single-chain Fv fragments (scFv) and their divalent (di-scFv) and trivalent (tri-scFv) forms. Such fragments can be generated through a variety of methods known in the art.

[0029] The terms "full length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in a substantially intact form, as opposed to an antigen-binding fragment thereof. Specifically, whole antibodies include those having heavy and light chains, including the Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0030] The term "complementarity determining region" or "CDR" is used in the context of this disclosure to refer to the portions of the two variable chains (heavy and light) of an antibody that recognize and bind to a specific antigen. CDRs are the most variable parts of the variable chains and provide the binding protein with its specificity. Generally, the variable heavy (V H ) chain and variable light (V L ) chains each have three CDRs.

[0031] As used herein, "variable region" refers to the portion of the light and / or heavy chain of an antibody as defined herein that specifically binds to an antigen, and includes, for example, the amino acid sequences of the CDRs, i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, a variable region includes three CDRs as well as three or four FRs (e.g., FR1, FR2, FR3, and optionally, FR4). H refers to the variable region of the heavy chain. L refers to the variable region of the light chain.

[0032] In one example, the amino acid positions assigned to the CDRs and FRs are defined according to the Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system" or "Kabat").

[0033] Other conventions involving modified or alternative numbering systems for variable domains include IMGT (Lefranc, et al. (2003), Dev Comp Immunol 27:55-77), Chothia (Chothia C, Lesk AM (1987), J Mol Biol 196:901-917; Chothia, et al. (1989), Nature 342:877-883), and AHo (Honegger A, Pluckthun A (2001) J Mol Biol 309:657-670). For convenience, exemplary binding proteins of the present disclosure may also be labeled according to IMGT.

[0034] As used herein, the term "constant region" refers to a portion of an antibody heavy or light chain other than the variable region. In the heavy chain, the constant region generally comprises multiple constant domains and a hinge region; for example, an IgG constant region comprises the following connecting components: constant heavy chain C H 1. Linker, C H 2, and C H In the heavy chain, the constant region comprises Fc. In the light chain, the constant region generally comprises one constant domain (CL1).

[0035] The terms "fragment crystallizable" or "Fc" or "Fc region" or "Fc portion" (which can be used interchangeably herein) refer to the region of an antibody comprising at least one constant domain, which is generally (but not necessarily) glycosylated and capable of binding to one or more Fc receptors and / or components of the complement cascade. The heavy chain constant region can be selected from any of the five isotypes: α, δ, ε, γ, or μ. Exemplary heavy chain constant regions are gamma 1 (IgG1), gamma 2 (IgG2), and gamma 3 (IgG3), or hybrids thereof.

[0036] A "constant domain" is a domain in an antibody whose sequence is very similar in antibodies of the same type, e.g., IgG or IgM or IgE. The constant region of an antibody generally contains multiple constant domains, e.g., the constant region of a gamma, alpha, or delta heavy chain contains two constant domains.

[0037] The term "antibody heavy chain" is used herein to refer to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. "Antibody light chain" is used herein to refer to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0038] The term "naked" is used to refer to a binding protein of the present disclosure that is not conjugated to another compound, e.g., a toxic compound or a radioactive label. For example, the term "naked" can be used to refer to a binding protein that is not conjugated to another compound. Thus, in one example, a binding protein of the present disclosure is "naked." In other words, a binding protein of the present disclosure may be unconjugated.

[0039] In contrast, the term "conjugated" is used in the context of the present disclosure to refer to a binding protein described herein that is conjugated to another compound, e.g., a toxic compound such as a cytotoxic agent or a radiolabel. Thus, in one example, a binding protein of the present disclosure is "conjugated."

[0040] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic agents include radioisotopes (e.g., At 211 , 1 131 , 1 125 , Y 90 ,Re 186 ,Re 188 , Sm153 , radioactive isotopes of Bi, P, Pb, and Lu), chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants; and various anti-tumor or anti-cancer agents disclosed below.

[0041] Terms such as "host cell," "host cell line," and "host cell culture" are used interchangeably in the context of this disclosure to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0042] An "isolated nucleic acid," according to the present disclosure, is a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained within a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0043] As used herein, the term "expression vector" refers to a vector containing a recombinant nucleic acid sequence comprising at least one expression control sequence operably linked to the nucleic acid sequence to be expressed. An expression vector contains all the necessary cis-acting elements required for expression. Examples of expression vectors include, but are not limited to, plasmids, cosmids, and viruses that encode the recombinant polynucleotide to be expressed. In another example, an expression vector contains a transposable element capable of integration into a genome, e.g., the PiggyBac expression system. In another example, an expression vector is a viral vector, e.g., a retroviral vector and a lentiviral vector, which allows the expression vector contents to be integrated into the host genome.

[0044] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and does not take into account any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared.

[0045] The binding proteins according to the present disclosure and compositions comprising same can be administered to a subject to treat a variety of indications. Terms such as "subject," "patient," or "individual" are terms that can be used interchangeably in this disclosure, depending on the context. In one example, the subject is a mammal. The mammal may be a companion animal, such as a dog or cat, or a livestock animal, such as a horse or cow. In one example, the subject is a human. For example, the subject may be an adult. In another example, the subject may be a child. In another example, the subject may be an adolescent.

[0046] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include a reduction in the rate of disease progression, an improvement or alleviation of the disease state, and remission or improved prognosis. An individual is successfully "treated," for example, if one or more symptoms associated with a disease are alleviated or eliminated.

[0047] As used herein, the term "prevention" includes providing protection against the occurrence or recurrence of a disease in an individual, who may be susceptible to or at risk of developing a disease or disease recurrence, but who has not yet been diagnosed with the disease or recurrence.

[0048] An "effective amount" refers to at least an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" refers to an amount necessary to treat a disease or condition as described below. The effective amount may vary depending on the disease or condition to be treated, as well as on the weight, age, racial background, sex, health, and / or physical condition and other factors related to the subject being treated. Typically, an effective amount will fall within a relatively broad range (e.g., a "dosage" range) that can be determined through routine trial and experimentation by a physician.

[0049] A "therapeutically effective amount" is at least the minimum concentration required to bring about a measurable improvement in a particular disorder (e.g., cancer). The therapeutically effective amount herein may vary depending on factors such as the patient's condition, age, sex, and weight, and the ability of the binding protein to induce a desired response in an individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the binding protein are outweighed by the therapeutically beneficial effects. In the case of cancer, a therapeutically effective amount of the binding protein may reduce the number of cancer cells; reduce the size of initial tumors; inhibit (i.e., slow to a certain extent, and in some cases, stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to a certain extent, and in some cases, stop) tumor metastasis; inhibit or delay tumor growth or tumor progression to a certain extent; and / or alleviate one or more symptoms associated with cancer. To the extent the binding protein may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by assessing survival, time to disease progression (TTP), response rate (RR), duration of response, and / or quality of life.

[0050] Human Binding Proteins The present disclosure relates to "human" binding proteins. In one example, a "human" binding protein of the present disclosure can include amino acid residues not encoded by human sequences, e.g., randomly introduced mutations or site-specific mutations in vitro (specific mutations, including conservative substitutions or mutations, at a small number of residues in the protein, e.g., at 1, 2, 3, 4, or 5 of the residues in the protein). These "human binding proteins" need not necessarily be produced as a result of a human immune response; rather, they can be produced using recombinant means (e.g., by screening phage display libraries) and / or by transgenic animals (e.g., mice) containing nucleic acids encoding human antibody constant regions and / or human antibody variable regions, and / or using guided selection (e.g., as described in U.S. Pat. No. 5,565,332). The term also encompasses affinity-matured forms of such antibodies. In one example, the term encompasses human antibodies.

[0051] Monoclonal antibodies are another exemplary form of binding protein contemplated by the present disclosure. The term "monoclonal antibody" or "MAb" refers to a homogeneous antibody population capable of binding to the same antigen(s), e.g., the same epitope within the antigen. The term is not limited regarding the source of the antibody or the method by which it is made.

[0052] The present disclosure also contemplates deimmunized antibodies or antigen-binding fragments thereof, e.g., as described in WO2000 / 34317 and WO2004 / 108158. Deimmunized antibodies and fragments have one or more epitopes, e.g., B cell or T cell epitopes, removed (i.e., mutated), thereby reducing the likelihood that a subject will mount an immune response to the antibody or protein. For example, antibodies of the present disclosure are analyzed to identify one or more B cell or T cell epitopes, and one or more amino acid residues within the epitope are mutated, thereby reducing the immunogenicity of the antibody.

[0053] antibody fragments Single Domain Antibodies In some examples, the binding proteins of the disclosure are or comprise single domain antibodies (used interchangeably with the terms "domain antibody" or "dAb"). A single domain antibody is a single polypeptide chain that comprises all or part of the heavy chain variable domain of an antibody.

[0054] Single-chain Fv (scFv) fragments Those skilled in the art will appreciate that an scFv is a V H Area and V L The V domain, as well as the scFv, are capable of forming the desired structure for antigen binding (i.e., the V domain of a single polypeptide chain). H and V L associate with each other to form Fv)V H and V L It will be appreciated that the single-chain variable fragment may comprise a polypeptide linker between the fragments. Single-chain variable fragments lack the constant Fc region found in intact antibody molecules, thereby potentially reducing immunogenicity. Exemplary linkers contain more than 12 amino acid residues, with (Gly4Ser)3 being one of the more preferred linkers for scFvs.

[0055] The present disclosure also contemplates disulfide-stabilized Fvs (or diFvs or dsFvs), in which a single cysteine ​​residue is located at the V H FR and V L The cysteine ​​residues are introduced into the FR of the Fv and linked by disulfide bonds to generate a stable Fv.

[0056] In another example, the disclosure encompasses dimeric scFvs (di-scFvs), i.e., proteins comprising two scFv molecules non-covalently or covalently linked, for example, by a leucine zipper domain (e.g., derived from Fos or Jun), or trimeric scFvs (tri-scFvs). In another example, the two scFvs are linked by a peptide linker of sufficient length to allow both scFvs to form and bind to antigen, as described, for example, in U.S. Patent Published Application No. 20060263367.

[0057] Diabodies, triabodies, and tetrabodies In some examples, the antigen-binding fragments of the present disclosure are or include diabodies, triabodies, tetrabodies, or higher order protein complexes such as those described in WO98 / 044001 and / or WO94 / 007921.

[0058] For example, a diabody is a protein that includes two related polypeptide chains, each of which has the structure V L -XV H or V H -XV L X is a V of a single polypeptide chain H and V L a linker that contains insufficient residues to allow the V of one polypeptide chain to associate (or form an Fv), or is absent H is the V of the other polypeptide chain. L to form an antigen-binding site, i.e., an Fv molecule capable of specifically binding to one or more antigens.

[0059] Immunoglobulins and immunoglobulin fragments Examples of binding proteins of the present disclosure are proteins (e.g., antibody mimetics) that comprise the variable region of an immunoglobulin, such as a T cell receptor or a heavy chain immunoglobulin (e.g., IgNAR, camelid antibody).

[0060] V-like protein An example of a binding protein of the present disclosure is a T cell receptor. T cell receptors have two V domains that combine into a structure similar to the Fv module of an antibody. Novotny et al., Proc Natl Acad Sci USA 88:8646-8650, 1991, describe how the two V domains of a T cell receptor (designated alpha and beta) can be fused and expressed as a single polypeptide chain, and how surface residues can be modified to reduce hydrophobicity, much like an antibody scFv. Other publications describing the production of single-chain or multimeric T cell receptors containing two V-alpha and V-beta domains include WO1999 / 045110 or WO2011 / 107595.

[0061] Other non-antibody proteins containing antigen-binding domains include proteins with V-like domains, which are generally monomeric. Examples of proteins containing such V-like domains include CTLA-4, CD28, and ICOS. Further disclosure of such V-like domain-containing proteins is contained in WO1999 / 045110.

[0062] Affibody In a further example, the binding protein of the present disclosure is an affibody. Affibodies are scaffolds derived from the Z domain (antigen-binding domain) of Staphylococcus aureus protein A that can be engineered to bind to antigens. The Z domain consists of a three-helix bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details, see EP1641818.

[0063] Abima In a further example, the binding protein of the present disclosure is an avimer. Avimers are multidomain proteins derived from the A-domain scaffold family. The natural domains of approximately 35 amino acids adopt a defined disulfide bond structure. Diversity is generated by shuffling the natural variants represented by the A-domain family. For further details, see WO2002 / 088171.

[0064] Other binding proteins Other examples of binding proteins encompassed by the present disclosure include: (i) peptide display scaffolds such as Affimer and Adviron (WO2009136182; Tiede et al. (2014) Protein Eng Des Sel 27, 145-155); and (ii) centyrins (Jacobs et al. (2012) Protein Eng Des Sel.25, 107-117; Diem et al. (2014) Protein Eng Des sel. 27, 49-429).

[0065] Lambda myeloma antigen (LMA) binding protein The binding proteins defined herein have an antigen binding domain that binds or specifically binds to lambda myeloma antigen (LMA). In one example, the binding proteins defined herein have an antigen binding domain that preferentially binds to LMA over free light chain. In one example, an anti-LMA binding protein according to the present disclosure comprises a heavy chain variable region (V) having CDR1 as set forth in SEQ ID NO: 1, CDR2 as set forth in SEQ ID NO: 2, and CDR3 as set forth in SEQ ID NO: 3. H In another example, the anti-LMA binding protein comprises a light chain variable region (V) having a CDR1 as set forth in SEQ ID NO:6, a CDR2 as set forth in SEQ ID NO:7, and a CDR3 as set forth in SEQ ID NO:8 or SEQ ID NO:13. LIn another example, the anti-LMA binding protein comprises a light chain variable region (V) having a CDR1 as set forth in SEQ ID NO:6, a CDR2 as set forth in SEQ ID NO:7, and a CDR3 as set forth in SEQ ID NO:8. L In another example, the anti-LMA binding protein comprises a light chain variable region (V) having a CDR1 as set forth in SEQ ID NO:6, a CDR2 as set forth in SEQ ID NO:7, and a CDR3 as set forth in SEQ ID NO:13. L Thus, in another example, the anti-LMA binding protein comprises a VHQ having a CDR1 as set forth in SEQ ID NO:1, a CDR2 as set forth in SEQ ID NO:2, and a CDR3 as set forth in SEQ ID NO:3. H and a V having a CDR1 as set forth in SEQ ID NO:6, a CDR2 as set forth in SEQ ID NO:7, and a CDR3 as set forth in SEQ ID NO:8. L In another example, the anti-LMA binding protein comprises a V having a CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3. H and a V having a CDR1 as set forth in SEQ ID NO:6, a CDR2 as set forth in SEQ ID NO:7, and a CDR3 as set forth in SEQ ID NO:13. L In these example embodiments, the LMA binding protein binds to LMA isotype 2 and LMA isotype 3.

[0066] In another example, the anti-LMA binding protein comprises a V polypeptide comprising an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:4. H In another example, the anti-LMA binding protein comprises a V that comprises an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:9 or SEQ ID NO:14. L For example, V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 9. In another example, V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 14. H comprises an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 5. In another example, V Lcomprises an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 15. For example, V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 10. In another example, V L comprises an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 15. Thus, in another example, an anti-LMA binding protein may comprise a V that comprises an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 4. H and V comprising an amino acid sequence at least 95% identical to the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 15. L For example, V H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:4, and V L comprises an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 10. For example, V H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:4, and V L comprises the amino acid sequence set forth in SEQ ID NO: 15. In these examples, V H and / or V L may be at least 96%, at least 97%, at least 98%, or at least 99% identical to the listed SEQ ID NO.

[0067] In another example, the anti-LMA binding protein comprises a V H In another example, the anti-LMA binding protein comprises a V that comprises the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:14. L For example, V L can comprise the amino acid sequence set forth in SEQ ID NO: 9. In another example, V L can comprise the amino acid sequence set forth in SEQ ID NO: 14. In another example, V H comprises the amino acid sequence set forth in SEQ ID NO: 5. In another example, V L comprises the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 15. For example, V L can comprise the amino acid sequence set forth in SEQ ID NO: 10. In another example, V Lcomprises the amino acid sequence set forth in SEQ ID NO: 15. Thus, in another example, the anti-LMA binding protein comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 4. H and V comprising the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 15. L For example, V H may comprise the amino acid sequence set forth in SEQ ID NO: 4, and V L comprises the amino acid sequence set forth in SEQ ID NO: 10. For example, V H may comprise the amino acid sequence set forth in SEQ ID NO: 4, and V L comprises the amino acid sequence set forth in SEQ ID NO: 15. In these examples, the binding protein specifically binds to LMA. For example, the binding protein may bind preferentially to LMA over free lambda light chain.

[0068] In another example, the anti-LMA binding protein comprises a heavy chain variable region (V) having a CDR1 as set forth in SEQ ID NO: 31, a CDR2 as set forth in SEQ ID NO: 32, and a CDR3 as set forth in SEQ ID NO: 33. H In another example, the anti-LMA binding protein comprises a heavy chain variable region (V) having a CDR1 as set forth in SEQ ID NO: 61, a CDR2 as set forth in SEQ ID NO: 62, and a CDR3 as set forth in SEQ ID NO: 63. H In another example, the anti-LMA binding protein comprises a light chain variable region (V) having a CDR1 as set forth in SEQ ID NO:36 or SEQ ID NO:41, a CDR2 as set forth in SEQ ID NO:37 or SEQ ID NO:42, and a CDR3 as set forth in SEQ ID NO:38 or SEQ ID NO:43. L For example, an anti-LMA binding protein may comprise a VHQ having a CDR1 as set forth in SEQ ID NO:36, a CDR2 as set forth in SEQ ID NO:37, and a CDR3 as set forth in SEQ ID NO:38. L In another example, the anti-LMA binding protein may comprise a V having a CDR1 as set forth in SEQ ID NO:41, a CDR2 as set forth in SEQ ID NO:42, and a CDR3 as set forth in SEQ ID NO:43. LThus, in an example, the anti-LMA binding protein may comprise a heavy chain variable region (V) having CDR1 as set forth in SEQ ID NO: 31, CDR2 as set forth in SEQ ID NO: 32, and CDR3 as set forth in SEQ ID NO: 33. H ), and V comprising CDR1 as set forth in SEQ ID NO: 36 or SEQ ID NO: 41, CDR2 as set forth in SEQ ID NO: 37 or SEQ ID NO: 42, and CDR3 as set forth in SEQ ID NO: 38 or SEQ ID NO: 43 L For example, the anti-LMA binding protein may comprise a heavy chain variable region (V) having a CDR1 as set forth in SEQ ID NO: 31, a CDR2 as set forth in SEQ ID NO: 32, and a CDR3 as set forth in SEQ ID NO: 33. H ), and a V comprising CDR1 as set forth in SEQ ID NO: 36, CDR2 as set forth in SEQ ID NO: 37, and CDR3 as set forth in SEQ ID NO: 38. L In another example, the anti-LMA binding protein may comprise a heavy chain variable region (V) having a CDR1 as set forth in SEQ ID NO: 31, a CDR2 as set forth in SEQ ID NO: 32, and a CDR3 as set forth in SEQ ID NO: 33. H ), and a V comprising CDR1 as set forth in SEQ ID NO: 41, CDR2 as set forth in SEQ ID NO: 42, and CDR3 as set forth in SEQ ID NO: 43 L may include:

[0069] In another example, the anti-LMA binding protein comprises a heavy chain variable region (V) having a CDR1 as set forth in SEQ ID NO: 61, a CDR2 as set forth in SEQ ID NO: 62, and a CDR3 as set forth in SEQ ID NO: 63. H ), and V comprising CDR1 as set forth in SEQ ID NO: 66 or SEQ ID NO: 71, CDR2 as set forth in SEQ ID NO: 67 or SEQ ID NO: 72, and CDR3 as set forth in SEQ ID NO: 68 or SEQ ID NO: 73 L For example, the anti-LMA binding protein may comprise a heavy chain variable region (V) having a CDR1 as set forth in SEQ ID NO: 61, a CDR2 as set forth in SEQ ID NO: 62, and a CDR3 as set forth in SEQ ID NO: 63. H), and a V comprising CDR1 as set forth in SEQ ID NO: 66, CDR2 as set forth in SEQ ID NO: 67, and CDR3 as set forth in SEQ ID NO: 68. L In another example, the anti-LMA binding protein may comprise a heavy chain variable region (V) having a CDR1 as set forth in SEQ ID NO: 61, a CDR2 as set forth in SEQ ID NO: 62, and a CDR3 as set forth in SEQ ID NO: 63. H ), and a V comprising CDR1 as set forth in SEQ ID NO: 71, CDR2 as set forth in SEQ ID NO: 72, and CDR3 as set forth in SEQ ID NO: 73 L In these example embodiments, the LMA binding protein binds to LMA isotypes 1, 2 and isotype 3.

[0070] In another example, V H comprises an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 64. For example, V H can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 34. H can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 64. L comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 39, 44, 69, or 74. In another example, V H comprises an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 64, and V L comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 39, 44, 69, or 74. For example, V H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 34, and V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 39. H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 34, and V Lcan comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 44. H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 64, and V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 39. H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 64, and V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 44. H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 64, and V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 69. H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 64, and V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 74. H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:5, and V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 15. In another example, V H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:5, and V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 10. In another example, V H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:5, and V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 15. In another example, V H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 34, and V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 45. H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 34, and VL can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 40. H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 34, and V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 45. H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 65, and V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 70 or SEQ ID NO: 75. H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 65, and V L can comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 70. H may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 65, and V L may comprise an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 75. In these examples, V H and / or V L can be at least 96%, at least 97%, at least 98%, or at least 99% identical to the listed SEQ ID NO. H comprises the amino acid sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 64. For example, V H comprises the amino acid sequence set forth in SEQ ID NO: 34. H comprises the amino acid sequence set forth in SEQ ID NO: 64. L comprises the amino acid sequence set forth in any one of SEQ ID NOs: 39, 44, 69, or 74. In another example, V H comprises the amino acid sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 64, and V L comprises the amino acid sequence set forth in any one of SEQ ID NOs: 39, 44, 69, or 74. For example, V H may comprise the amino acid sequence set forth in SEQ ID NO: 34, and V Lcan comprise the amino acid sequence set forth in SEQ ID NO: 39. H comprises the amino acid sequence set forth in SEQ ID NO: 34, and V L comprises the amino acid sequence set forth in SEQ ID NO: 44. H comprises the amino acid sequence set forth in SEQ ID NO: 64, and V L comprises the amino acid sequence set forth in SEQ ID NO: 39. H comprises the amino acid sequence set forth in SEQ ID NO: 64, and V L comprises the amino acid sequence set forth in SEQ ID NO: 44. H comprises the amino acid sequence set forth in SEQ ID NO: 64, and V L comprises the amino acid sequence set forth in SEQ ID NO: 69. H comprises the amino acid sequence set forth in SEQ ID NO: 64, and V L comprises the amino acid sequence set forth in SEQ ID NO: 74. H comprises the amino acid sequence set forth in SEQ ID NO: 5, and V L comprises the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 15. In another example, V H comprises the amino acid sequence set forth in SEQ ID NO: 5, and V L comprises the amino acid sequence set forth in SEQ ID NO: 10. In another example, V H comprises the amino acid sequence set forth in SEQ ID NO: 5, and V L comprises the amino acid sequence set forth in SEQ ID NO: 15. In another example, V H comprises the amino acid sequence set forth in SEQ ID NO: 34, and V L comprises the amino acid sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 45. H comprises the amino acid sequence set forth in SEQ ID NO: 34, and V L comprises the amino acid sequence set forth in SEQ ID NO: 40. H comprises the amino acid sequence set forth in SEQ ID NO: 34, and V L comprises the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 45. H comprises the amino acid sequence set forth in SEQ ID NO: 65, and V Lcomprises the amino acid sequence set forth in SEQ ID NO: 70 or SEQ ID NO: 75. H comprises the amino acid sequence set forth in SEQ ID NO: 65, and V L comprises the amino acid sequence set forth in SEQ ID NO: 70. H comprises the amino acid sequence set forth in SEQ ID NO: 65, and V L comprises the amino acid sequence set forth in SEQ ID NO:75.

[0071] In one example, the above-mentioned binding protein is an antibody. For example, the present disclosure encompasses antibodies having the above-mentioned combinations of CDRs. For example, the antibody of the present disclosure may comprise a heavy chain variable region (V) having CDR1 as set forth in SEQ ID NO: 1, CDR2 as set forth in SEQ ID NO: 2, and CDR3 as set forth in SEQ ID NO: 3. H In another example, the antibody may comprise a light chain variable region (V) having a CDR1 as set forth in SEQ ID NO:6, a CDR2 as set forth in SEQ ID NO:7, and a CDR3 as set forth in SEQ ID NO:8 or SEQ ID NO:13. L In another example, the antibody comprises a light chain variable region (V) having a CDR1 as set forth in SEQ ID NO:6, a CDR2 as set forth in SEQ ID NO:7, and a CDR3 as set forth in SEQ ID NO:8. L In another example, the antibody comprises a light chain variable region (V) having a CDR1 as set forth in SEQ ID NO:6, a CDR2 as set forth in SEQ ID NO:7, and a CDR3 as set forth in SEQ ID NO:13. L Thus, in another example, the antibody comprises a VHQ having a CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3. H and a V having a CDR1 as set forth in SEQ ID NO:6, a CDR2 as set forth in SEQ ID NO:7, and a CDR3 as set forth in SEQ ID NO:8. L In another example, the antibody comprises a V having a CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3. Hand a V having a CDR1 as set forth in SEQ ID NO:6, a CDR2 as set forth in SEQ ID NO:7, and a CDR3 as set forth in SEQ ID NO:13. L In these example embodiments, the antibody binds to LMA isotype 2 and isotype 3.

[0072] In another example, the binding proteins encompassed by the present disclosure include those exemplified above. H and V L In one example, the CDRs are defined using Kabat. In another example, the CDRs are defined using IMGT.

[0073] Referring to the binding proteins having the above-referenced % sequence identity, in various examples, binding proteins encompassed by the present disclosure can have at least one, at least two, at least three, at least four, or at least five amino acid substitutions compared to the referenced sequence identifier number. Exemplary substitutions include conservative amino acid substitutions as set forth in Table A below. [Table 1-A]

[0074] The term "4G7" is used in the context of this disclosure to refer to a monoclonal antibody against a human free lambda light chain that binds to an epitope comprising the amino acid sequence shown below: SEQ ID NOs: 91 (KADGSPVK) and 93 (SHR) (isotype 1), SEQ ID NOs: 92 (KADSSPVK) and 93 (SHR) (isotype 2), or SEQ ID NOs: 92 (KADSSPVK) and 94 (SHK) (isotype 3). 4G7 is commercially available from a variety of sources (e.g., Abcam, Cambridge, United Kingdom, #ab54380; Yamasa Corporation, Choshi, Japan, #7642).

[0075] In one example, an anti-LMA binding protein encompassed by the present disclosure may bind to the same epitope as 4G7. For example, an anti-LMA binding protein encompassed by the present disclosure may bind to an epitope comprising the amino acid sequence shown below: SEQ ID NOs: 91 and 93 (lambda isotype 1), SEQ ID NOs: 92 and 93 (lambda isotype 2), or - SEQ ID NOs: 92 and 94 (lambda isotype 3).

[0076] In another example, an anti-LMA binding protein encompassed by the present disclosure binds to an epitope comprising the amino acid sequence shown below: SEQ ID NOs: 91 and 93 (lambda isotype 1), SEQ ID NOs: 92 and 93 (lambda isotype 2), or SEQ ID NOs: 92 and 94 (lambda isotype 3), It does not bind to soluble lambda light chains.

[0077] In another example, the binding protein binds to SEQ ID NOs: 92 and 93 (lambda isotype 2) and SEQ ID NOs: 92 and 94 (lambda isotype 3).

[0078] A variety of lambda light chain isotypes have been described, which are defined by amino acid differences in the constant region of the molecule.

[0079] Lambda light chain isotypes are represented with varying frequency. For example, in subjects with multiple myeloma, approximately 14% express isotype 1, approximately 64% express isotype 2, and approximately 23% express isotype 3. Thus, in some instances, it may be desirable to administer an antibody according to the present disclosure that binds to one or more or all lambda light chain isotypes. In one example, the binding protein binds to lambda isotype 2 and isotype 3. In one example, the binding protein does not bind to isotype 1.

[0080] In another example, the binding protein has improved manufacturability compared to 4G7.

[0081] Improved manufacturability includes deamidation sites, aspartate isomerization sites, oxidation sites such as methionine and tryptophan, free cysteine ​​thiol groups, reduced number of N&O-glycosylation sites, presence of a C-terminal lysine, and / or post-translational modifications related to isoelectric point or improved chemical stability.

[0082] In one example, the binding protein has fewer asparagines in the V compared to 4G7. H and / or V L Included.

[0083] In one example, the binding protein contains fewer methionines in the V compared to 4G7. H and / or V L Included.

[0084] In one example, the binding protein contains less tryptophan in V compared to 4G7. H and / or V L Included.

[0085] In one example, the binding protein contains fewer aspartates in the V compared to 4G7. H and / or V L Included.

[0086] In one example, the physical stability of the binding protein is greater than that of 4G7.

[0087] Physical stability can include the tendency for aggregation in solution. The term "aggregation" is used in the context of this disclosure to refer to protein self-association, which can occur in multiple environments, from cell culture and fermentation, to isolation, purification, and formulation processes. For example, the term "aggregation" can be used to describe the formation of inclusion bodies; the accumulation of proteins in the "insoluble" fraction after cell fractionation; the appearance of turbidity in a sample, protein precipitation, or particle formation; or the formation of small soluble oligomers, among others.

[0088] Thus, in the example mentioned above, the physical stability of the binding protein can be based on its physical stability in solution, with precipitation of the binding protein from solution indicating that the binding protein has become unstable. To assess physical stability, a solution containing a binding protein according to the present disclosure or 4G7 can be incubated at 4°C and visually assessed for precipitation at 2 weeks, 4 weeks, 12 weeks, 6 months, and 12 months.

[0089] In another example, the binding protein has reduced immunogenicity in a human subject compared to 4G7. For example, the binding protein may have reduced immunogenicity compared to 4G7 when immunogenicity is measured by enzyme-linked immunosorbent assay (ELISA). In another example, the binding protein may have reduced immunogenicity compared to 4G7 when immunogenicity is measured via surface plasmon resonance.

[0090] In another example, the binding protein has greater specificity for LMA than for 4G7.

[0091] In another example, the binding protein has lower cross-reactivity (i.e., the ability of the binding protein to react with similar antigenic sites on different proteins) compared to 4G7. In this example, the cross-reactivity of the binding protein can be measured using various methods. In one example, the cross-reactivity is assessed via ELISA.

[0092] In another example, the binding protein has a higher binding affinity for LMA than for 4G7.

[0093] In another example, the binding protein has a higher binding affinity for LMA than for 4G7 relative to the light chain.

[0094] In the examples mentioned above, the affinity of the binding protein for LMA can be measured using a variety of methods. In one example, the dissociation constant (K D ) or association constant (K A) or equilibrium constant (K D ) is determined. In one example, these constants for a binding protein are measured by a radiolabeled or fluorescently labeled LMA binding assay. This assay equilibrates the binding protein with a minimal concentration of labeled LMA in the presence of a titration series of unlabeled LMA. After washing to remove unbound LMA, the amount of label is determined.

[0095] Affinity measurements can be determined by standard techniques for following antibody reactions, such as immunoassays, surface plasmon resonance (SPR) (Rich and Myszka Curr. Opin. Biotechnol 11:54, 2000; Englebienne Analyst. 123:1599, 1998), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art.

[0096] In one example, the constant is measured using a surface plasmon resonance assay, for example, using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized LMA. An exemplary SPR method is described in U.S. Patent No. 7,229,619.

[0097] In another example, the affinity of a binding protein for an LMA can be measured using isothermal titration microcalorimetry.

[0098] Generation of binding proteins Recombinant expression In one example, the binding proteins described herein are peptides or polypeptides (e.g., antibodies or antigen-binding fragments thereof). In one example, the binding proteins are recombinant.

[0099] In the case of a recombinant peptide or polypeptide, the nucleic acid encoding it can be cloned into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulin or antibody protein, such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells.

[0100] Suitable molecular cloning techniques are known in the art and are described, for example, in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art. See U.S. Patent No. 4,816,567 or U.S. Patent No. 5,530,101.

[0101] After isolation, the nucleic acid is inserted operably linked to a promoter into an expression construct or expression vector for further cloning (amplification of the DNA) or expression in a cell-free system or cell. Accordingly, another example of the present disclosure provides an expression construct comprising an isolated nucleic acid of the present disclosure and one or more additional nucleotide sequences. Suitably, the expression construct is in the form of, or contains genetic components of, a plasmid, bacteriophage, cosmid, yeast, or bacterial artificial chromosome, as understood in the art. The expression construct may be suitable for maintaining and propagating the isolated nucleic acid in bacteria or other host cells, for manipulation by recombinant DNA technology, and / or for expressing the nucleic acid or binding protein of the present disclosure.

[0102] Many vectors for intracellular expression are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding a binding protein (e.g., obtained from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, α-factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0103] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate-early promoter (CMV-IE), the human elongation factor 1-alpha promoter (EF1), small nuclear RNA promoters (U1a and U1b), the alpha-myosin heavy chain promoter, the simian virus 40 promoter (SV40), the Rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the beta-actin promoter; hybrid regulatory elements containing the CMV enhancer / beta-actin promoter or immunoglobulin or antibody promoters or their active fragments. Examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0104] Representative promoters suitable for expression in yeast cells, e.g., yeast cells selected from the group including Pichia pastoris, Saccharomyces cerevisiae, and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0105] Means for introducing isolated nucleic acids or expression constructs containing the same into cells for expression are known to those of skill in the art. The technique used for a given cell depends on the known and successful technique. Means for introducing recombinant DNA into cells include microinjection, DEAE-dextran-mediated transfection, liposome-mediated transfection, e.g., by using Lipofectamine (Gibco, MD, USA) and / or Cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, particle bombardment, e.g., by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA), among others.

[0106] Host cells used to produce binding proteins (e.g., antibodies or antigen-binding fragments) may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPML-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing the other cell types discussed herein are known in the art.

[0107] Those skilled in the art will appreciate from the foregoing that the present disclosure also provides isolated nucleic acids encoding binding proteins of the present disclosure (e.g., peptide or polypeptide binding proteins or antibodies or antigen-binding fragments thereof).

[0108] The present disclosure also provides an expression construct comprising the isolated nucleic acid of the present disclosure operably linked to a promoter. In one example, the expression construct is an expression vector.

[0109] In one example, the expression construct of the disclosure comprises a polypeptide (e.g., V H a nucleic acid encoding a polypeptide (e.g., a polypeptide encoding a polypeptide of interest) operably linked to a promoter; L The present invention relates to a nucleic acid encoding a

[0110] The present disclosure also provides a host cell comprising an expression construct according to the present disclosure.

[0111] The present disclosure also provides isolated cells that express the binding proteins of the present disclosure, or recombinant cells that have been genetically modified to express the binding proteins.

[0112] Protein isolation Purification methods for binding proteins according to the present disclosure are known in the art. When the peptide or polypeptide is secreted into the medium, the supernatant from such an expression system can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0113] The binding protein prepared from the cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., Protein A affinity chromatography or Protein G chromatography), or any combination of the above. These methods are known in the art and are described, for example, in WO99 / 57134 or Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0114] Conjugates In one example, the binding protein of the present disclosure is conjugated to another compound. The binding protein can be directly or indirectly bound to the compound (e.g., a linker can be included in the case of indirect binding). Examples of compounds include radioisotopes (e.g., iodine-131, yttrium-90, or indium-111), detectable labels (e.g., fluorophores or fluorescent nanocrystals or quantum dots), therapeutic compounds (e.g., chemotherapeutic or anti-inflammatory), colloids (e.g., gold), toxins (e.g., ricin or tetanus toxoid), nucleic acids, agents that extend the half-life of the compound in a subject (e.g., polyethylene glycol or other water-soluble polymers with this activity), and mixtures thereof.

[0115] Methods for attaching drugs or other small molecule pharmaceuticals to antibodies are well known and include, but are not limited to, N-succinimidyl(4-iodoacetyl)-aminobenzoate; sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate; 4-succinimidyl-oxycarbonyl-(2-pyridyldithio)toluene; sulfosuccinimidyl-6-[α-methyl∀-(pyridyldithiol)-toluamido]hexanoate; N-succinimidyl-3-(-2-pyridyldithio)-propionate; succinimidyl-6-[3(-(-2-pyridyldithio)-propionate] The use of bifunctional chemical linkers may include the use of bifunctional linkers such as sulfosuccinimidyl-6-[3-(-(-2-pyridyldithio)-propionamido]hexanoate; sulfosuccinimidyl-6-[3-(-(-2-pyridyldithio)-propionamido]hexanoate; 3-(2-pyridyldithio)-propionyl hydrazide, Ellmans' reagent, dichlorotriazine acid, S-(2-thiopyridyl)-L-cysteine, and the like. Additional bifunctional linker molecules are described, for example, in U.S. Pat. Nos. 5,349,066, 5,618,528, 4,569,789, 4,952,394, and 5,137,877.

[0116] Linkers can be cleavable or non-cleavable. A highly stable linker can reduce the amount of payload shed during circulation, thereby improving the safety profile and allowing more payload to reach target cells. Linkers can be based on chemical motifs, including disulfides, hydrazones, or peptides (cleavable), or thioethers (non-cleavable), to control the distribution and delivery of active agents to target cells. Cleavable and non-cleavable linkers have been proven safe in preclinical and clinical trials (see, for example, brentuximab vedotin, which contains an enzyme-sensitive linker cleavable by cathepsins, and trastuzumab emtansine, which contains a stable non-cleavable linker). In one example, the linker is a peptide linker cleavable by Edman degradation (Bachor, et al., Molecular diversity, 17(3):605-11 (2013)).

[0117] In one example, the binding protein is conjugated to a nanoparticle or microparticle (e.g., as reviewed in Kogan et al., Nanomedicine (Lond). 2:287-306, 2007). The nanoparticle may be a metal nanoparticle. The particle may be a polymer particle, a liposome, a micelle, a microbubble, or other carrier and delivery vehicle known in the art.

[0118] Some exemplary compounds that can be conjugated to the binding proteins of the present disclosure are listed in Table B. [Table 1-B]

[0119] In one example, a binding protein of the present disclosure is conjugated to a chemotherapeutic agent.

[0120] composition Suitably, in compositions or methods for administering a binding protein according to the present disclosure to a subject, the binding protein is combined with a pharmaceutically acceptable carrier, as understood in the art. In one example, the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising a binding protein of the present disclosure combined with a pharmaceutically acceptable carrier. In another example, the present disclosure provides a kit comprising a pharmaceutically acceptable carrier suitable for combining or mixing with a binding protein disclosed herein prior to administration to a subject. In this example, the kit may further include instructions for use.

[0121] In general terms, the term "carrier" is used to refer to a solid or liquid filler, binder, diluent, encapsulating material, emulsifier, wetting agent, solvent, suspending agent, coating, or lubricant that can be safely administered to a subject, e.g., a human subject. Depending on the particular route of administration, a variety of acceptable carriers known in the art may be used, as described below, e.g., in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991).

[0122] For example, suitable carriers may be selected from the group comprising sugars (e.g., sucrose, maltose, trehalose, glucose), starch, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, oils including vegetable oils, synthetic oils and synthetic mono- or diglycerides, lower alcohols, polyols, alginic acid, phosphate buffers, lubricants such as sodium or magnesium stearate, isotonic saline, and pyrogen-free water. In one example, the carrier is not HO.

[0123] In one example, the carrier is compatible with or suitable for parenteral administration. Parenteral administration includes administration routes that do not go through the digestive tract. Examples of parenteral administration include injection, infusion, etc. Examples of administration by injection include intravenous, intraarterial, intramuscular, and subcutaneous injection. In another example, the composition can be delivered via a depot or sustained-release formulation that can be delivered intradermally, intramuscularly, or subcutaneously.

[0124] In one example, the LMA-binding proteins disclosed herein are utilized to detect the site(s) of cancer. The method typically involves administering to a subject in need thereof an effective amount of a detectable agent using diagnostic imaging or nuclear medicine techniques and detecting the agent. In such methods, the agent is typically conjugated to the LMA-binding protein or encapsulated in a delivery vehicle that is conjugated to the LMA-binding protein. The diagnostic imaging or nuclear medicine technique can be, for example, PET-CT, bone scan, MRI, CT, echocardiography, ultrasound, and X-ray.

[0125] In one example, the binding proteins and compositions comprising them can be used in the manufacture of a medicament for the treatment of a condition characterized by abnormal proliferation of LMA-expressing cells, such as an LMA-expressing cancer. In another example, the disclosure relates to the binding proteins or compositions comprising them for use in treating a condition. Examples of conditions to be treated are discussed below.

[0126] Condition to be treated In one example, the disclosure includes a method of treating an LMA-expressing cancer, the method comprising administering an anti-LMA binding protein as defined herein. For example, the disclosure includes a method of treating a B-cell malignancy in which the malignant B cells express LMA. In another example, the disclosure includes a method of treating multiple myeloma and related conditions. The terms "multiple myeloma" or "myeloma," in the context of this disclosure, are used to refer to cancers of plasma cells. In the context of this disclosure, these terms include secretory myeloma, non-secretory myeloma, light chain-only myeloma, smoldering myeloma, and related conditions. Examples of related conditions include plasmacytoma, amyloidosis, and monoclonal gammopathy of undetermined significance. In one example, the multiple myeloma is lambda multiple myeloma.

[0127] Thus, in one example, the present disclosure encompasses a method of treating amyloidosis. In one example, such a method comprises administering a binding protein disclosed herein having high affinity for free lambda light chains. For example, a V binding protein having a CDR1 as set forth in SEQ ID NO: 31, a CDR2 as set forth in SEQ ID NO: 32, and a CDR3 as set forth in SEQ ID NO: 33. H and a V having a CDR1 as set forth in SEQ ID NO: 36, a CDR2 as set forth in SEQ ID NO: 37, and a CDR3 as set forth in SEQ ID NO: 38. L In another example, a binding protein comprising the amino acid sequence set forth in SEQ ID NO: 34 can be administered. H and V having the amino acid sequence shown in SEQ ID NO: 39 L In another example, a V binding protein having a CDR1 as set forth in SEQ ID NO: 66, a CDR2 as set forth in SEQ ID NO: 67, and a CDR3 as set forth in SEQ ID NO: 68 can be administered. H and a V having CDR1 as set forth in SEQ ID NO: 71, CDR2 as set forth in SEQ ID NO: 72, and CDR3 as set forth in SEQ ID NO: 73. L In another example, a binding protein comprising the amino acid sequence set forth in SEQ ID NO: 64 can be administered. Hand V having the amino acid sequence shown in SEQ ID NO: 74 L In another example, a V binding protein having a CDR1 as set forth in SEQ ID NO: 61, a CDR2 as set forth in SEQ ID NO: 62, and a CDR3 as set forth in SEQ ID NO: 63 can be administered. H and a V having a CDR1 as set forth in SEQ ID NO: 66, a CDR2 as set forth in SEQ ID NO: 67, and a CDR3 as set forth in SEQ ID NO: 68. L A binding protein comprising:

[0128] Subjects with multiple myeloma can be characterized into various subject populations. Exemplary populations are described in (Rajkumar et al. 2011).

[0129] In one example, the subject's multiple myeloma can be characterized as progressive disease (Rajkumar et al. 2011). In other words, the methods of the present disclosure relate to treating progressive multiple myeloma in a subject. Exemplary indicators of "progressive disease" include an increase of about 25% from the minimum response value in any one of the following: serum M component (an absolute increase of 0.5 g / dL or more), and / or urine M component (an absolute increase must be 200 mg / 24 hours or more). Other exemplary indicators include a clear progression of new bone lesions or soft tissue plasmacytomas or a clear increase in the size of existing bone lesions or soft tissue plasmacytomas that can be attributed solely to multiple myeloma; and a progression of hypercalcemia (corrected serum calcium greater than 11.5 mg / dL). In one example, the subject's multiple myeloma has relapsed and is characterized as progressive disease. In this example, the subject's multiple myeloma may also be refractory to therapy.

[0130] In one example, the subject's multiple myeloma has relapsed. "Relapsed myeloma" is used to refer to previously treated myeloma that has progressed and requires the initiation of salvage therapy, but does not meet the criteria for any "primary refractory myeloma."

[0131] In another example, the subject has primary refractory myeloma. "Primary refractory myeloma" is used to refer to refractory disease in patients who have not achieved more than a minimal response to any treatment.

[0132] In another example, the subject has refractory myeloma. The term "refractory myeloma" is used to refer to disease that is refractory to first-line or salvage therapy or progresses within 60 days of the last therapy. In one example, the subject's multiple myeloma is refractory to anti-cancer therapy. The term "refractory" in this context refers to an anti-cancer treatment line that is no longer therapeutically effective against the subject's multiple myeloma. For example, a subject treated by the methods of the present disclosure may be refractory to at least one proteasome inhibitor. A "line of treatment" is defined as one or more cycles of a planned treatment program. This may consist of one or more planned cycles of monotherapy or combination therapy, and a series of treatments administered in a planned manner. For example, a planned treatment approach of induction therapy, followed by autologous stem cell transplantation, followed by maintenance therapy, is considered one line of treatment.

[0133] In another example, the subject is refractory to at least two prior lines of therapy, hi another example, the subject may be refractory to at least three, at least four, at least five, or at least six prior lines of therapy.

[0134] In another example, the subject has relapsed and refractory myeloma, which is used to refer to disease that is refractory during salvage therapy or progresses within 60 days of the last treatment in patients who have achieved a minimal response (MR) or better at some point in the disease course before progressing.

[0135] In one example, multiple myeloma treated according to the present disclosure is characterized by stable disease at the time of the first administration. In other words, the subject may be in a plateau phase at the time of the first administration. Exemplary criteria for stable disease may include stabilization of M protein without further tumor regression despite continued treatment, few or no symptoms of myeloma, and / or no need for transfusions (Blade et al. 1998).

[0136] In another example, the methods of the present disclosure can be used to treat B-cell lymphoma and macroglobulinemia.

[0137] In another example, the methods of the present disclosure can be used to treat POEMS. As used herein, "POEMS syndrome" refers to a rare blood disorder that damages nerves and affects many other parts of the body. "POEMS" refers to these signs and symptoms: polyneuropathy: numbness, tingling, and weakness in the legs and hands over time, as well as difficulty breathing; organomegaly: enlargement of the spleen, liver, or lymph nodes; endocrine disorders: abnormal hormone levels that can lead to hypothyroidism (hyperthyroidism), diabetes, sexual problems, fatigue, swelling of the hands and feet, and problems with metabolism and other essential functions; monoclonal plasma proliferative disorders: abnormal bone marrow cells (plasma cells) that produce proteins that can be found in the bloodstream; and skin changes: more color than normal on the skin, sometimes thicker skin, and increased hair on the face and / or legs.

[0138] In another example, the binding proteins defined herein can be administered to a subject to treat an autoimmune disorder. In one example, the autoimmune disorder is characterized by the abnormal proliferation of plasma cell precursors that express LMA as membrane free light chain (mFLC). For example, the binding proteins defined herein can be administered to a subject to treat an autoimmune disorder, such as rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, multiple sclerosis, Crohn's disease, immune thrombocytopenic purpura, pemphigus vulgaris, autoimmune urticaria, celiac disease, dermatitis herpetiformis, acute rheumatic fever, Graves' disease, myasthenia gravis, Sjögren's syndrome, Goodpasture's syndrome, poststreptococcal glomerulonephritis, contact dermatitis, autoimmune thyroiditis, Hashimoto's thyroiditis, Addison's disease, autoimmune hemolytic anemia, pernicious anemia, antineutrophil cytoplasmic antibody (ANCA) vasculitis, polyarteritis nodosa, autoimmune hepatitis, and primary biliary cirrhosis. For example, the methods of the present disclosure can be used to treat rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, and multiple sclerosis. Thus, in one example, the methods of the present disclosure can be used to treat rheumatoid arthritis. In another example, the methods of the present disclosure can be used to treat systemic lupus erythematosus. In another example, the binding proteins of the present disclosure can be used to treat diabetes mellitus. In another example, the binding proteins of the present disclosure can be used to treat multiple sclerosis.

[0139] In another example, a binding protein according to the present disclosure can be used to reduce lambda free light chain levels in a subject (i.e., to reduce the amount of lambda light chain in a subject that is not expressed on the cell membrane, e.g., lambda light chain in serum). Such a method includes administering a binding protein disclosed herein that has high affinity for free lambda light chain. For example, a V binding protein having a CDR1 as set forth in SEQ ID NO: 31, a CDR2 as set forth in SEQ ID NO: 32, and a CDR3 as set forth in SEQ ID NO: 33 can be used. H and a V having a CDR1 as set forth in SEQ ID NO: 36, a CDR2 as set forth in SEQ ID NO: 37, and a CDR3 as set forth in SEQ ID NO: 38. LIn another example, a binding protein comprising the amino acid sequence set forth in SEQ ID NO: 34 can be administered. H and V having the amino acid sequence shown in SEQ ID NO: 39 L In another example, a V binding protein having a CDR1 as set forth in SEQ ID NO: 66, a CDR2 as set forth in SEQ ID NO: 67, and a CDR3 as set forth in SEQ ID NO: 68 can be administered. H and a V having CDR1 as set forth in SEQ ID NO: 71, CDR2 as set forth in SEQ ID NO: 72, and CDR3 as set forth in SEQ ID NO: 73. L In another example, a binding protein comprising the amino acid sequence set forth in SEQ ID NO: 64 can be administered. H and V having the amino acid sequence shown in SEQ ID NO: 74 L In another example, a V binding protein having a CDR1 as set forth in SEQ ID NO: 61, a CDR2 as set forth in SEQ ID NO: 62, and a CDR3 as set forth in SEQ ID NO: 63 can be administered. H and a V having a CDR1 as set forth in SEQ ID NO: 66, a CDR2 as set forth in SEQ ID NO: 67, and a CDR3 as set forth in SEQ ID NO: 68. L A binding protein comprising:

[0140] In another example, the methods of the disclosure include treating an LMA-expressing cancer or other disorder disclosed herein by administering a binding protein of the disclosure having high affinity for free lambda light chains in combination with a binding protein of the disclosure having low affinity for free lambda light chains. For example, a V comprising the amino acid sequence set forth in SEQ ID NO: 64. H and V having the amino acid sequence shown in SEQ ID NO: 74 L The binding protein having the V H and V having the amino acid sequence shown in SEQ ID NO:9 L In another example, a V antibody can be administered in combination with a binding protein having the amino acid sequence set forth in SEQ ID NO: 64. Hand V having the amino acid sequence shown in SEQ ID NO: 74 L The binding protein having the V H and V having the amino acid sequence shown in SEQ ID NO:9 L The compound may be administered in combination with a binding protein having the formula:

[0141] Antibodies administered in combination as part of the practice of the methods of the present disclosure may be administered simultaneously or sequentially. [Example]

[0142] Example 1: Lambda myeloma antigen (LMA) on lambda multiple myeloma cells To confirm the presence of lambda myeloma antigen (LMA) on the surface of lambda multiple myeloma cells, the murine monoclonal antibody 4G7 against lambda Bence Jones protein (BJP) was used in ELISA, surface plasmon resonance (SPR), and Western blot assays. Compared with all free lambda light chains of BJP and the murine monoclonal antibody 3D12, 4G7 exhibited pan-reactivity with a broad range of lambda human multiple myeloma cell lines, encompassing the three major lambda light chain isotypes (Tables 1 and 2). The interaction could be inhibited by lambda free light chains, but not by IgG / lambda, demonstrating the presence of lambda light chains on the cell surface of the lambda human multiple myeloma cell line RPMI8226. 4G7 also detected LMA in bone marrow mononuclear cell populations from lambda multiple myeloma patients that were positive for CD38 and CD138 as determined by flow cytometry analysis (Table 3). Epitope excision experiments identified two peptides as components of the 4G7 monoclonal antibody epitope on the lambda multiple myeloma cell line (Fig. 1 ). [Table 1] [Table 2] [Table 3]

[0143] Example 2: Generation of human anti-LMA antibodies HuMAb-mice (Medarex) were immunized with Ig free lambda light chains and lambda myeloma antigen (LMA)-positive cell lines. Human anti-LMA antibodies were screened for binding to Ig free lambda light chains and Ig bound lambda light chains using ELISA to identify LMA-specific candidates. Human anti-LMA antibodies were purified and characterized for binding to LMA-positive cell lines using flow cytometry and for affinity for Ig free lambda light chains using surface plasmon resonance (SPR) analysis. The identified human anti-LMA antibody candidates were 1A11, 7F11, 10B3, 18E8, 18E11, and 18F9.

[0144] Example 3: Human anti-LMA antibodies bind to lambda light chains To determine the binding affinity of anti-LMA antibodies to lambda light chains, surface plasmon resonance (SPR) experiments were performed. Anti-human Ig light chains were immobilized on a BIACORE CM5 chip and used to capture human anti-LMA antibody candidates (20 μg / ml). After candidate capture, the tested lambda light chains were injected (20 μL / min) and SPR was determined (relative units: RU).

[0145] Human anti-LMA antibodies (clones 1A11, 7F11, 10B3, 18E8, 18E11, and 18F9) showed high and selective affinity for purified lambda light chain BJP (but not κBJP (KAP960M)) (Figure 2A), cynomolgus monkey lambda light chains (CYNO1, CYNO3), and the supernatants of lambda light chain-transfected HEK cells, LP-1 lambda light chain isolate (LP-1 ISO), and RPMI-8226 lambda light chain isolate (RPMI-8226 ISO) (Figure 3). Similarly, human anti-LMA antibodies (6A1, 13H3, and 4A1) showed high selective affinity for human multiple myeloma cell lines expressing lambda light chain isotypes, but not for the kappa light chain-expressing JJN3 cell line (Figure 4), whereas clones 18E8, 7F11, and 18F9 showed selective affinity for the commercially available lambda light chain (Bethyl) (Figure 2B).

[0146] The human anti-LMA antibody 7F11 showed lower selective affinity for purified lambda light chain BJP compared with the other clones tested (Figures 2A and 2B), and showed lower affinity for cynomolgus monkey lambda light chains (CYNO1, CYNO3; Figure 3) and supernatants from lambda light chain-transfected HEK cells, the LP-1 lambda light chain isolate (LP-1 ISO), and the RPMI-8226 lambda light chain isolate (RPMI-8226 ISO) (Figure 4).

[0147] Example 4: Human anti-LMA antibodies bind to LMA-positive human myeloma cell lines Flow cytometric analysis was performed to determine the binding of human anti-LMA antibodies to human myeloma cell lines expressing different lambda isotypes of LMA.

[0148] A solution of human anti-LMA antibody (50 μg / mL) or mouse anti-LMA antibody 4G7 was cultured on a human myeloma cell line (5x10 5Human anti-LMA antibodies (1A11, 7F11, 10B3, 18E8, 18E11, and 18F9) showed selective staining of all human myeloma cell lines tested (RPMI-8226 (lambda isotype 2), U266 (lambda isotype 2), JJN3 (kappa), and OPM-2 (lambda isotype 3)), with no or weak staining of the JJN3 (kappa) cell line (Figure 5A) and weak staining of KMS-18 (lambda isotype 1) (Figure 5B). Human anti-LMA antibodies (6A1, 4A1, and 13H3) showed weak staining of all LMA-positive human myeloma cell lines tested.

[0149] Example 5: Antibody-dependent cytotoxicity (ADCC) Peripheral blood mononuclear cell (PMBC) preparations, or specific cell populations contained in PMBC preparations, such as natural killer (NK) cells or monocytes, were analyzed in ADCC assays. Blood was layered onto Ficoll, the gradient was centrifuged, and PBMCs were collected from the interface of the gradient.

[0150] Specific cell populations were isolated from PMBC preparations using magnetically labeled antibody preparations (Miltenyi Biotec, Germany) to remove unwanted cells. Effector and target cells were mixed and incubated for 16 hours at 37°C in RPMI supplemented with 10% fetal bovine serum. The extent of cell lysis was determined by measuring the level of released intracellular lactate dehydrogenase (LDH) (CytoTox-ONE Homogenous Membrane Integrity Assay Kit; Promega, USA).

[0151] The human anti-LMA antibodies exhibited varying degrees of effector function, with clone 10B3 exhibiting the strongest ADCC among the antibodies tested (FIG. 6).

[0152] Example 6: Complement-dependent cytotoxicity (CDC) Target cells were incubated in RPMI supplemented with 10% fetal bovine serum in the presence of complement (either purified or human serum containing complement) and antibody at 37°C for 30 minutes to 12 hours. The degree of cell lysis was determined by measuring the level of released intracellular lactate dehydrogenase (LDH) (CytoTox-ONE Homogenous Membrane Integrity Assay Kit; Promega, USA). The metabolic status of the cells was measured using Alamar Blue (Invitrogen, USA).

[0153] The human anti-LMA antibodies exhibited varying degrees of effector function, with clone 10B3 exhibiting the strongest CDC activity among the antibodies tested (FIG. 7).

[0154] Example 7: Human anti-LMA antibody 7F11 is selective for LMA but not for free lambda light chains The human anti-LMA antibodies 10B3 and 18E8 bind both soluble lambda light chains and LMA-positive cells and can promote ADCC and CDC when used to target lambda-expressing RPMI8226 cells (Table 5). Interestingly, the human anti-LMA antibody 7F11 is selective for LMA (membrane-bound light chains) and can promote ADCC and CDC when used to target lambda-expressing RPMI8226 cells but not soluble lambda light chains (Table 5). Analysis of the 7F11 sequence revealed unique heavy chain CDRs compared to other human antibodies bound to free light chains (Figure 8). The distinct binding properties of the human anti-LMA antibody 7F11 compared to 10B3 and 18E8 allow for its selective use to deplete free lambda light chains and / or target LMA-positive cells. [Table 5]

[0155] Table 5 shows that 7F11 does not bind to cells expressing lambda light chain isotype 1, whereas 4G7, 18E8, and 10B3 do.

[0156] Example 8: Human anti-LMA antibody staining in human tissues Human LP-1 (multiple myeloma) cells, JJN3 (plasma cell leukemia), human tonsil specimens, and human cerebellum specimens were contacted with the 10B3 and 7F11 antibodies, and antibody staining was compared to control antibody staining from HuIgG1-lambda.

[0157] LP-1 cells express LMA isotype 1. 10B3 stained LP-1 cells, but 7F11 did not. These results are consistent with the results above, which show that 10B3, but not 7F11, binds to lambda isotype 1. No staining of JJN3 cells was observed with either 10B3 or 7F11.

[0158] Tonsils are the primary site for microbial and environmental antigens to be administered in the body and therefore house lymphoid cells (approximately 55% of lymphoid cells are B cells). Weak to strong staining of follicular germinal centers, interfollicular germinal centers, and rare mononuclear cells in the interfollicular region was observed with 10B3 and 7F11 (Figures 9 and 10; human tonsil stained with 10B3, with higher affinity than 7F11). These results confirm that both 10B3 and 7F11 can bind to LMA-expressing cells in humans.

[0159] Importantly, no staining of human cerebellum was observed with 10B3 or 7F11. Human cerebellum is known to be an effective control for assessing nonspecific binding. The lack of staining with both 10B3 and 7F11 highlights the specificity of these antibodies for LMA and reduces the risk of nonspecific binding in vivo.

[0160] Example 9: Anti-LMA antibody affinity Anti-human Fc IgG was immobilized on a CM5 sensor chip (Protein A; Fc2 was captured with antibody, and Fc1 was set as a blank). Anti-LMA antibodies (7F11-VL1; 10B3-VL1; 10B3-VL2) were flowed over the chip surface at 25°C (running buffer: 1x HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4)), and binding characteristics were recorded using a Biacore 8k. Assay characteristics are summarized in Table 6. Affinity measurements are shown in Table 7.

[0161] 10B3-VL1 bound with high affinity to all lambda free light chain antigens. Only 7F11 bound to the lambda free light chain antigens of Sigma, 134c, and 788a, but with low affinity. These findings are consistent with Example 7, which shows that 7F11 is selective for LMA, but not for free lambda light chains. 10B3-VL2 did not bind to lambda light chains, suggesting that modifications to the J gene of this antibody abolished its affinity for lambda free light chains.

[0162] Importantly, no binding to kappa light chains was observed for any of the antibodies tested, further confirming the specificity of these antibodies for lambda light chains.

[0163] In summary, 7F11 has no / low affinity for binding to soluble lambda light chains but binds to LMA+ cell lines of isotypes 2 and 3. In contrast, 10B3-VL1 binds to all soluble lambda light chains with high affinity and binds to all LMA+ cell lines (isotypes 1, 2, and 3). 10B3-VL2, which has a different J gene from 10B3-VL1, does not bind to soluble lambda light chains. [Table 6] [Table 7]

[0164] It will be understood by those skilled in the art that numerous variations and / or modifications may be made to the present disclosure as set forth in the specific embodiments without departing from the spirit or scope of the present disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0165] All of the above publications are incorporated herein in their entirety.

[0166] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure and is not to be construed as an admission that any or all of these matters formed part of the prior art base or were common general knowledge in the art relevant to the present disclosure as they existed before the priority date of each claim of this application.

[0167] This application claims priority to AU 2018900534, filed February 20, 2018, the entire contents of which are incorporated herein by reference.

[0168] (References) TIFF0007798956000009.tif224170

Claims

1. An anti-lambda myeloma antigen (LMA) binding protein having an antigen-binding domain, wherein the antigen-binding domain binds to LMA and comprises a heavy chain variable region (V Ｈ ) and the light chain variable region (V Ｌ ) and V Ｈ comprises a complementarity determining region (CDR) 1 set forth in SEQ ID NO: 31, a CDR2 set forth in SEQ ID NO: 32, and a CDR3 set forth in SEQ ID NO: 33, Ｌ an anti-LMA binding protein comprising a CDR1 set forth in SEQ ID NO:36 or SEQ ID NO:41, a CDR2 set forth in SEQ ID NO:37 or SEQ ID NO:42, and a CDR3 set forth in SEQ ID NO:38 or SEQ ID NO:

43.

2. The V Ｈ The binding protein of claim 1, wherein said binding protein comprises the amino acid sequence set forth in SEQ ID NO:

35.

3. The V Ｌ 3. The binding protein of claim 1 or claim 2, wherein said binding protein comprises the amino acid sequence set forth in SEQ ID NO: 40 or SEQ ID NO:

45.

4. An anti-lambda myeloma antigen (LMA) binding protein having an antigen-binding domain, wherein the antigen-binding domain binds to LMA and comprises a heavy chain variable region (V Ｈ ) and the light chain variable region (V Ｌ ) and V Ｈ comprises the amino acid sequence set forth in SEQ ID NO: 35, Ｌ An anti-LMA binding protein comprising the amino acid sequence set forth in SEQ ID NO:40 or SEQ ID NO:

45.

5. The binding protein of any one of claims 1 to 4, wherein the binding protein specifically binds to LMA.

6. The binding protein of any one of claims 1 to 5, which is an antibody.

7. The V Ｈ and V Ｌ The binding protein of any one of claims 1 to 6, wherein is a single polypeptide chain.

8. (i) single chain Fv fragments (scFv), (ii) dimeric scFv (di-scFv), (iii) trimeric scFv (tri-scFv), (iv) the constant region of an antibody, Fc, or heavy chain constant domain C Ｈ 2 and / or C Ｈ Any one of (i), (ii), or (iii) linked to 3 8. The binding protein of claim 7, wherein:

9. The V Ｈ and V Ｌ The binding protein of any one of claims 1 to 6, wherein are separate polypeptide chains.

10. (i) diabodies, (ii) triabodies, (iii) tetrabodies, (iv) Fab, (v)F(ab’) ２ 、 (vi) Fv, (vii) the constant region of an antibody, Fc, or heavy chain constant domain C Ｈ 2 and / or C Ｈ one of (i) to (vi) linked to 3; or (viii) intact antibody 10. The binding protein of claim 9, wherein 11. A binding protein according to any one of claims 1 to 10, which is a human binding protein.

12. An anti-LMA binding protein having an antigen-binding domain, wherein the antigen-binding domain binds to LMA and comprises a heavy chain variable region (V Ｈ ) and the light chain variable region (V Ｌ ) and V Ｈ is encoded by the nucleotide sequence set forth in SEQ ID NO: 50, Ｌ An anti-LMA binding protein, wherein the anti-LMA binding protein is encoded by the nucleotide sequence set forth in SEQ ID NO:

55.

13. A nucleic acid encoding the binding protein of any one of claims 1 to 12.

14. An expression construct comprising the nucleic acid of claim 13.

15. 15. An isolated or recombinant cell that expresses a binding protein according to any one of claims 1 to 12, comprising a nucleic acid according to claim 13 or an expression construct according to claim 14.

16. A composition comprising a pharmaceutical carrier and a binding protein according to any one of claims 1 to 12.

17. 17. Use of a binding protein according to any one of claims 1 to 12 or a composition according to claim 16 in the manufacture of a medicament for treating an LMA-expressing cancer.

18. 17. The binding protein of any one of claims 1 to 12 or the composition of claim 16 for use in treating an LMA-expressing cancer.

19. 18. The use of claim 17, wherein the LMA-expressing cancer is multiple myeloma, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma (DLBCL), POEMS syndrome, or amyloidosis.

20. 19. The binding protein or composition of claim 18, wherein the LMA-expressing cancer is multiple myeloma, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma (DLBCL), POEMS syndrome, or amyloidosis.

Images