Binding Protein 1

Abstract

The present disclosure relates to cell-permeable anti-DNA binding proteins. Compositions containing these binding proteins can be useful for delivering agents to cells and for treating diseases such as cancer.

Description

Technical Field

[0001] The present disclosure relates to cell - permeable anti - DNA - binding proteins. Compositions containing these binding proteins can be useful for delivering agents to cells and for treating diseases such as cancer.

Background Art

[0002] The development of cell - permeable anti - DNA - binding proteins as therapeutic agents for human diseases has significant clinical potential, particularly due to their ability to selectively impair DNA repair pathways and / or deliver various therapeutic payloads to target cells.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Therefore, improved cell - permeable anti - DNA - binding proteins are needed.

Means for Solving the Problems

[0004] The inventors have identified modifications of cell - permeable anti - DNA - binding proteins that surprisingly increase nuclear penetration. In some cases, these modifications can also improve physical stability and reduce immunogenicity.

[0005] Thus, in a first example, the present disclosure provides a cell - permeable anti - DNA - binding protein having an antigen - binding domain, wherein the antigen - binding domain binds or specifically binds to DNA and has a heavy - chain variable region (V H ) having complementarity - determining region (CDR) 1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2 or SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4, and a light - chain variable region (V L ) having CDR1 as shown in SEQ ID NO: 5 or SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8. Relates to the cell - permeable anti - DNA - binding protein, including this. In this example, the CDRs are defined using Kabat.

[0006] In another example, the present disclosure provides a cell - permeable anti - DNA - binding protein having an antigen - binding domain, wherein the antigen - binding domain binds to or specifically binds to DNA and: - A heavy - chain variable region (V H ) having complementarity - determining regions (CDRs) 1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10 or SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12 - A light - chain variable region (V L ) having CDR1 as shown in SEQ ID NO: 13 or SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 Relates to the cell - permeable anti - DNA - binding protein, including this. In this example, the CDRs are defined using IMGT.

[0007] In another example, the binding protein according to the present disclosure is (i) A V H containing a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 17 - 23 (ii) A V L containing a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 24 - 29, or (iii) A V H containing a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 17 - 23 and a V L containing a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 24 - 29. For example, the binding protein may contain a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 17 - 23. In another example, the binding protein is a V Lmay include. In another example, the binding protein includes a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 17 to 23 for V H and a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 24 to 29 for V L and may include.

[0008] In another example, V H and V L are separated by a linker. For example, the linker may include (Gly 4 Ser) 3 and may include. In another example, the linker includes the amino acid sequence as shown in SEQ ID NO: 30.

[0009] In one example, V H and V L are in a single polypeptide chain. For example, the binding protein may be (i) a single-chain Fv fragment (scFv), (ii) a dimeric scFv (di-scFv), (iii) a trimeric scFv (tri-scFv), (iv) the constant region of an antibody, Fc or the heavy-chain constant domain C H 2 and / or C H 3 linked to any one of (i), (ii) or (iii) and may be. For example, the binding protein may be a di-scFv. In this example, the scFv may be separated by a linker. For example, the linker may include the amino acid sequence as shown in SEQ ID NO: 31.

[0010] In another example, V H and V L are in separate polypeptide chains. For example, the binding protein may be (i) a diabody, (ii) a triabody, (iii) a tetrabody, (iv) a Fab, (v) an F(ab’) 2 , (vi) Fv, (vii) the constant region of the antibody, Fc or heavy chain constant domain C H 2 and / or C H linked to C2 and / or C3, one of (i)-(vi), or, (viii) an intact antibody can be.

[0011] Thus, the V of Fv H and V L can be formed from a single peptide chain (e.g., scFv) or from two separate peptide chains.

[0012] In one example, the binding protein is humanized.

[0013] In another example, the present disclosure provides a cell-permeable anti-DNA Fv fragment having an antigen-binding domain, wherein the antigen-binding domain binds to or specifically binds to DNA and - CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2 or SEQ ID NO: 3, V having CDR3 as shown in SEQ ID NO: 4 H and V having CDR1 as shown in SEQ ID NO: 5 or SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7 and CDR3 as shown in SEQ ID NO: 8 L , - CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10 or SEQ ID NO: 11, V having CDR3 as shown in SEQ ID NO: 12 H and V having CDR1 as shown in SEQ ID NO: 13 or SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15 and CDR3 as shown in SEQ ID NO: 16 L , - V containing a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOS: 17-23 H and V containing a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOS: 24-29L Relating to the cell-permeable anti-DNA Fv fragment comprising at least one of them. In this example, the Fv fragment may be a diabody-scFv. In one example, the Fv fragment may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 32 to 47. For example, the Fv fragment may comprise an amino acid sequence as shown in SEQ ID NO: 41.

[0014] In some embodiments, the Fv is naked. In another example, the Fv fragment may be conjugated to another compound.

[0015] In one example, the Fv is humanized. For example, the Fv may be a humanized diabody-scFv.

[0016] In another example, the present disclosure relates to a nucleic acid sequence encoding the binding protein mentioned above. Exemplary nucleic acid sequences are shown in SEQ ID NOs: 51 to 66. The nucleic acid sequences of the present disclosure can be codon-optimized to increase the expression level for synthesizing the protein. In another example, the present disclosure relates to an expression vector comprising the nucleic acid sequence according to the present disclosure. For example, the expression vector may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 51 to 66 or its codon-optimized sequence.

[0017] In another example, the present disclosure relates to a host cell comprising the binding protein, nucleic acid or vector mentioned above, or its codon-optimized sequence.

[0018] In another example, the present disclosure relates to a method of treating cancer. For example, a method of treating cancer comprising administering to a subject an Fv fragment comprising an amino acid sequence as shown in any one of SEQ ID NOs: 32, 36, 41, or 43. For example, an Fv fragment comprising an amino acid sequence as shown in SEQ ID NO: 32 may be administered to the subject. In another example, an Fv fragment comprising an amino acid sequence as shown in SEQ ID NO: 36 may be administered to the subject. In another example, an Fv fragment comprising an amino acid sequence as shown in SEQ ID NO: 41 may be administered to the subject. In another example, an Fv fragment comprising an amino acid sequence as shown in SEQ ID NO: 43 may be administered to the subject. In one example, the cancer is colon cancer, brain cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, or pancreatic cancer. For example, the cancer may be colon cancer or brain cancer. In one example, the cancer is brain cancer. In one example, the brain cancer is glioblastoma.

[0019] In another example, the present disclosure relates to the use of a binding protein such as an Fv fragment, composition, vector, or host cell according to the present disclosure in the manufacture of a medicament for treating cancer. In another example, the present disclosure relates to a binding protein such as an Fv fragment, composition, vector, or host cell according to the present disclosure for use in treating cancer.

[0020] The following experimental results also illustrate that the binding proteins disclosed herein can act together with a poly(ADP-ribose) polymerase (PARP) inhibitor to kill cancer cells. Thus, in another example, the present disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a binding protein or Fv fragment as defined herein and a PARP inhibitor.

[0021] In one example, the PARP inhibitor is olaparib.

[0022] In one example, the cancer is substantially HDR - deficient. In another example, the cancer is substantially BRCA2 - deficient. In another example, the cancer is substantially PTEN - deficient. In one example, the cancer is colon cancer, brain cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, or pancreatic cancer. For example, the cancer may be colon cancer or brain cancer. In one example, the cancer is brain cancer. In one example, the brain cancer is glioblastoma. In one example, the cancer is resistant to PARP inhibition. For example, the cancer may be resistant to treatment with olaparib. In another example, the cancer is triple - negative breast cancer.

[0023] In another example, the disclosure relates to a therapeutic combination comprising a binding protein or Fv fragment as defined herein and a PARP inhibitor, wherein the therapeutic combination is administered simultaneously or sequentially. In another example, the disclosure - a binding protein or Fv comprising the CDR of SEQ ID NO: 41, or - a binding protein comprising the amino acid sequence shown in SEQ ID NO: 41, and a PARP inhibitor, wherein the therapeutic combination is administered simultaneously or sequentially. For example, the binding protein or Fv may comprise heavy - chain CDRs as shown in SEQ ID NOs: 1, 3, and 4 and light - chain CDRs as shown in SEQ ID NOs: 6, 7, and 8. In these examples, the therapeutic combination may be used to treat cancer. Further, in these examples, the PARP inhibitor may be olaparib.

[0024] Any example herein shall be construed to apply to any other example, with necessary modifications, unless otherwise specifically stated.

[0025] The disclosure is not limited in scope by the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the disclosure as described herein.

[0026] Throughout this specification, unless specifically stated otherwise or the context otherwise requires, references to a single step, composition of matter, group of steps or group of compositions of matter shall be construed to include one and more (i.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

[0027] In the following of this specification, the present disclosure will be described according to the following non-limiting examples and with reference to the accompanying drawings.

Brief Description of the Drawings

[0028]

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Mode for Carrying Out the Invention

[0029] Highlights of the Sequence Listing SEQ ID NO: 1 - Heavy Chain CDR1 KABAT SEQ ID NO: 2 - Heavy Chain CDR2 (Variants 2 - 4, 6 - 8, 10 - 12) KABAT SEQ ID NO: 3 - Heavy Chain CDR2 (Variants 13 - 19) KABAT SEQ ID NO: 4 - Heavy Chain CDR3 KABAT SEQ ID NO: 5 - Light Chain CDR1 (Variants 2 - 4, 6 - 8, 10 - 12) KABAT SEQ ID NO: 6 - Light Chain CDR1 (Variants 13 - 19) KABAT SEQ ID NO: 7 - Light Chain CDR2 KABAT SEQ ID NO: 8 - Light Chain CDR3 KABAT SEQ ID NO: 9 - Heavy Chain CDR1 IMGT SEQ ID NO: 10 - Heavy Chain CDR2 (Variants 2 - 4, 6 - 8, 10 - 12) IMGT SEQ ID NO: 11 - Heavy Chain CDR2 (Variants 13 - 19) IMGT SEQ ID NO: 12 - Heavy Chain CDR3 IMGT SEQ ID NO: 13 - Light Chain CDR1 (Variants 2 - 4, 6 - 8, 10 - 12) IMGT SEQ ID NO: 14 - Light Chain CDR1 (Variants 13 - 19) IMGT SEQ ID NO: 15 - Light Chain CDR2 IMGT SEQ ID NO: 16 - Light Chain CDR3 IMGT SEQ ID NO: 17 - Heavy Chain Variable Region (Variants 2, 6 and 10) SEQ ID NO: 18 - Heavy Chain Variable Region (Variants 3, 7 and 11) SEQ ID NO: 19 - Heavy Chain Variable Region (Variants 4, 8 and 12) SEQ ID NO: 20 - Heavy Chain Variable Region (Variants 6 and 10) SEQ ID NO: 21 - Heavy Chain Variable Region (Variants 13, 16 and 19) SEQ ID NO: 22 - Heavy Chain Variable Region (Variants 14 and 17) Accession No. 23 - Heavy Chain Variable Region (Variants 15 and 18) Accession No. 24 - Light Chain Variable Region (Variants 2, 3, and 4) Accession No. 25 - Light Chain Variable Region (Variants 6, 7, and 8) Accession No. 26 - Light Chain Variable Region (Variants 10, 11, and 12) Accession No. 27 - Light Chain Variable Region (Variants 13, 14, and 15) Accession No. 28 - Light Chain Variable Region (Variants 16, 17, and 18) Accession No. 29 - Light Chain Variable Region (Variant 19) Accession No. 30 - Linker Array 1 Accession No. 31 - Linker Array 2 Accession No. 32 - Variant 2 Accession No. 33 - Variant 3 Accession No. 34 - Variant 4 Accession No. 35 - Variant 6 Accession No. 36 - Variant 7 Accession No. 37 - Variant 8 Accession No. 38 - Variant 10 Accession No. 39 - Variant 11 Accession No. 40 - Variant 12 Accession No. 41 - Variant 13 Accession No. 42 - Variant 14 Accession No. 43 - Variant 15 Accession No. 44 - Variant 16 Accession No. 45 - Variant 17 Accession No. 46 - Variant 18 Accession No. 47 - Variant 19 Accession No. 48 - Heavy Chain Variable Region Mouse (D31N) Anti-DNA Binding Antibody Accession No. 49 - Light Chain Variable Region Mouse (D31N) Anti-DNA Binding Antibody Accession No. 50 - (D31N) Mouse Prototype Produced from P. pastoris Accession No. 51 - DNA Sequence Variant 2 Accession No. 52 - DNA Sequence Variant 3 Accession No. 53 - DNA Sequence Variant 4 Accession No. 54 - DNA Sequence Variant 6 Accession No. 55 - DNA Sequence Variant 7 Accession No. 56 - DNA Sequence Variant 8 Accession No. 57 - DNA Sequence Variant 10 Accession No. 58 - DNA Sequence Variant 11 Accession No. 59 - DNA Sequence Variant 12 Accession No. 60 - DNA Sequence Variant 13 Accession No. 61 - DNA Sequence Variant 14 Accession No. 62 - DNA Sequence Variant 15 Accession No. 63 - DNA Sequence Variant 16 Accession No. 64 - DNA Sequence Variant 17 Accession No. 65 - DNA Sequence Variant 18 Accession No. 66 - DNA Sequence Variant 19 Accession No. 67 - (GGGGS) 3 Linker Accession No. 68 - 3E10 Human IgG1 L2345A / L235A Heavy Chain Full-Length Sequence Accession No. 69 - 3E10 Human IgG1 Constant Heavy Region 1 Accession No. 70 - 3E10 Human IgG1 Hinge Region Accession No. 71 - 3E10 Human IgG1 L2345A / L235A Constant Heavy Region 2 Accession No. 72 - 3E10 Human IgG1 Constant Heavy Region 3 Accession No. 73 - 3E10 Human IgG1 N297D Heavy Chain Full-Length Sequence Accession No. 74 - 3E10 Human IgG1 N297D Constant Heavy Region 2 Accession No. 75 - 3E10 Human IgG1 L2345A / L235A / N297D Heavy Chain Full-Length Sequence Accession No. 76 - 3E10 Human IgG1 L2345A / L235A / N297D Constant Heavy Region 2 Accession No. 77 - Unmodified Constant Heavy Chain Region 2 Accession No. 78 - Light Chain Full-Length Sequence

[0030] Detailed Description of the Invention General Techniques and Selected Definitions Unless otherwise specifically defined, all technical and scientific terms used herein shall be construed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in the fields of molecular biology, biochemistry, antibodies, antibody fragments such as single-chain variable fragments, and clinical research).

[0031] The term "cell permeable" is used in the context of the present disclosure to refer to an anti-DNA binding protein, such as an antigen-binding fragment, that is transported to the nucleus of a live mammalian cell and binds to DNA (e.g., single-stranded and / or double-stranded DNA). In one example, a cell-permeable anti-DNA binding protein is transported to the nucleus of a cell without the aid of a carrier or conjugate.

[0032] The term "anti-DNA binding protein" is used in the context of the present disclosure to refer to a protein that can bind to DNA. Exemplary binding proteins include immunoglobulins, antibodies, and antigen-binding fragments. Other examples of binding proteins are discussed below.

[0033] The term "immunoglobulin" will be understood to include any anti-DNA binding protein that contains an immunoglobulin domain. Exemplary immunoglobulins are antibodies. Additional proteins included within the term "immunoglobulin" include domain antibodies, camelid antibodies, and antibodies derived from cartilaginous fish (i.e., immunoglobulin new antigen receptor (IgNAR)). In general, camelid antibodies and IgNAR lack H but contain L and are often referred to as heavy-chain immunoglobulins. Other "immunoglobulins" include T cell receptors.

[0034] The term "antibody" is used in the context of the present disclosure to refer to immunoglobulin molecules that are immunologically reactive with a particular antigen and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). The term "antibody" also includes antigen-binding forms of antibodies that have antigen-binding ability (e.g., Fab’, F(ab’) rd , Fab, Fv and rIgG) as described in Pierce Catalogue and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.), Kuby, J., Immunology, 3 2 . This term is also used to refer to recombinant single-chain Fv fragments (scFv), and even their bivalent (di-scFv) and trivalent (tri-scFV) forms. The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrahedrabodies. Examples of bivalent and bispecific molecules are described in Kostelny et al. (1992) J Immunol 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579, Hollinger et al., 1993, supra, Gruber et al. (1994) J.Immunol.:5368, Zhu et al. (1997) Protein Sci 6:781, Hu et al. (1996) Cancer Res.56:3055, Adams et al. (1993) Cancer Res.53:4026, and McCartney, et al. (1995) Protein Eng.8:301.

[0035] The "antigen-binding fragment" of an antibody comprises 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, single-chain antibody molecules formed from antibody fragments, and multispecific antibodies. For example, the term antigen-binding fragment can be used to refer to a recombinant single-chain Fv fragment (scFv), and even its bivalent (di-scFv) and trivalent (tri-scFv) forms. Such fragments can be generated by various methods known in the art. For example, the di-scFv included in the present disclosure can be generated and purified by the method described in Example 1 below.

[0036] The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment of an antibody. Specifically, whole antibodies include those having heavy and light chains, including the Fc region. The constant domain can be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or an amino acid sequence variant thereof.

[0037] As used herein, "variable region" refers to the portion of the light and / or heavy chains of an antibody that specifically binds an antigen, as defined herein, and includes, for example, the CDRs, i.e., CDR1, CDR2, and CDR3, as well as the amino acid sequences of the framework regions (FR). For example, the variable region includes three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. V H refers to the variable region of the heavy chain. V L refers to the variable region of the light chain.

[0038] As used herein, the term "complementary determining region" (synonyms are CDR, i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of the antibody variable region whose presence is a major contributor to specific antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2, and CDR3. In one example, the amino acid positions assigned to the CDRs and FRs are defined according to 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").

[0039] Other rules, including correction 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 Mal 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, examples of the binding proteins of the present disclosure may sometimes be labeled according to IMGT. These examples are so clearly indicated. See, for example, SEQ ID NOs: 9-16.

[0040] "Framework region" (synonym is FR) is the variable domain residue other than the CDR residue.

[0041] As used herein, the term "constant region" refers to the portion of the heavy or light chain of an antibody other than the variable region. In the heavy chain, the constant region generally includes a plurality of constant domains and a hinge region. For example, the IgG constant region is the following linked components, constant heavy chain C H 1, linker, C H 2 and C HIt includes 3. In the heavy chain, the constant region includes Fc. In the light chain, the constant region generally includes one constant domain (CL1).

[0042] The terms "crystalline fragment" or "Fc" or "Fc region" or "Fc portion" (which may be used interchangeably herein) refer to the region of an antibody that includes at least one constant domain, which is generally (but not necessarily) glycosylated and can bind to one or more Fc receptors and / or components of the complement cascade. The heavy chain constant region can be selected from any of 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.

[0043] A "constant domain" is a domain in an antibody, and its sequence is very similar in antibodies of the same type, e.g., IgG or IgM or IgE. The constant region of an antibody generally includes multiple constant domains. For example, the constant region of a gamma, alpha, or delta heavy chain includes two constant domains.

[0044] 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 radiolabel. For example, the term "naked" can be used to refer to a binding protein such as a di-scFv that is not conjugated to another compound. Thus, in one example, the binding protein of the present disclosure is "naked". In other words, the binding protein of the present disclosure does not have to be conjugated.

[0045] In contrast, the term "conjugated" is used in the context of the present disclosure to refer to a binding protein of the present disclosure that is conjugated to another compound, e.g., a toxic compound such as a cytotoxic drug or a radiolabel. Thus, in one example, the binding protein of the present disclosure is "conjugated".

[0046] As used herein, the term "cytotoxic drug" refers to a substance that inhibits or interferes with cellular function and / or causes cell death or destruction. Cytotoxic drugs include, but are not limited to, radioisotopes (e.g., radioisotopes of At 211 、1 131 、1 125 、Y 90 、Re 186 、Re 188 、Sm 153 、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 inhibitors, enzymes such as nucleolytic enzymes and fragments thereof, antibiotics, small molecule toxins or enzymatically active toxins derived from bacteria, fungi, plants, or animals, including fragments and / or variants thereof, and various antitumor or anticancer agents disclosed below.

[0047] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably in the context of the present disclosure to refer to a cell (including progeny of such cells) into which foreign nucleic acid has been introduced. Host cells include "transformants" and "transformed cells", including primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical to the parental cell in nucleic acid content and may include mutations. Mutant progeny having the same function or biological activity as selected or screened in the original transformed cell are included herein.

[0048] An "isolated nucleic acid" according to the present disclosure is a nucleic acid molecule that has been separated from the components of its natural environment. An isolated nucleic acid includes nucleic acid molecules contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0049] The "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 with each other and, if necessary, introducing gaps to achieve the maximum percent sequence identity, and where any conservative substitutions are not considered part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by various methods within the skill of one of ordinary skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0050] As used herein, the term "binds" with respect to the interaction of a binding protein and an antigen means that the interaction is dependent on the presence of a particular structure on the antigen (e.g., an antigenic determinant or epitope). For example, a binding protein generally recognizes and binds to a specific antigenic structure rather than to the antigen itself. For example, if a binding protein binds to epitope "A", in a reaction containing label "A" and its binding protein, the presence of a molecule containing epitope "A" (or free unlabeled "A") will reduce the amount of label "A" that binds to the binding protein.

[0051] As used herein, the term "specifically binds" is to be interpreted to mean that the binding interaction between a binding protein and DNA is governed by the detection of DNA by the binding protein. Thus, the binding protein preferentially binds to or preferentially recognizes DNA even when present in a mixture of other molecules or organisms.

[0052] In one instance, the binding protein reacts or associates with DNA more frequently, more rapidly, for a longer duration, and / or with a higher affinity than it does with an alternative antigen or cell. By reading this definition, it is understood that, for example, a binding protein that specifically binds to DNA may or may not specifically bind to a second antigen. Thus, "specific binding" does not necessarily require exclusive binding or undetectable binding to another antigen. The term "specifically binds" may be used interchangeably herein with "selectively binds". Generally, references to binding herein mean specific binding, and each term is understood to give clear support to the other terms. Methods for determining specific binding will be apparent to those skilled in the art. For example, a binding protein of the disclosure is contacted with DNA or an alternative antigen. Next, the binding of the binding protein to DNA or the alternative antigen is determined, and a binding protein that binds to DNA as described above rather than to the alternative antigen is considered to specifically bind to DNA.

[0053] The binding proteins and compositions containing them according to the present disclosure can be administered to a subject for treating various indications. Terms such as "subject", "patient" or "individual" are terms that can be used interchangeably in the context of the present disclosure. In one instance, the subject is a mammal. The mammal can be a companion animal such as a dog or a cat, or a domestic animal such as a horse or a cow. In one instance, the subject is a human. For example, the subject can be an adult. In another example, the subject can be a pediatric. In another example, the subject can be an adolescent.

[0054] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the clinical pathology in an individual or cell being treated. Desirable effects of treatment include slowing the rate of progression of a disease, improving or alleviating a condition, and achieving remission or improving the prognosis. For example, if one or more symptoms associated with a disease are reduced or eliminated, the individual is "treated" successfully.

[0055] As used herein, the term "prevent" includes effecting prevention in an individual with respect to the occurrence or recurrence of a disease. An individual may be susceptible to, or have a risk of, the occurrence of a disease or the recurrence of a disease, but has not yet been diagnosed as having the disease or recurrence.

[0056] The term "treatment" is used herein in the context of referring to the medical management of a patient for the purpose of curing, ameliorating, or stabilizing a disease, pathological condition, or disorder. The term "treatment" includes active treatment, i.e., treatment directed particularly to the amelioration of a disease, pathological condition, or disorder, and causal treatment, i.e., treatment directed to the removal of the cause of a related disease, pathological condition, or disorder. In addition, the term "treatment" includes palliative treatment, i.e., treatment designed for the relief of symptoms rather than the cure of a disease, pathological condition, or disorder, prophylactic treatment, i.e., treatment directed to minimizing or partially or completely inhibiting the occurrence of a related disease, pathological condition, or disorder, and supportive treatment, i.e., treatment used to supplement another specific therapy directed to the amelioration of a related disease, pathological condition, or disorder.

[0057] "Effective amount" refers to an amount effective, at a minimum, at the dosage and for the period required to achieve the desired therapeutic or prophylactic result. An effective amount can be administered in one or more administrations. In some examples of the present disclosure, the term "effective amount" means an amount necessary to effect treatment of the following diseases or conditions. The effective amount can vary depending on the disease or condition being treated and also depending on the body weight, age, ethnic background, gender, health status and / or physical condition of the subject being treated and other factors. Typically, the effective amount will fall within a relatively broad range (e.g., a "dosage" range) that can be determined by routine trials and experimentation by a physician. The effective amount can be administered as a single dose or as one or more repeated doses over the treatment period.

[0058] "Therapeutically effective amount" is at least the minimum concentration required to obtain a measurable improvement in a particular disorder (e.g., cancer). The therapeutically effective amount herein can vary depending on factors such as the patient's medical condition, age, gender and weight, as well as the ability of the binding protein to induce the desired response in an individual. A therapeutically effective amount is also an amount where the therapeutic beneficial effect outweighs any toxicity or adverse effect of the binding protein. In the case of cancer, the therapeutically effective amount of the binding protein can reduce the number of cancer cells, shrink the size of the primary tumor, inhibit the invasion of cancer cells into surrounding organs (i.e., can slow down to some extent and in some cases, stop), inhibit tumor metastasis (i.e., can slow down to some extent and in some cases, stop), inhibit tumor growth or tumor progression to some extent or delay it, and / or alleviate one or more of the symptoms associated with cancer to some extent. The binding protein can, to some extent, block the growth of existing cancer cells and / or kill them, which can be cytostatic and / or cytotoxic. In cancer treatment, the in vivo efficacy can be measured, for example, by evaluating survival time, time to progression (TTP), response rate (RR), response duration, and / or quality of life.

[0059] Deimmunized, chimeric, humanized, synhumanized, primatized and human antibodies or antigen-binding fragments Monoclonal antibodies are one example of the exemplary forms of binding proteins contemplated by the present disclosure. The term "monoclonal antibody" or "MAb" refers to a homogeneous population of antibodies that can bind to the same antigen(s), e.g., the same epitope within the antigen. This term is not intended to be limited with respect to the source of the antibody or the method by which the antibody is made.

[0060] In one example, the binding proteins encompassed by the present disclosure may be "humanized." A "humanized antibody" is an immunoglobulin molecule that contains minimal sequences derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues of the recipient's complementarity determining regions (CDRs) have been replaced with residues from CDRs (donor antibodies) of non-human species such as mice, rats or rabbits that have the desired specificity, affinity and capacity. In some examples, Fv framework residues of the human immunoglobulin are replaced with the corresponding non-human residues. Humanized antibodies may also include residues not found in the recipient antibody or in the transferred CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, typically two variable domains, wherein all or substantially all of the CDR regions correspond to those of the non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of the human immunoglobulin consensus sequence. In one example, a humanized antibody will also include at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)).

[0061] In one example, a "human" binding protein of the present disclosure can include amino acid residues not encoded by a human sequence, such as mutations introduced by random or site-directed mutagenesis in vitro (in particular, conservative substitutions or mutations in a small number of residues of the protein, such as 1, 2, 3, 4, or 5 of the residues of the protein). These "human antibodies" do not necessarily have to be produced as a result of a human immune response. Rather, they can be generated using recombinant means (e.g., screening of phage display libraries) and / or by transgenic animals (e.g., mice) containing nucleic acids encoding human antibody constant and / or variable regions and / or using guided selection (e.g., as described in U.S. Patent No. 5,565,332). The term also encompasses affinity matured forms of such antibodies.

[0062] In another example, a binding protein encompassed by the present disclosure may be synthetically humanized. The term "synthetically humanized" refers to an antibody prepared by the method described in WO2007 / 019620. A synthetically humanized antibody contains the variable region of the antibody, where the variable region contains FRs from New World primate antibody variable regions and CDRs from non-New World primate antibody variable regions.

[0063] In another example, a binding protein of the present disclosure may be primatized. A "primatized antibody" contains the variable region(s) from an antibody produced after immunization of a non-human primate (e.g., cynomolgus monkey). In one example, the variable region of the non-human primate antibody is linked to a human constant region to produce a primatized antibody. Exemplary methods for producing primatized antibodies are described in U.S. Patent No. 6,113,898.

[0064] In one example, the binding protein of the present disclosure is a chimeric antibody or fragment. The term "chimeric antibody" or "chimeric antigen-binding fragment" refers to an antibody or fragment in which one or more of the variable domains are from a particular species (e.g., murine such as mouse or rat) or belong to a particular antibody class or subclass, while the remainder of the antibody or fragment is from a different species (e.g., human or non-human primate) or belongs to a different antibody class or subclass. In one example, the chimeric antibody contains V H and / or V L derived from a non-human antibody (e.g., a mouse antibody), and the remaining regions of the antibody are derived from a human antibody.

[0065] The present disclosure also contemplates deimmunized antibodies or antigen-binding fragments thereof as described, for example, in WO2000 / 34317 and WO2004 / 108158. Deimmunized antibodies and fragments have one or more epitopes, e.g., B-cell epitopes 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, the antibodies of the present disclosure are analyzed to identify one or more B or T cell epitopes, and one or more amino acid residues within the epitope are mutated to thereby reduce the immunogenicity of the antibody.

[0066] Antibody fragment Single-domain antibody In some examples, the binding protein of the present disclosure is or includes a single-domain antibody (used interchangeably with the term "domain antibody" or "dAb"). A single-domain antibody is a single polypeptide chain that includes all or part of the variable domain of an antibody heavy chain.

[0067] Single-chain Fv (scFv) fragment Those skilled in the art will appreciate that an scFv is a V in a single polypeptide chain H and V L regions, and for antigen binding (i.e., the V of the single polypeptide chain H and V Lsuch that the scFv forms a structure that enables it to form a desirable structure for the association of V to each other to form an Fv H and V L will be recognized to include a polypeptide linker between and. A single-chain variable fragment lacks the constant Fc region present in a full antibody molecule and can thus have reduced immunogenicity. Exemplary linkers contain more than 12 amino acid residues, where (Gly 4 Ser) 3 is one of the more preferred linkers for scFv. Another example of a suitable linker is shown in SEQ ID NO: 31.

[0068] The present disclosure also contemplates disulfide-stabilized Fv (or diFv or dsFv) in which a single cysteine residue is introduced into the FR of V H and the FR of V L and those cysteine residues are linked by disulfide bonds to yield a stable Fv.

[0069] In another example, the present disclosure encompasses dimeric scFv (di-scFV), i.e., a protein comprising two scFv molecules linked by non-covalent or covalent means, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun), or trimeric scFV (tri-scFv). In another example, two scFv are linked by a peptide linker of sufficient length such that, as described in, e.g., U.S. Patent Application Publication No. 20060263367, both scFv are formed and are capable of binding to an antigen.

[0070] Diabody, triabody, tetrabody In some examples, the antigen-binding fragment of the present disclosure is a higher-order protein complex such as, or includes, a diabody, triabody, tetrabody or those described in WO98 / 044001 and / or WO94 / 007921.

[0071] For example, a diabody is a protein comprising two associated polypeptide chains, where each polypeptide chain has the structural formula V L -X-V H or V H -X-V L wherein X is a linker that contains residues insufficient to allow V H and V L in a single polypeptide chain to associate (or form an Fv), or is absent, and V of one polypeptide chain H binds to V of the other polypeptide chain L to form an antigen-binding site, i.e., forms an Fv molecule that can specifically bind to one or more antigens. V L and V H may be the same in each polypeptide chain, or V L and V H may be different in each polypeptide chain such that they form a bispecific diabody (i.e., contain two Fvs with different specificities).

[0072] Other antibodies and antibody fragments Other examples of binding proteins included in the present disclosure include (i) "key and hole" bispecific proteins as described in U.S. Patent No. 5,731,168, (ii) heteroconjugate proteins as described, for example, in U.S. Patent No. 4,676,980, (iii) heteroconjugate proteins produced using chemical cross-linkers as described, for example, in U.S. Patent No. 4,676,980, and (iv) Fab 3 (e.g., as described in European Patent No. 19930302894) are included.

[0073] Immunoglobulins and immunoglobulin fragments Examples of the binding proteins of the present disclosure are proteins that include variable regions of immunoglobulins such as T cell receptors (e.g., antibody mimics) or heavy chain immunoglobulins (e.g., IgNAR, camelid antibodies).

[0074] V-like protein One example of the binding protein of the present disclosure is a T cell receptor. A T cell receptor has two V domains that combine to form a structure similar to the Fv module of an antibody. Novotny et al., Proc Natl Acad Sci USA 88:8646-8650, 1991 describes how the two V domains of the T cell receptor (referred to as alpha and beta) are fused and expressed as a single-chain polypeptide, and further how surface residues are altered to directly reduce the hydrophobicity similar to that of an antibody scFv. Other publications that describe the production of single-chain T cell receptors or multimeric T cell receptors containing two V-alpha and V-beta domains include WO1999 / 045110 or WO2011 / 107595.

[0075] Other non-antibody proteins containing antigen-binding domains include proteins having V-like domains, which are generally monomeric. Examples of such proteins containing V-like domains include CTLA-4, CD28, and ICOS. Further disclosure of such proteins containing V-like domains is included in WO1999 / 045110.

[0076] Affibody In a further example, the binding protein of the present disclosure is an Affibody. An Affibody is a scaffold derived from the Z domain (antigen-binding domain) of protein A of Staphylococcus aureus that can be engineered to bind to an antigen. The Z domain consists of a bundle of three helices of approximately 58 amino acids. The library is generated by randomization of surface residues. For further details, see European Patent No. 1641818.

[0077] Abimer In yet another example, the binding protein of the present disclosure is an affimer. An affimer is a multi-domain protein derived from the A domain scaffold family. The native domain of about 35 amino acids adopts a defined disulfide bond structure. Diversity is generated by shuffling the native variants exhibited by the A domain family. For further details, see WO2002 / 088171.

[0078] Binding protein In one example, the anti-DNA binding protein according to the present disclosure has a heavy chain variable region (V H ) comprising CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2 or SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4. For example, the anti-DNA binding protein may comprise a V H having CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 4. In another example, the anti-DNA binding protein may comprise a V H having CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4.

[0079] In another example, the anti-DNA binding protein has a light chain variable region (V L ) comprising CDR1 as shown in SEQ ID NO: 5 or SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8. For example, the anti-DNA binding protein may comprise a V L having CDR1 as shown in SEQ ID NO: 5, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8. In another example, the anti-DNA binding protein may comprise a V L having CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8.

[0080] In another example, the anti-DNA binding protein has a V with CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2 or SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4 H and a V with CDR1 as shown in SEQ ID NO: 5 or SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 L For example, the anti-DNA binding protein may include a V with CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 4 H and a V with CDR1 as shown in SEQ ID NO: 5, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 L In another example, the anti-DNA binding protein may include a V with CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 4 H and a V with CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 L In another example, the anti-DNA binding protein may include a V with CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4 H and a V with CDR1 as shown in SEQ ID NO: 5, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 L In another example, the anti-DNA binding protein may include a V with CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4 H and a V with CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 L and may include.

[0081] The exemplary binding proteins described above may also have CDRs assigned using the IMGT system. Thus, in another example, an anti-DNA binding protein has CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10 or SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12, and has a V H region. For example, an anti-DNA binding protein may have CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10, and CDR3 as shown in SEQ ID NO: 12, and has a V H region. In another example, an anti-DNA binding protein may have CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12, and has a V H region.

[0082] In another example, an anti-DNA binding protein has CDR1 as shown in SEQ ID NO: 13 or SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16, and has a V L region. For example, an anti-DNA binding protein may have CDR1 as shown in SEQ ID NO: 13, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16, and has a V L region. In another example, an anti-DNA binding protein may have CDR1 as shown in SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16, and has a V L region.

[0083] In another example, an anti-DNA binding protein has CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10 or SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12, and has a V H region, as well as CDR1 as shown in SEQ ID NO: 13 or SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16, and has a VL including. For example, the anti-DNA binding protein has a V having CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10, and CDR3 as shown in SEQ ID NO: 12 H and a V having CDR1 as shown in SEQ ID NO: 13, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L may be included. In another example, the anti-DNA binding protein has a V having CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10, and CDR3 as shown in SEQ ID NO: 12 H and a V having CDR1 as shown in SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L may be included. In another example, the anti-DNA binding protein has a V having CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12 H and a V having CDR1 as shown in SEQ ID NO: 13, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L may be included. In another example, the anti-DNA binding protein has a V having CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12 H and a V having CDR1 as shown in SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L may be included.

[0084] In another example, the anti-DNA binding protein includes a V that includes a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 17 to 23 H including. For example, the anti-DNA binding protein includes a V that includes a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 18 Hmay include. In another example, the anti-DNA binding protein includes a V that contains a sequence that is at least 95% identical to the sequence as shown in SEQ ID NO: 23 H may include. In another example, the anti-DNA binding protein includes a V that contains a sequence that is at least 95% identical to the sequence as shown in any one of SEQ ID NOS: 24 to 29 L includes. For example, the anti-DNA binding protein includes a V that contains a sequence that is at least 95% identical to the sequence as shown in SEQ ID NO: 25 L may include. In another example, the anti-DNA binding protein includes a V that contains a sequence that is at least 95% identical to the sequence as shown in SEQ ID NO: 27 L may include. In another example, the anti-DNA binding protein includes a V that contains a sequence that is at least 95% identical to the sequence as shown in any one of SEQ ID NOS: 17 to 23 H and a V that contains a sequence that is at least 95% identical to the sequence as shown in any one of SEQ ID NOS: 24 to 29 L includes. For example, the anti-DNA binding protein includes a V that contains a sequence that is at least 95% identical to the sequence as shown in SEQ ID NO: 18 H and a V that contains a sequence that is at least 95% identical to the sequence as shown in SEQ ID NO: 25 L may include. In another example, the anti-DNA binding protein includes a V that contains a sequence that is at least 95% identical to the sequence as shown in SEQ ID NO: 23 H and a V that contains a sequence that is at least 95% identical to the sequence as shown in SEQ ID NO: 27 L may include. 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 recited SEQ ID NOs.

[0085] In another example, the anti-DNA binding protein includes a V that contains the sequence as shown in any one of SEQ ID NOS: 17 to 23 H includes. For example, the anti-DNA binding protein includes a V that contains the sequence as shown in SEQ ID NO: 18 Hmay include. In another example, the anti-DNA binding protein may include a V having a sequence as set forth in SEQ ID NO: 23 H may include. In another example, the anti-DNA binding protein may include a V having a sequence as set forth in any one of SEQ ID NOs: 24 to 29 L includes. For example, the anti-DNA binding protein includes a V having a sequence as set forth in SEQ ID NO: 25 L may include. In another example, the anti-DNA binding protein may include a V having a sequence as set forth in SEQ ID NO: 27 L may include. In another example, the anti-DNA binding protein may include a V having a sequence as set forth in any one of SEQ ID NOs: 17 to 23 H and a V having a sequence as set forth in any one of SEQ ID NOs: 24 to 29 L includes. For example, the anti-DNA binding protein includes a V having a sequence as set forth in SEQ ID NO: 18 H and a V having a sequence as set forth in SEQ ID NO: 25 L may include. In another example, the anti-DNA binding protein may include a V having a sequence as set forth in SEQ ID NO: 23 H and a V having a sequence as set forth in SEQ ID NO: 27 L may include.

[0086] In one example, the anti-DNA binding protein can be a cell-permeable anti-DNA Fv fragment having an antigen-binding domain that binds or specifically binds to DNA. For example, the Fv may include a V having an amino acid sequence as set forth in SEQ ID NO: 48 H and a V having an amino acid sequence as set forth in SEQ ID NO: 49 L and can bind to the same epitope as a binding protein having. In another example, the Fv can bind to the same epitope as a di-scFv having an amino acid sequence as set forth in SEQ ID NO: 50. In one example, the Fv has a CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2 or SEQ ID NO: 3, and a CDR3 as set forth in SEQ ID NO: 4 and has a V HIt includes. For example, Fv has CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 4 in V H It may include. In another example, Fv has CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4 in V H It may include.

[0087] In another example, Fv has CDR1 as shown in SEQ ID NO: 5 or SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 in V L It includes. For example, Fv has CDR1 as shown in SEQ ID NO: 5, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 in V L It may include. In another example, the anti-DNA binding protein has CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 in V L It may include.

[0088] In another example, Fv has CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2 or SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4 in V H and V having CDR1 as shown in SEQ ID NO: 5 or SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 L It includes. For example, Fv has CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 4 in V H and V having CDR1 as shown in SEQ ID NO: 5, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 Lmay include. In another example, the Fv has a V with CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 4 H and a V having CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 L may include. In another example, the Fv has a V with CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4 H and a V having CDR1 as shown in SEQ ID NO: 5, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 L may include. In another example, the Fv has a V with CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4 H and a V having CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 L may include.

[0089] The Fvs exemplified above may also have CDRs assigned using the IMGT system. Thus, in another example, the Fv has a V with CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10 or SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12 H including. For example, the Fv has a V with CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10, and CDR3 as shown in SEQ ID NO: 12 H may include. In another example, the Fv has a V with CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12 H may include.

[0090] In another example, the Fv has a V with CDR1 as shown in SEQ ID NO: 13 or SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L and includes. For example, the Fv has a V with CDR1 as shown in SEQ ID NO: 13, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L and may include. In another example, the Fv has a V with CDR1 as shown in SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L and may include.

[0091] In another example, the Fv has a V with CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10 or SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12 H as well as a V with CDR1 as shown in SEQ ID NO: 13 or SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L and includes. For example, the Fv has a V with CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10, and CDR3 as shown in SEQ ID NO: 12 H as well as a V with CDR1 as shown in SEQ ID NO: 13, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L and may include. In another example, the Fv has a V with CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10, and CDR3 as shown in SEQ ID NO: 12 H as well as a V with CDR1 as shown in SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L and may include. In another example, the Fv has a V with CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12H and a V having CDR1 as shown in SEQ ID NO: 13, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L may be included. In another example, the Fv has a V having CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12 H and a V having CDR1 as shown in SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L may be included.

[0092] In another example, the Fv includes a V having a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOS: 17 to 23 H For example, the Fv may include a V having a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 18 H In another example, the Fv may include a V having a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 21 H In another example, the Fv may include a V having a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 23 H In another example, the Fv includes a V having a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOS: 24 to 29 L For example, the Fv may include a V having a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 25 L In another example, the Fv may include a V having a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 27 L In another example, the Fv includes a V having a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOS: 17 to 23 H and a V having a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOS: 24 to 29 L For example, the Fv may include a V having a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 18 Hand a V comprising a sequence that is at least 95% identical to the sequence as set forth in SEQ ID NO: 25 L may be included. In another example, the Fv comprises a V comprising a sequence that is at least 95% identical to the sequence as set forth in SEQ ID NO: 21 H and a V comprising a sequence that is at least 95% identical to the sequence as set forth in SEQ ID NO: 27 L may be included. In another example, the Fv comprises a V comprising a sequence that is at least 95% identical to the sequence as set forth in SEQ ID NO: 23 H and a V comprising a sequence that is at least 95% identical to the sequence as set forth in SEQ ID NO: 27 L may be included. In these examples, the V H and / or the V L may be at least 96%, at least 97%, at least 98% or at least 99% identical to the recited SEQ ID NOs. In these examples, the Fv may have the combinations mentioned above with respect to the CDRs. For example, the Fv may comprise a V comprising a sequence that is at least 95% identical to the sequence as set forth in SEQ ID NO: 21 H and a V comprising a sequence that is at least 95% identical to the sequence as set forth in SEQ ID NO: 27 L may be included, wherein the V H has CDR1 as set forth in SEQ ID NO: 1, CDR2 as set forth in SEQ ID NO: 3 and CDR3 as set forth in SEQ ID NO: 4, and the V L has CDR1 as set forth in SEQ ID NO: 6, CDR2 as set forth in SEQ ID NO: 7 and CDR3 as set forth in SEQ ID NO: 8.

[0093] In another example, the Fv comprises a V comprising a sequence as set forth in any one of SEQ ID NOs: 17 - 23 H For example, the Fv may comprise a V comprising a sequence as set forth in SEQ ID NO: 18 H may be included. In another example, the Fv may comprise a V comprising a sequence as set forth in SEQ ID NO: 21 H may be included. In another example, the Fv may comprise a V comprising a sequence as set forth in SEQ ID NO: 23 Hmay include. In another example, Fv includes a V having the sequence as shown in any one of SEQ ID NOs: 24 to 29 L includes. For example, Fv includes a V having the sequence as shown in SEQ ID NO: 25 L may include. In another example, Fv includes a V having the sequence as shown in SEQ ID NO: 27 L may include. In another example, Fv includes a V having the sequence as shown in any one of SEQ ID NOs: 17 to 23 H and a V having the sequence as shown in any one of SEQ ID NOs: 24 to 29 L includes. For example, Fv includes a V having the sequence as shown in SEQ ID NO: 18 H and a V having the sequence as shown in SEQ ID NO: 25 L may include. In another example, Fv includes a V having the sequence as shown in SEQ ID NO: 21 H and a V having the sequence as shown in SEQ ID NO: 27 L may include. In another example, Fv includes a V having the sequence as shown in SEQ ID NO: 23 H and a V having the sequence as shown in SEQ ID NO: 27 L may include.

[0094] In another example, Fv includes a V having the amino acid sequence as shown in SEQ ID NO: 48 H and a V having the amino acid sequence as shown in SEQ ID NO: 49 L has improved manufacturability as compared to a binding protein having. In another example, Fv has improved manufacturability as compared to a di-scFv having the amino acid sequence as shown in SEQ ID NO: 50.

[0095] The improved manufacturability includes an increase in post-translational modification or chemical stability associated with a decrease in the number of deamidation sites, aspartate isomerization sites, oxidation sites such as methionine and tryptophan, free cysteine thiol groups, N&O-glycosylation sites, C-terminal lysine and / or isoelectric points.

[0096] In one example, the Fv contains a V having the amino acid sequence as set forth in SEQ ID NO: 48 H and a V having the amino acid sequence as set forth in SEQ ID NO: 49 L and contains less asparagine in V H and / or V L compared to the binding protein having them. In another example, the Fv contains less asparagine in V H and / or V L compared to the diabody-scFv having the amino acid sequence as set forth in SEQ ID NO: 50.

[0097] In one example, the Fv contains a V having the amino acid sequence as set forth in SEQ ID NO: 48 H and a V having the amino acid sequence as set forth in SEQ ID NO: 49 L and contains less methionine in V H and / or V L compared to the binding protein having them. In another example, the Fv contains less methionine in V H and / or V L compared to the diabody-scFv having the amino acid sequence as set forth in SEQ ID NO: 50.

[0098] In one example, the Fv contains a V having the amino acid sequence as set forth in SEQ ID NO: 48 H and a V having the amino acid sequence as set forth in SEQ ID NO: 49 L and contains less tryptophan in V H and / or V L compared to the binding protein having them. In another example, the Fv contains less tryptophan in V H and / or V L compared to the diabody-scFv having the amino acid sequence as set forth in SEQ ID NO: 50.

[0099] In one example, the Fv contains a V having the amino acid sequence as set forth in SEQ ID NO: 48 H and a V having the amino acid sequence as set forth in SEQ ID NO: 49 LCompared to the binding protein having [it], V H and / or V L contains less aspartic acid. In another example, the Fv contains less aspartic acid in V H and / or V L compared to the diabody-scFv having the amino acid sequence as shown in SEQ ID NO: 50.

[0100] In one example, the physical stability of the Fv is higher than that of the binding protein having V H containing the amino acid sequence as shown in SEQ ID NO: 48 and V L containing the amino acid sequence as shown in SEQ ID NO: 49. In another example, the physical stability of the Fv is higher than that of the diabody-scFv having the amino acid sequence as shown in SEQ ID NO: 50.

[0101] Physical stability can include the property of aggregating in solution. The term "aggregation" is used in the context of the present disclosure to refer to protein self-association that can occur in multiple environments from cell culture and fermentation to isolation, purification, and formulation processes. For example, the term "aggregation" can be used in particular to describe the formation of inclusion bodies, the accumulation of proteins in the "insoluble" fraction after cell fractionation, turbidity in a sample, the appearance of protein precipitation or particle formation, or the formation of small soluble oligomers.

[0102] Thus, in the examples mentioned above, the physical stability of the Fv can be based on its physical stability in solution, where precipitation of the Fv from the solution indicates that the Fv is unstable. To evaluate physical stability, a solution containing either the Fv according to the present disclosure, or the binding protein having V H containing the amino acid sequence as shown in SEQ ID NO: 48 and V L containing the amino acid sequence as shown in SEQ ID NO: 49, or the diabody-scFv containing the amino acid sequence as shown in SEQ ID NO: 50 can be incubated at 4°C and visually evaluated for precipitation at 2 weeks, 4 weeks, 12 weeks, 6 months, and 12 months.

[0103] In one example, the physical stability of the Fv according to the present disclosure is such that when the Fv is kept in solution at 4° C. for at least 4 weeks, the V contains the amino acid sequence as set forth in SEQ ID NO: 48 H and the V containing the amino acid sequence as set forth in SEQ ID NO: 49 L is higher than that of a binding protein having the V or a di-scFv containing the amino acid sequence as set forth in SEQ ID NO: 50. In one example, the physical stability of the Fv according to the present disclosure is such that when the Fv is kept in solution at 4° C. for at least 6 months, the V contains the amino acid sequence as set forth in SEQ ID NO: 48 H and the V containing the amino acid sequence as set forth in SEQ ID NO: 49 L is higher than that of a binding protein having the V or a di-scFv containing the amino acid sequence as set forth in SEQ ID NO: 50.

[0104] In another example, the Fv has reduced immunogenicity in a human subject compared to a binding protein having the V containing the amino acid sequence as set forth in SEQ ID NO: 48 H and the V containing the amino acid sequence as set forth in SEQ ID NO: 49 L For example, the Fv may have reduced immunogenicity compared to a binding protein having the V containing the amino acid sequence as set forth in SEQ ID NO: 48 when the immunogenicity is measured by enzyme-linked immunosorbent assay (ELISA). In another example, the Fv may have reduced immunogenicity compared to a binding protein having the V containing the amino acid sequence as set forth in SEQ ID NO: 48 when the immunogenicity is measured by surface plasmon resonance H and the V containing the amino acid sequence as set forth in SEQ ID NO: 49 L For example, the Fv may have reduced immunogenicity compared to a binding protein having the V containing the amino acid sequence as set forth in SEQ ID NO: 48 when the immunogenicity is measured by surface plasmon resonance. In another example, the Fv may have reduced immunogenicity compared to a binding protein having the V containing the amino acid sequence as set forth in SEQ ID NO: 48 when the immunogenicity is measured by surface plasmon resonance H and the V containing the amino acid sequence as set forth in SEQ ID NO: 49 L For example, the Fv may have reduced immunogenicity compared to a binding protein having the V containing the amino acid sequence as set forth in SEQ ID NO: 48 when the immunogenicity is measured by surface plasmon resonance.

[0105] In another example, the ability of the Fv to permeate cells is such that the V contains the amino acid sequence as set forth in SEQ ID NO: 48 H and the V containing the amino acid sequence as set forth in SEQ ID NO: 49L Higher than that of the binding protein having it. In another example, the ability of the Fv to permeate cells is higher than that of the diabody-scFv having the amino acid sequence as shown in SEQ ID NO: 50. In another example, the ability of the Fv to permeate the cell nucleus is the V containing the amino acid sequence as shown in SEQ ID NO: 48 H And the V containing the amino acid sequence as shown in SEQ ID NO: 49 L Higher than that of the binding protein having it. In another example, the ability of the Fv to permeate the cell nucleus is higher than that of the diabody-scFv having the amino acid sequence as shown in SEQ ID NO: 50. For example, the diabody-scFv may contain the amino acid sequence as shown in SEQ ID NO: 36. In another example, the diabody-scFv may contain the amino acid sequence as shown in SEQ ID NO: 41. In another example, the diabody-scFv may contain the amino acid sequence as shown in SEQ ID NO: 43. In the examples mentioned above, the ability of the binding protein to permeate cells or cell nuclei can be measured using a colorimetric assay. For example, cells are treated with either a control medium, a binding protein according to the present disclosure, or a V containing the amino acid sequence as shown in SEQ ID NO: 48 H And the V containing the amino acid sequence as shown in SEQ ID NO: 49 L Having a binding protein or a diabody-scFv having the amino acid sequence as shown in SEQ ID NO: 50 for 1 hour. Then, the cells are washed, fixed, blocked with 1% BSA-TBST, and then probed with Protein L for 1 hour. Then, the cells are washed and incubated with an anti-Protein L primary antibody for 1 hour. After another round of washing, the cells are incubated with an alkaline phosphatase-conjugated secondary antibody for 1 hour. Finally, the cells are washed and the signal is generated by the addition of NBT / BCIP. If distinct nuclear staining can be identified in any of the samples, the generation of the signal is stopped by the removal and washing of NBT / BCIP. Then, the nuclear and / or cell staining is measured using Image J.

[0106] In one example, an Fv that results in nuclear staining with a reciprocal intensity of at least 190 absorbance units (au) has a high ability to penetrate the cell nucleus. In one example, an Fv that results in nuclear staining with a reciprocal intensity of at least 200 au has a high ability to penetrate the cell nucleus. In one example, an Fv that results in nuclear staining with a reciprocal intensity of at least 210 au has a high ability to penetrate the cell nucleus. In one example, an Fv that results in nuclear staining with a reciprocal intensity of at least 220 au has a high ability to penetrate the cell nucleus. In another example, the ability of an Fv to penetrate the cell nucleus can be evaluated by measuring the fluorescence of individual cells. In one example, an Fv that results in nuclear staining with a reciprocal intensity of at least 190 au in at least 20 cells has a high ability to penetrate the cell nucleus. In another example, an Fv that results in nuclear staining with a reciprocal intensity of at least 190 au in at least 30 cells has a high ability to penetrate the cell nucleus. In another example, an Fv that results in nuclear staining with a reciprocal intensity of at least 190 au in at least 40 cells has a high ability to penetrate the cell nucleus. In another example, an Fv that results in nuclear staining with a reciprocal intensity of at least 200 au in at least 20 cells has a high ability to penetrate the cell nucleus. In another example, an Fv that results in nuclear staining with a reciprocal intensity of at least 200 au in at least 30 cells has a high ability to penetrate the cell nucleus. In another example, an Fv that results in nuclear staining with a reciprocal intensity of at least 200 au in at least 50 cells has a high ability to penetrate the cell nucleus. In another example, an Fv that results in nuclear staining with a reciprocal intensity of at least 200 au in at least 70 cells has a high ability to penetrate the cell nucleus. In another example, an Fv that results in nuclear staining with a reciprocal intensity of at least 200 au in at least 80 cells has a high ability to penetrate the cell nucleus.

[0107] In another example, the Fv contains a V for DNA with an amino acid sequence as shown in SEQ ID NO: 48 H and a V for DNA with an amino acid sequence as shown in SEQ ID NO: 49 LIt has higher specificity than the binding protein having it. In another example, the Fv has higher specificity than the di-scFv having the amino acid sequence as shown in SEQ ID NO: 50 for DNA.

[0108] In another example, the Fv comprises a V having the amino acid sequence as shown in SEQ ID NO: 48 H and a V having the amino acid sequence as shown in SEQ ID NO: 49 L and has low cross-reactivity (i.e., the ability of the Fv to react with similar antigenic sites on different proteins) compared to the binding protein having them. In another example, the Fv has low cross-reactivity with other targets compared to the di-scFv having the amino acid sequence as shown in SEQ ID NO: 50. In this example, the cross-reactivity of the Fv can be measured using various methods. In one example, the cross-reactivity is evaluated by ELISA.

[0109] In another example, the Fv for DNA comprises a V having the amino acid sequence as shown in SEQ ID NO: 48 H and a V having the amino acid sequence as shown in SEQ ID NO: 49 L and has higher binding affinity than the binding protein having them. In another example, the Fv for DNA has higher binding affinity than the di-scFv having the amino acid sequence as shown in SEQ ID NO: 50.

[0110] In the examples mentioned above, the affinity of the Fv for DNA can be measured using various methods. In one example, the dissociation constant (K D ) or the association constant (K A ) or the equilibrium constant (K D ) of the binding protein for DNA is determined. These constants for the binding protein are measured in one example by a radiolabeled or fluorescently labeled DNA binding assay. In this assay, the binding protein is equilibrated with a minimum concentration of labeled DNA (or its soluble form containing the extracellular region of DNA fused to, for example, the Fc region) in the presence of a titration series of unlabeled DNA. After washing to remove unbound DNA, the amount of label is determined.

[0111] The measurement of affinity can be determined by standard methodologies for 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.

[0112] In one example, the constant is measured by using a surface plasmon resonance assay, such as using a BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized DNA. Exemplary SPR methods are described in U.S. Patent No. 7,229,619.

[0113] In some embodiments, the binding affinity of the Fv for DNA is between about 5 nM and about 100 pM, 10 pM, 1 pM, 100 fM, 10 fM, or 1 fM.

[0114] In one example, the Fv encompassed by the present disclosure has a binding affinity for DNA comparable to about 5 nM or less, or about 4.9 nM, or about 4.8 nM, or about 4.7 nM, or about 4.6 nM, or about 4.7 nM, or about 4.6 nM, or about 4.5 nM, or about 4.4 nM, or about 4.3 nM, or about 4.2 nM, or about 4.1 nM, or about 4.0 nM, or about 3.9 nM, or about 3.8 nM, or about 3.7 nM, or about 3.6 nM, or about 3.5 nM, or about 3.4 nM, or about 3.3 nM, or about 3.2 nM, or about 3.1 nM, or about 3.0 nM.

[0115] In other examples, the present Fv may have a binding affinity comparable to about 100 pM, or about 150 pM, or about 200 pM, or about 250 pM, or about 300 pM, or about 350 pM, or about 400 pM, or about 450 pM, or about 466 pM for DNA when measured by surface plasmon resonance (e.g., using a BIAcore 3000 instrument).

[0116] In other examples, the affinity of the binding protein for DNA can be measured using Isothermal Titration Microcalorimetry.

[0117] In one example, the Fv contains a linker. Various suitable linkers and methods for designing them have already been described (e.g., U.S. Patent No. 4,946,778, WO1994 / 012520, and U.S. Patent No. 4,704,692). In one example, the Fv contains a glycine - serine (GS) linker. For example, the GS linker may contain (GGGGS) 3 (SEQ ID NO: 67). In one example, the Fv contains a linker having the sequence shown in SEQ ID NO: 30. In another example, the Fv contains a linker having the sequence shown in SEQ ID NO: 31. In another example, the Fv contains a linker having the sequences shown in SEQ ID NO: 30 and SEQ ID NO: 31.

[0118] In one example, the V of the Fv H and V L may be in a single polypeptide chain. In another example, the Fv lacks the Fc region. For example, the Fv may be a single - chain Fv fragment (scFv), a dimeric scFv (di - scFv), a trimeric scFv (tri - scFv). In one example, the Fv may be an scFv. In another example, the Fv is a di - scFv. In another example, the Fv is a tri - scFv.

[0119] In another example, the scFv, di - scFv or tri - scFv may be linked to the constant region of the antibody, Fc or the heavy - chain constant domain C H 2 and / or C H 3.

[0120] In one example, the disclosure includes a cell-permeable di-scFv having an antigen-binding domain that binds or specifically binds to DNA.

[0121] In one example, the di-scFv according to the present disclosure comprises an amino acid sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 32 to 47. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 32. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 33. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 34. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 35. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 36. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 37. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 38. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 39. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 40. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 41. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 42. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 43. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 44. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 45. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 46. In one example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 47. For example, the di-scFv comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in any one of SEQ ID NOs: 32, 36, 41 or 43.In these examples, the amino acid sequence can be at least 96%, at least 97%, at least 98% or at least 99% identical to the recited SEQ ID NO.

[0122] In one example, a diabody according to the present disclosure comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 32 to 47. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 32. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 33. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 34. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 35. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 36. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 37. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 38. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 39. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 40. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 41. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 42. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 43. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 44. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 45. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 46. In one example, the diabody comprises an amino acid sequence as set forth in SEQ ID NO: 47. For example, the diabody can comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 32, 36, 41 and 43.

[0123] In another example, the V of the binding protein H and V Lare in separate polypeptide chains. For example, the binding protein can be a diabody, a triabody, a tetrabody, a Fab, an F(ab’) 2 and the like. In another example, the binding protein can be a V H and V L in an Fv that contains them in separate polypeptide chains. In these examples, the binding protein may be linked to the constant region of the antibody, Fc or the heavy chain constant domain C H 2 and / or C H 3. In another example, the binding protein can be an intact antibody. Thus, in one example, the present disclosure encompasses antibodies having an antigen-binding domain that binds or specifically binds to DNA. For example, the antibody can bind to the same epitope as a binding protein having a V H comprising the amino acid sequence as shown in SEQ ID NO: 48 and a V L comprising the amino acid sequence as shown in SEQ ID NO: 49. In another example, the antibody can bind to the same epitope as a di-scFv having the amino acid sequence as shown in SEQ ID NO: 50. In one example, the antibody comprises a V H having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2 or SEQ ID NO: 3, and a CDR3 as shown in SEQ ID NO: 4. For example, the antibody can comprise a V H having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, and a CDR3 as shown in SEQ ID NO: 4. In another example, the antibody can comprise a V H having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 3, and a CDR3 as shown in SEQ ID NO: 4

[0124] In another example, the antibody has a CDR1 as shown in SEQ ID NO: 5 or SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7, and a CDR3 as shown in SEQ ID NO: 8 in a V LIt includes. For example, the antibody has a V with CDR1 as shown in SEQ ID NO: 5, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8. L It may include. In another example, the anti-DNA binding protein has a V with CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8. L It may include.

[0125] In another example, the antibody has a V with CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2 or SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4. H And a V with CDR1 as shown in SEQ ID NO: 5 or SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8. L It includes. For example, the antibody has a V with CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 4. H And a V with CDR1 as shown in SEQ ID NO: 5, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8. L It may include. In another example, the antibody has a V with CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 4. H And a V with CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8. L It may include. In another example, the antibody has a V with CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4. H And a V with CDR1 as shown in SEQ ID NO: 5, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8. LIt may include. In another example, the antibody has a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 3, and a CDR3 as shown in SEQ ID NO: 4 in V H and a CDR1 as shown in SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7, and a CDR3 as shown in SEQ ID NO: 8 in V L It may include.

[0126] The exemplary antibodies above may also have CDRs assigned using the IMGT system. Thus, in another example, the antibody has a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10 or SEQ ID NO: 11, and a CDR3 as shown in SEQ ID NO: 12 in V H It includes. For example, the antibody has a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10, and a CDR3 as shown in SEQ ID NO: 12 in V H It may include. In another example, the antibody has a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 11, and a CDR3 as shown in SEQ ID NO: 12 in V H It may include.

[0127] In another example, the antibody has a CDR1 as shown in SEQ ID NO: 13 or SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 15, and a CDR3 as shown in SEQ ID NO: 16 in V L It includes. For example, the antibody has a CDR1 as shown in SEQ ID NO: 13, a CDR2 as shown in SEQ ID NO: 15, and a CDR3 as shown in SEQ ID NO: 16 in V L It may include. In another example, the antibody has a CDR1 as shown in SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 15, and a CDR3 as shown in SEQ ID NO: 16 in V L It may include.

[0128] In another example, the antibody has a V with CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10 or SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12 H and a V with CDR1 as shown in SEQ ID NO: 13 or SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L For example, the antibody can include a V with CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10, and CDR3 as shown in SEQ ID NO: 12 H and a V with CDR1 as shown in SEQ ID NO: 13, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L In another example, the antibody can include a V with CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 10, and CDR3 as shown in SEQ ID NO: 12 H and a V with CDR1 as shown in SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L In another example, the antibody can include a V with CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12 H and a V with CDR1 as shown in SEQ ID NO: 13, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L In another example, the antibody can include a V with CDR1 as shown in SEQ ID NO: 9, CDR2 as shown in SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12 H and a V with CDR1 as shown in SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15, and CDR3 as shown in SEQ ID NO: 16 L can be included.

[0129] In another example, the antibody comprises a V that includes a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 17-23 H For example, the antibody may comprise a V that includes a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 18 H In another example, the antibody may comprise a V that includes a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 21 H In another example, the antibody may comprise a V that includes a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 23 H In another example, the antibody comprises a V that includes a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 24-29 L For example, the antibody may comprise a V that includes a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 25 L In another example, the antibody may comprise a V that includes a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 27 L In another example, the antibody comprises a V that includes a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 17-23 H as well as a V that includes a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 24-29 L For example, the antibody may comprise a V that includes a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 18 H and a V that includes a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 25 L In another example, the antibody may comprise a V that includes a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 21 H and a V that includes a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 27 L In another example, the antibody may comprise a V that includes a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 23 H and a V that includes a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 27 L In these examples, the V H and / or the VL can be at least 96%, at least 97%, at least 98% or at least 99% identical to the listed SEQ ID NOs. In these examples, the antibody can have the combinations of CDRs mentioned above. For example, the antibody can include a V having a sequence that is at least 95% identical to the sequence as shown in SEQ ID NO: 21 H and a V including a sequence that is at least 95% identical to the sequence as shown in SEQ ID NO: 27 L and in that case, the V H has CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4, and the V L has CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8.

[0130] In another example, the antibody includes a V having a sequence as shown in any one of SEQ ID NOs: 17 - 23 H For example, the antibody can include a V having a sequence as shown in SEQ ID NO: 18 H The antibody can include a V having a sequence as shown in SEQ ID NO: 21 H In another example, the antibody can include a V having a sequence as shown in SEQ ID NO: 23 H In another example, the antibody can include a V having a sequence as shown in any one of SEQ ID NOs: 24 - 29 L For example, the antibody can include a V having a sequence as shown in SEQ ID NO: 25 L In another example, the antibody can include a V having a sequence as shown in SEQ ID NO: 27 L In another example, the antibody can include a V having a sequence as shown in any one of SEQ ID NOs: 17 - 23 H and a V including a sequence as shown in any one of SEQ ID NOs: 24 - 29 L For example, the antibody can include a V having a sequence as shown in SEQ ID NO: 18 H and a V including a sequence as shown in SEQ ID NO: 25 Lmay include. In another example, the antibody includes a V having the sequence as set forth in SEQ ID NO: 21 H and a V having the sequence as set forth in SEQ ID NO: 27 L may include. In another example, the antibody includes a V having the sequence as set forth in SEQ ID NO: 23 H and a V having the sequence as set forth in SEQ ID NO: 27 L may include.

[0131] In another example, the antibody mentioned above may include a constant heavy region 1 having the sequence as set forth in SEQ ID NO: 69. In another example, the antibody mentioned above may include a constant heavy region 3 having the sequence as set forth in SEQ ID NO: 72. In another example, the antibody mentioned above may include a hinge region having the sequence as set forth in SEQ ID NO: 70. In these examples, the antibody includes a V having the amino acid sequence as set forth in SEQ ID NO: 27 L may include. For example, the antibody may include the amino acid sequence as set forth in SEQ ID NO: 78.

[0132] In another example, the antibody mentioned above may include a constant heavy region 1 having the sequence as set forth in SEQ ID NO: 69, a constant heavy region 3 having the sequence as set forth in SEQ ID NO: 72, a hinge region having the sequence as set forth in SEQ ID NO: 70, and a constant heavy region 2 having the sequence as set forth in any one of SEQ ID NOs: 71, 74, 76. In this example, the antibody includes a V having the amino acid sequence as set forth in SEQ ID NO: 27 L may include. For example, the antibody may include the amino acid sequence as set forth in SEQ ID NO: 78.

[0133] In another example, the antibody has the amino acid sequence as set forth in SEQ ID NO: 68. In another example, the antibody has the amino acid sequence as set forth in SEQ ID NO: 73. In another example, the antibody has the amino acid sequence as set forth in SEQ ID NO: 75. In another example, the antibody has the amino acid sequence as set forth in any one of SEQ ID NOs: 68, 73 or 75.

[0134] As is known in the art, antibodies can occur in different isotypes such as IgA, IgD, IgE, IgG, and IgM. In one example, the antibodies encompassed by the present disclosure are IgG. In another example, the antibodies encompassed by the present disclosure are IgM.

[0135] In one example, the physical stability of the antibody according to the present disclosure is higher than that of an Fv such as a scFv or a di-scFv having the corresponding V H and V L sequences. In one example, the physical stability of the antibody according to the present disclosure is higher than that of a di-scFv containing the amino acid sequence as shown in SEQ ID NO: 50 when the antibody is kept in solution at 4°C for at least 4 weeks. In one example, the physical stability of the antibody according to the present disclosure is higher than that of a di-scFv containing the amino acid sequence as shown in SEQ ID NO: 50 when the antibody is kept in solution at 4°C for at least 6 months.

[0136] In another example, the antibody has reduced immunogenicity in a human subject compared to a binding protein having a V H comprising the amino acid sequence as shown in SEQ ID NO: 48 and a V L comprising the amino acid sequence as shown in SEQ ID NO: 49. For example, the antibody may have reduced immunogenicity compared to a binding protein having a V H comprising the amino acid sequence as shown in SEQ ID NO: 48 and a V L comprising the amino acid sequence as shown in SEQ ID NO: 49 when the immunogenicity is measured by enzyme-linked immunosorbent assay (ELISA). In another example, the antibody may have reduced immunogenicity compared to a binding protein having a V H comprising the amino acid sequence as shown in SEQ ID NO: 48 and a V L comprising the amino acid sequence as shown in SEQ ID NO: 49 when the immunogenicity is measured by surface plasmon resonance.

[0137] In another example, the antibody has reduced immunogenicity compared to a diabody comprising the amino acid sequence as set forth in SEQ ID NO: 50 in a human subject. For example, the antibody may have reduced immunogenicity compared to a diabody comprising the amino acid sequence as set forth in SEQ ID NO: 50 when immunogenicity is measured by enzyme-linked immunosorbent assay (ELISA). In another example, the antibody may have reduced immunogenicity compared to a diabody comprising the amino acid sequence as set forth in SEQ ID NO: 50 when immunogenicity is measured by surface plasmon resonance.

[0138] In one example, the antibody has a modified Fc region. For example, the antibody Fc region may include an amino acid sequence as shown in SEQ ID NO: 71. In another example, the antibody Fc region includes an amino acid sequence as shown in SEQ ID NO: 74. In another example, the antibody includes an Fc region that includes an amino acid sequence as shown in SEQ ID NO: 76. In another example, the antibody includes an Fc region that includes an amino acid sequence as shown in any one of SEQ ID NOs: 71, 74, or 76. In another example, the antibody includes an Fc region that includes an amino acid sequence as shown in SEQ ID NO: 77 having three amino acid substitutions. In this example, two of the amino acid substitutions are between amino acids 1 and 10 of SEQ ID NO: 77. In another example, two of the amino acid substitutions are between amino acids 5 and 10 of SEQ ID NO: 77. In another example, two of the amino acid substitutions are at positions 7 and 8 of SEQ ID NO: 77. In these examples, the third amino acid substitution is between amino acids 65 and 75 of SEQ ID NO: 77. In another example, the third amino acid substitution is between amino acids 68 and 72 of SEQ ID NO: 77. In another example, the third amino acid substitution is between amino acids 65 and 75 of SEQ ID NO: 77. In one example, the antibody includes an Fc region that includes an amino acid sequence as shown in SEQ ID NO: 77 having an L7A mutation. In another example, the antibody includes an Fc region that includes an amino acid sequence as shown in SEQ ID NO: 77 having an L8A mutation. In another example, the antibody includes an Fc region that includes an amino acid sequence as shown in SEQ ID NO: 77 having an N70D mutation. In another example, the antibody includes an Fc region that includes an amino acid sequence as shown in SEQ ID NO: 77 having L7A and L8A mutations. In another example, the antibody includes an Fc region that includes an amino acid sequence as shown in SEQ ID NO: 77 having L7A and N70D mutations. In another example, the antibody includes an Fc region that includes an amino acid sequence as shown in SEQ ID NO: 77 having L8A and N70D mutations. In another example, the antibody includes an Fc region that includes an amino acid sequence as shown in SEQ ID NO: 77 having L7A, L8A, and N70D mutations.

[0139] Above, variant forms of the sequences of the present disclosure that have at least 95% sequence identity with a reference sequence, as well as heavy and light chain polypeptide sequences and their CDRs, have generally been presented, but variants with lower identity are clearly also disclosed herein. Thus, in some examples, the DNA-binding protein comprises a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence of any of the polypeptides of SEQ ID NOs: 32-47. In some embodiments, the DNA-binding protein comprises a variable heavy chain and / or light chain that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence of the heavy and / or light chain of any of SEQ ID NOs: 32-47 (e.g., any of SEQ ID NOs: 17-29). In some embodiments, the DNA-binding protein comprises one or more CDRs that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence of any of the CDRs of SEQ ID NOs: 32-47 (e.g., any of SEQ ID NOs: 1-16).

[0140] Production of the binding protein Recombinant expression In one example, the binding protein as described herein is a peptide or polypeptide (e.g., an antibody or an antigen-binding fragment thereof). In one example, the binding protein is recombinant.

[0141] In the case of recombinant peptides or polypeptides, the nucleic acids encoding them can be cloned into an expression vector and then transfected into host cells such as E. coli cells, yeast cells, insect cells, or mammalian cells such as simian COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce immunoglobulins or antibody proteins.

[0142] 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 updates to the present 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 the construction of recombinant nucleic acids. Methods for generating recombinant antibodies are also known in the art. See U.S. Patent No. 4,816,567 or U.S. Patent No. 5,530,101.

[0143] After isolation, the nucleic acid is operably linked and inserted into a promoter in an expression construct or expression vector for further cloning (DNA amplification) or for expression in a cell-free system or in cells. 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 a plasmid, bacteriophage, cosmid, yeast, or bacterial artificial chromosome, or comprises genetic components thereof, as understood in the art. The expression construct may be suitable for the maintenance and propagation of the isolated nucleic acid in bacteria or other host cells, for manipulation by recombinant DNA techniques, and / or for the expression of the nucleic acid or binding protein of the present disclosure.

[0144] Many vectors are available for expression in cells. 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., derived 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).

[0145] Exemplary promoters active in mammalian cells include the cytomegalovirus-mediated immediate early promoter (CMV-IE), the human elongation factor 1-α promoter (EF1), the small nuclear RNA promoters (U1a and U1b), the α-myosin heavy chain promoter, the simian virus 40 promoter (SV40), the Rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the β-actin promoter, the CMV enhancer / β-actin promoter, or hybrid regulatory elements containing immunoglobulin or antibody promoters or active fragments thereof. Examples of useful mammalian host cell lines are the simian kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651), the human fetal-derived kidney line (293 or 293 cells subcloned for growth in suspension culture), the baby hamster kidney cells (BHK, ATCC CCL10), or the Chinese hamster ovary cells (CHO).

[0146] For example, typical promoters suitable for expression in yeast cells, such as yeast cells selected from the group including Pichia pastoris, Saccharomyces cerevisiae, and S. pombe, include, but are not limited to, the ADH1 promoter, GAL1 promoter, GAL4 promoter, CUP1 promoter, PHO5 promoter, nmt promoter, RPR1 promoter, or TEF1 promoter.

[0147] Means for introducing an isolated nucleic acid or an expression construct containing the same into a cell for expression are known to those skilled in the art. The techniques used for a given cell are by known and successful techniques. Means for introducing recombinant DNA into cells include, inter alia, microinjection, transfection mediated by DEAE-dextran, liposome-mediated transfection, such as by using Lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and microparticle bombardment, such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0148] Host cells used to produce a binding protein (e.g., an antibody or antigen-binding fragment) can 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 other cell types discussed herein are known in the art.

[0149] One skilled in the art will understand from the above description that the present disclosure also provides an isolated nucleic acid encoding a binding protein of the present disclosure (e.g., a peptide or polypeptide binding protein or an antibody or an antigen-binding fragment thereof).

[0150] The present disclosure also provides an expression construct comprising an isolated nucleic acid of the present disclosure operably linked to a promoter. In one example, the expression construct is an expression vector.

[0151] In one example, the expression construct of the present disclosure comprises a nucleic acid encoding a polypeptide (e.g., including V H operably linked to a promoter) and a nucleic acid encoding another polypeptide (e.g., including V L operably linked to a promoter).

[0152] The present disclosure also provides a host cell comprising an expression construct according to the present disclosure.

[0153] The present disclosure also provides an isolated cell expressing a binding protein of the present disclosure or a recombinant cell genetically engineered to express a binding protein.

[0154] Isolation of Protein Methods for purifying a binding protein according to the present disclosure are known in the art and / or described herein. An example is shown in Example 1 below.

[0155] When a 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, e.g., an Amicon or Millipore Pellicon ultrafiltration device. To inhibit proteolysis, a protease inhibitor such as PMSF may be included in any of the foregoing steps, and an antibiotic may be included to prevent the growth of exogenous contaminants.

[0156] Binding proteins prepared from cells can be purified, for example, using 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 foregoing. 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).

[0157] Conjugate In one example, the binding proteins of the disclosure are conjugated to another compound. The binding protein can be directly or indirectly bound to the compound (e.g., in the case of indirect binding, a linker may be included). 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 agents or anti-inflammatory agents), colloids (e.g., gold), toxins (e.g., ricin or tetanus toxoid), nucleic acids, peptides (e.g., serum albumin-binding peptides), proteins (e.g., proteins containing the antigen-binding domain of an antibody or serum albumin), agents that increase the half-life of the compound in a subject (e.g., polyethylene glycol or other water-soluble polymers having this activity), and mixtures thereof.

[0158] Methods for conjugating drugs or other small molecule pharmaceuticals to antibodies are well known and can include the use of bifunctional chemical linkers such as N-succinimidyl (4-iodoacetyl)-aminobenzoate, sulfosuccinimidyl (4-iodoacetyl)-aminobenzoate, 4-succinimidyl-oxycarbonyl--(2-pyridyldithio)toluene, sulfosuccinimidyl-6-[α-methyl-∀-(pyridyldithiol)-toluamide]hexanoate, N-succinimidyl-3-(-2-pyridyldithio)-propionate, succinimidyl-6-[3(-(-2-pyridyldithio)-propionamide]hexanoate, sulfosuccinimidyl-6-[3(-(-2-pyridyldithio)-propionamide]hexanoate, 3-(2-pyridyldithio)-propionyl hydrazide, Ellman's reagent, dichlorotriazine acid, S-(2-thiopyridyl)-L-cysteine, etc. Additional bifunctional linking molecules are discussed, for example, in U.S. Patent Nos. 5,349,066, 5,618,528, 4,569,789, 4,952,394, and 5,137,877.

[0159] Linkers may be cleavable or non-cleavable. Highly stable linkers can reduce the amount of payload that is decreased in circulation and thus improve the safety profile and ensure that most of the payload reaches the target cells. Linkers may be based on chemical motifs including disulfide, hydrazone or peptide (cleavable), or thioether (non-cleavable) and can control the distribution and delivery of the active agent to the target cells. Cleavable or non-cleavable types of linkers have been demonstrated to be safe in preclinical and clinical trials (see, for example, Brentuximab Vedotin, which includes an enzyme-sensitive linker cleavable by cathepsin, and Trastuzumab Emtansine, which includes a stable and non-cleavable linker). In certain embodiments, the linker is a peptide linker cleavable by Edman degradation (Bachor, et al., Molecular diversity, 17(3):605-11(2013)).

[0160] A cleavable linker can maintain the active agent within the cell or target microenvironment. As a result, the entire antibody, linker, and active agent enter the targeted cell, where the antibody is degraded down to the amino acid level. The complex that results between the amino acids of the antibody, the linker, and the active agent becomes the actual drug. In contrast, a cleavable linker is catalyzed by an enzyme in the target cell or microenvironment, where it releases the active agent. Once cleaved, the payload can escape from the targeted cell and attack adjacent cells (also referred to as "bystander killing"). In the case of the binding proteins of the present disclosure, cleavage of the linker can result in two active agents, the antibody itself and its payload, which can have different mechanisms of action in the target cell or microenvironment.

[0161] In some embodiments, one or more additional molecules are present between the active agent and the cleavage site. Other considerations include site-directed conjugation (TDC) (Axup, Proceedings of the National Academy of Sciences, 109(40):16101-6 (2012)) as well as conjugation techniques such as those described in Lyon, et al., Bioconjugate Chem., 32(10):1059-1062 (2014), and Kolodych, et al., Bioconjugate Chem., 26(2):197-200 (2015), which can improve stability and the therapeutic index, and alpha-emitting immunoconjugates (Wulbrand, et al., Multhoff, Gabriele, ed., PLoS ONE. 8(5):e64730 (2013)).

[0162] In one example, the binding protein is conjugated to a nanoparticle or microparticle (e.g., as outlined in Kogan et al., Nanomedicine (Lond). 2:287-306, 2007). The nanoparticle can be a metal nanoparticle. The particle can be a polymeric particle, liposome, micelle, microbubble, and other carriers and delivery vehicles known in the art.

[0163] When the delivery vehicle is a polymer particle, the binding protein can be directly coupled to the particle or to an adapter element such as a fatty acid incorporated in the polymer. The ligand can be bound to the surface of the polymer particle via a functional chemical group (carboxylic acid, aldehyde, amine, sulfhydryl and hydroxyl) present on the surface of the particle and a functional chemical group (carboxylic acid, aldehyde, amine, sulfhydryl and hydroxyl) present on the ligand to be bound. The functional group may be introduced by crosslinking the particle and the ligand with a homo- or hetero-bifunctional crosslinker after the preparation of the particle. In this procedure, a group of crosslinkers (such as CDT, EDAC, glutaraldehyde as discussed in more detail below) or any other crosslinker that couples the ligand to the particle surface by appropriate chemistry and chemical modification of the particle surface after preparation may be used.

[0164] The binding protein may be indirectly bound to the polymer particle via an adapter element that interacts with the polymer particle. The adapter element can be bound to the polymer particle in at least two ways. The first method is by introducing a stabilizer having a functional chemical group during the preparation of micro- and nanoparticles, for example during the preparation of an emulsion of microparticles. For example, adapter elements such as fatty acids, hydrophobic or amphiphilic peptides and polypeptides can be inserted into the particles during emulsion preparation. In a second embodiment, the adapter element may be an amphiphilic molecule such as a fatty acid or lipid that adsorbs and binds passively to the particle surface, thereby introducing a functional end group for tethering the binding protein. The adapter element may associate with micro- and nanoparticles by various interactions including, but not limited to, hydrophobic interactions, electrostatic interactions and covalent bonds.

[0165] Suitable polymers include ethyl cellulose and other natural or synthetic cellulose derivatives. Polymers such as hydroxypropyl methylcellulose or polyethylene oxide that dissolve slowly in an aqueous environment to form a gel may also be suitable as the particle material. Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyrate (PHB) and its copolymers, poly-4-hydroxybutyrate (P4HB) and its copolymers, polycaprolactone and its copolymers, and combinations thereof.

[0166] Some exemplary compounds that can be conjugated to the binding proteins of the present disclosure are listed in Table 1. [Table 1]

[0167] In one example, the binding protein of the present disclosure is conjugated to a chemotherapeutic agent.

[0168] In one example, the binding protein of the present disclosure is conjugated to a maytansinoid, such as DM1 or DM4.

[0169] In another example, the binding protein of the present disclosure is conjugated to an auristatin, such as MMAE or MMAD.

[0170] In another example, the binding protein of the present disclosure is conjugated to an enzyme, such as MTM1, GAA or AGL.

[0171] In another example, the binding protein of the present disclosure is conjugated to MBNL.

[0172] In another example, the binding protein of the present disclosure is conjugated to a heat shock protein (HSP). In various examples, the binding protein of the present disclosure is conjugated to an HSP from the family of HSP33, HSP70, HSP90, HSP100, small HSP (sHSP), or a combination thereof. For example, the binding protein of the present disclosure may be conjugated to HSP72. Thus, in one example, the present disclosure includes an Fv conjugated to an HSP from the HSP70 family. In another example, the present disclosure includes an Fv conjugated to HSP72.

[0173] In another example, the binding protein of the present disclosure is conjugated to a PARP inhibitor disclosed herein. For example, the binding protein of the present disclosure may be conjugated to olaparib.

[0174] In one aspect of the above example, the binding protein conjugate can be used to deliver the conjugated payload to cells. Exemplary cells include heart cells such as cardiomyocytes, lung cells such as alveolar cells, and nerve cells such as neurons. Other exemplary cells include cancer cells or virus-infected cells.

[0175] In some embodiments, one or more of the above-described compounds are expressly excluded from being conjugated to the binding protein of the present disclosure. For example, the binding protein may be naked.

[0176] Composition Suitably, in a composition or method for administering a binding protein according to the present disclosure, 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 prior to administration to a subject. In this example, the kit may further include instructions for use.

[0177] In general name, "carrier" is used to refer to a solid or liquid bulking agent, binder, diluent, encapsulating substance, emulsifier, wetting agent, solvent, suspending agent, coating agent or lubricant that can be safely administered to a subject, for example, a human subject. Depending on the individual route of administration, various acceptable carriers known in the art as described, for example, in Remington’s Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991) can be used.

[0178] For example, suitable carriers can be selected from the group including sugars (e.g., sucrose, maltose, trehalose, glucose), starch, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils and oils containing synthetic mono- or di-glycerides, lower alcohols, polyols, alginic acid, phosphate buffer solutions, lubricants such as sodium or magnesium stearate, isotonic saline and pyrogen-free water. In one example, the carrier is not H 2 O.

[0179] In one example, the carrier is compatible with or suitable for parenteral administration. Parenteral administration includes any route of administration that does not pass 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 by a depot or sustained release formulation that can be delivered intradermally, intramuscularly or subcutaneously.

[0180] In some embodiments, the binding protein is encapsulated or incorporated into nanoparticles, microparticles, or other delivery vehicles such as, but not limited to, those discussed above.

[0181] In some embodiments, a DNA-binding protein is utilized to detect one or more sites of cancer, tissue damage, injury, infection, or ischemia. This method typically involves administering to a subject in need thereof an effective amount of an agent detectable using imaging diagnostics or nuclear medicine techniques, and detecting the agent. In such methods, the agent is typically conjugated to the DNA-binding protein or encapsulated in a delivery vehicle conjugated to the DNA-binding protein. Imaging diagnostics or nuclear medicine techniques may be, for example, PET-CT, bone scan, MRI, CT, echocardiogram, ultrasound, and X-ray.

[0182] In one example, the binding protein and compositions containing it can be used in the manufacture of a medicament for treating a condition. In another example, the present disclosure relates to a binding protein or a composition containing it for use in the treatment of a condition. Examples of conditions to be treated are discussed below.

[0183] The methods and uses typically involve administering to a subject in need thereof an effective amount of the binding protein. In some embodiments, the subject has cancer cells or virus-infected cells or transformed cells. In some embodiments, the subject has a disease or disorder characterized by exogenous or extracellular DNA including, but not limited to, ischemia, tissue damage, injury, or infection. The methods and uses may include combination therapy with a second, third, or additional active agent. For example, the binding proteins of the present disclosure can be used in combination with standard chemotherapy, radiation therapy, and other anti-cancer treatments. Radiation therapy (also known as radiotherapy) is the medical use of ionizing radiation as part of cancer treatment to control malignant cells.

[0184] Combination therapy Data compiled by the inventors of the present disclosure indicate that the disclosed binding proteins act in concert with poly(ADP-ribose) polymerase (PARP) inhibitors to kill cancer cells. For example, supra-additive cell death was observed in HDR-deficient cancer cells treated with the di-scFv and a PARP inhibitor.

[0185] Accordingly, in another example, the present disclosure encompasses a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a binding protein and a PARP inhibitor disclosed herein. In another example, the present disclosure relates to a therapeutic combination comprising a binding protein and a PARP inhibitor disclosed herein, wherein the combination is administered simultaneously or sequentially. In another example, the present disclosure relates to a therapeutic combination comprising a binding protein and a PARP inhibitor disclosed herein for use in the treatment of cancer.

[0186] In one example, the PARP inhibitor is selected from the group consisting of olaparib, niraparib, veliparib, rucaparib, talazoparib, and BGB-290. For example, the PARP inhibitor may be olaparib.

[0187] Examples of binding proteins suitable for administration with a PARP inhibitor are shown above. In one example, the binding protein has a V with 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: 4 H and a V having CDR1 as set forth in SEQ ID NO: 5, CDR2 as set forth in SEQ ID NO: 7, and CDR3 as set forth in SEQ ID NO: 8 L In another example, the binding protein has a V with 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: 4 H and a V having CDR1 as set forth in SEQ ID NO: 6, CDR2 as set forth in SEQ ID NO: 7, and CDR3 as set forth in SEQ ID NO: 8 LIt includes. In another example, the binding protein has CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4 of V H and CDR1 as shown in SEQ ID NO: 5, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 of V L It includes. In another example, the binding protein has CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4 of V H and CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 of V L It includes. In these examples, the binding protein may be an Fv. In one example, the binding protein may be a di-scFv.

[0188] A subject having one or more of the conditions discussed below can be treated by administering the binding protein and PARP inhibitor disclosed herein. In one example, the subject has pancreatic cancer. In another example, the subject has colon cancer. In one example, the subject has a cancer substantially deficient in BRCA2.

[0189] In another example, the combination therapy mentioned above can be used to treat a subject having a cancer resistant to PARP inhibitor therapy.

[0190] In one example, the binding protein and PARP inhibitor are administered as a single composition.

[0191] In another example, the binding protein and the PARP inhibitor are administered as separate compositions. For example, the binding protein and the PARP inhibitor can be administered simultaneously. In another example, the binding protein and the PARP inhibitor can be administered sequentially. In this example, the administration of the binding protein and the PARP inhibitor is carried out over a specified period (usually several minutes, several hours or several days). In one example, the period between successive administrations can be several days as long as the therapeutic effect of the second therapeutic agent is obtained or a still sufficient level of the first therapeutic agent is present when the second therapeutic agent is administered. In one example, after administration of the binding protein, continuous administration of the PARP inhibitor follows. In another example, after administration of the PARP inhibitor, continuous administration of the binding protein follows.

[0192] The therapeutic combinations according to the present disclosure can be administered by a variety of routes. Exemplary routes of administration include intravenous administration as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, intrathecal, oral routes.

[0193] In one example, the binding protein and the PARP inhibitor are administered by the same route. For example, both the binding protein and the PARP inhibitor can be administered intravenously by continuous infusion. In another example, the binding protein and the PARP inhibitor are administered by different routes. For example, the binding protein can be administered intravenously by continuous infusion and the PARP inhibitor can be administered orally.

[0194] In some examples, the administration of the binding protein or Fv fragment as defined herein and the PARP inhibitor achieves a greater result than when the binding protein or Fv fragment and the PARP inhibitor are administered alone or in isolation. For example, the result achieved by this combination can exceed the sum of the results achieved by the individual components alone.

[0195] In one example, administration of a combination of a binding protein or Fv fragment as defined herein and a PARP inhibitor is effective in reducing cancer cell proliferation or survival to a greater extent in a subject having cancer than when the same amounts of the individual components are administered to the subject alone. For example, the reduction in cancer cell proliferation or survival in a subject having cancer can exceed the sum of the results achieved by the individual components alone. In some examples, in a subject having cancer, this combination is effective for reducing tumor burden, reducing tumor progression, or a combination thereof, which can exceed the sum of the results achieved by the individual components alone.

[0196] Condition to be treated In one example, a binding protein according to the present disclosure can be administered to a subject to treat various conditions.

[0197] In some examples of the present disclosure, the methods described herein are methods for treating cancer. The term "cancer" typically refers to, or describes, a physiological state in mammals characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, lung cancers including squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancers including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanoma, nodular melanoma, multiple myeloma and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, as well as post-transplant lymphoproliferative disorder (PTLD), and furthermore, abnormal vascular proliferation associated with nevus syndromes, edema (such as those associated with brain tumors), Meigs syndrome, brain, and furthermore, head and neck cancers, and related metastases. In another example, the term "cancer" encompasses triple negative breast cancer. Thus, in one example, the present disclosure relates to methods for treating breast, ovarian, colon, prostate, lung, brain, skin, liver, stomach, pancreas or blood-based cancers. In another example, the present disclosure relates to the treatment of glioblastoma. In this example, glioblastoma can be treated by administering a binding protein disclosed herein such as a di-scFv having SEQ ID NO: 41 or an antibody having the heavy and light chain variable regions defined in SEQ ID NO: 41.

[0198] In other examples, the methods described herein are used to treat cancers associated with mutations in BRCA1, BRCA2, PALB2, or RAD51B, RAD51C, RAD51D or related genes. In other examples, the methods described herein are used to treat cancers associated with mutations in genes associated with DNA mismatch repair, such as MSH2, MLH1, PMS2, and related genes. In other examples, the methods described herein are used to treat cancers having silenced DNA repair genes, such as BRCA1, MLH1, or RAD51B, RAD51C, or RAD51D.

[0199] In another example, the methods described herein are used to kill cells in which the DNA repair process is damaged. For example, cells with damaged DNA repair may aberrantly express genes related to DNA repair, DNA synthesis, or homologous recombination. Exemplary genes include XRCC1, ADPRT (PARP-1), ADPRTL2, (PARP-2), POLYMERASE BETA, CTPS, MLH1, MSH2, FANCD2, PMS2, p53, p21, PTEN, RPA, RPAl, RPA2, RPA3, XPD, ERCC1, XPF, MMS19, RAD51, RAD51B, RAD51C, RAD51D, DMC1, XRCCR, XRCC3, BRCA1, BRCA2, PALB2, RAD52, RAD54, RAD50, MREU, NB51, WRN, BLM, KU70, KU80, ATM, ATR CPIK1, CHK2, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, RAD1, and RAD9. In one example, the methods described herein can be used to kill HDR-deficient cells. In another example, the methods described herein are used to kill cells having a mutated tumor suppressor gene. For example, the cells may have one or more mutations in BRCA1 or BRCA2. For example, the cells may be BRCA2-deficient colon cancer cells.

[0200] In one example, the methods described herein are methods for treating cancers that are substantially HDR - deficient. In one example, the methods described herein are methods for treating cancers that are substantially BRCA2 - deficient. For example, BRCA2 - deficient colon cancer can be treated. In another example, the methods described herein are methods for treating cancers that are substantially PTEN - deficient. For example, PTEN - deficient brain cancer can be treated. In another example, the methods described herein are methods for treating cancers that are resistant to PARP inhibition.

[0201] In other examples of the present disclosure, the methods described herein are used to treat virus - transformed cells, such as cells infected with oncoviruses. The term "oncovirus" is used in the context of the present disclosure to refer to a virus that can replicate in tumor cells and can reduce their growth. In one example, the oncovirus can naturally replicate in tumor cells and can reduce their growth. Examples of such viruses include Newcastle disease virus, vesicular stomatitis, myxoma, reovirus, sindbis, measles, and coxsackievirus. In another example, the oncovirus is engineered to replicate in tumor cells and reduce their growth. Exemplary viruses suitable for such engineering include adenovirus, herpes simplex virus (HSV), lentivirus, vaccinia, and vesicular stomatitis virus (VSV).

[0202] Other exemplary oncoviruses include human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), human T - lymphotropic virus (HTLV), Kaposi's sarcoma - associated herpesvirus (HHV - 8), Merkel cell polyomavirus, Epstein - Barr virus (EBV), human immunodeficiency virus (HIV), and human cytomegalovirus (CMV).

[0203] In other examples of the present disclosure, the methods described herein are used to kill cells transformed with latent viruses. Exemplary latent viruses include CMV, EBV, herpes simplex virus (types 1 and 2), and varicella zoster virus.

[0204] In other examples of the present disclosure, the methods described herein are used to treat active viral infections caused by viruses that cause cancer, immunodeficiency, hepatitis, encephalitis, pneumonitis, or respiratory diseases. Exemplary viruses include the oncoviruses, parvoviruses, poxviruses, and herpesviruses mentioned above.

[0205] In other examples of the present disclosure, the methods described herein are used to treat Colorado tick fever (caused by Coltivirus, an RNA virus), West Nile fever (encephalitis caused by Flavivirus, occurring mainly in the Middle East and Africa), yellow fever, rabies (caused by several different strains of neurotropic viruses of the family Rhabdoviridae), viral hepatitis, gastroenteritis (viral) - Norwalk and Norwalk-like viruses, rotavirus, calicivirus, and acute viral gastroenteritis caused by astrovirus, poliomyelitis, influenza (flu) caused by orthomyxoviruses that can undergo frequent antigenic variations, measles, paramyxovirus, mumps, respiratory syndromes including viral pneumonia caused by various viruses collectively referred to as acute respiratory viruses and acute respiratory syndromes including croup, and respiratory diseases caused by respiratory syncytial virus.

[0206] In other examples of the present disclosure, the methods described herein are used to treat nucleotide repeat disorders or exon splicing disorders. In other examples of the present disclosure, the methods described herein are used to treat disorders associated with abnormal microsatellite expansions, such as myotonic dystrophy. For example, the methods of the present disclosure can be used to treat myotonic dystrophy. Myotonic dystrophy type 1 (DM1, trinucleotide (CTG) n expansion (n = 50 to > 3000) in the 3'-untranslated region of the myotonic dystrophy-protein kinase (DMPK) gene) and type 2 (DM2, tetranucleotide (CCTG) n expansion (n = 75 to about 11,000) in the first intron of the zinc finger protein 9 (ZNF9) gene). In other examples of the present disclosure, the methods described herein are used to treat neurofibromatosis. In other examples of the present disclosure, the methods described herein are used to treat Huntington's disease. In other examples of the present disclosure, the methods described herein are used to treat myotubular myopathy. In other examples of the present disclosure, the methods described herein are used to treat glycogen storage disorders. In other examples of the present disclosure, the methods described herein are used to treat Pompe disease. In other examples of the present disclosure, the methods described herein are used to treat Forbes-Cori disease. In other examples of the present disclosure, the methods described herein are used to treat Lafora disease.

[0207] In other examples of the present disclosure, the methods described herein are used to increase muscleblind-like (MBNL) activity in cells in vitro or in a subject by administering a binding protein according to the present disclosure conjugated to an MBNL polypeptide. In other examples of the present disclosure, the methods described herein are used for enzyme or protein replacement therapy.

[0208] In other examples of the present disclosure, the methods described herein are used to increase HSP activity in vitro in cells or in a subject by administering a binding protein according to the present disclosure conjugated to an HSP from the HSP70 family. In other examples of the present disclosure, the methods described herein are used to increase HSP72 activity in vitro in cells or in a subject by administering a binding protein according to the present disclosure conjugated to an HSP72 polypeptide.

Example

[0209] Example 1 - Expression and Purification of Di-scFV Variants A single gene GS vector (using Lonza’s GS Xceed™ Gene Expression System) was established, sequenced, linearized, and used to generate stable pools for each variant. After cryopreservation, the expanded stable pools were each seeded into a 200 mL culture volume and subjected to abridged fed batch overgrow with a single bolus feed on day 4 and harvested on day 8. Supernatant titers were determined by Protein L Octet. Clarified supernatant for ion exchange purification was obtained by centrifugation followed by filter sterilization using a 0.22 μm filter. An ion exchange purification method was developed using the dimeric version of the mouse antibody as a reference.

[0210] A pre-packed 5 mL HiTrap Capto S column (GE Healthcare, 17-544122) was used to purify the clarified supernatant with an AKTA purifier (running at 5 mL / min). The column was equilibrated with 50 mM sodium phosphate pH 6 before and after sample loading, and the product was eluted with a linear gradient of 0 - 1 M NaCl. Quantification of bound and unbound material by Protein L Octet showed that approximately 57% of the material remained in the unbound fraction. Repeating the chromatography using the unbound fraction again, approximately 64% of the starting material remained in the unbound fraction.

[0211] The purification of the remaining supernatant was carried out using two consecutive steps of ion exchange chromatography with a linear elution gradient of 0 - 1 M NaCl. After purification, the product was quantified and concentrated to approximately 1 mg / mL by ultrafiltration using an Amicon Ultra-15 filter (Millipore, UFC903024).

[0212] Duplicate samples were analyzed by SE-HPLC on an Agilent 1200 series HPLC system using a Zorbax GF-250 9.4 mm ID×25 cm column (Agilent) and by SDS-PAGE analysis. The yields and titers of the expression cultures are summarized in Table 1. The SDS-PAGE analysis of the variants is shown in Figures 1 and 2.

Table 2

[0213] Example 2 - Nuclear Penetration of Variants Alkaline Phosphatase-Based Assay for Nuclear Penetration DLD1 colon cancer cells were treated with each of the control medium or the indicated variants for 1 hour. The cells were then washed, fixed, blocked with 1% BSA-TBST, and then probed with Protein L for 1 hour. The cells were then washed and incubated with an anti-Protein L primary antibody for 1 hour. After another round of washing, the cells were incubated with an alkaline phosphatase-conjugated secondary antibody for 1 hour. Finally, the cells were washed and the signal was generated by the addition of NBT / BCIP. Representative images are shown in Figure 3. The darkly stained areas indicate the positions of the variants.

[0214] Raw density integrated values reflecting nuclear alkaline phosphatase staining in DLD1 cells from the experiment in Figure 3 were obtained by analysis using ImageJ. Box and whisker plots of the value distributions are shown in Figure 4 for each variant.

[0215] A histogram plot of cell number versus nuclear staining intensity (expressed in arbitrary units as inverse intensity) is shown in FIG. 5. Most of the variants other than variants 12 and 14 show improved nuclear penetration compared to the parental yeast, as evidenced by the right shift of the histogram peak. In addition, the narrowing of the distribution observed in the histograms for many of the humanized variants indicates improved uniformity of nuclear penetration compared to the parental yeast. Variants 13 and 15 in particular showed a marked right shift and narrowing of the distribution compared to the parental yeast.

[0216] Immunofluorescence-based assay of nuclear penetration DLD1 colon cancer cells were treated with each of the control medium or the indicated variants for 1 hour. The cells were then washed, fixed, blocked with 1% BSA-TBST, and then probed with Protein L for 1 hour. The cells were then washed and incubated with an anti-Protein L primary antibody for 1 hour. After another round of washing, the cells were incubated with an Alexa488-conjugated secondary antibody for 1 hour. Finally, the cells were washed and the signal was visualized by fluorescence microscopy. Representative images are shown in FIG. 6. The green signal indicates the location of the variant.

[0217] Raw density integrated values reflecting the Alexa488 fluorescence signal in DLD1 cells from the experiment of FIG. 6 were obtained by analysis using ImageJ. Box-and-whisker plots of the value distributions are shown in FIG. 7 for each variant.

[0218] Example 2 - Accumulation of DNA damage Pairs of PTEN-proficient and PTEN-deficient U251 human glioma cells were treated for 24 hours with control medium or medium containing variant 10, 11, 13, 15, or 16. The cells were then washed, fixed, blocked, and then probed overnight with an anti-phospho-53BP1 antibody. The cells were then washed and incubated with an AlexaFluor555-conjugated secondary antibody. Finally, the cells were washed, counterstained with DAPI, and visualized under a fluorescence microscope. Images were saved and evaluated by CellProfiler to determine the average number of phospho-53BP1 foci per cell. The new variants increased the number of foci in PTEN-deficient cells but not in PTEN-proficient cells. Representative images are shown in panel A of FIG. 8, and the quantitative analysis by CellProfiler is shown in panel B of FIG. 8.

[0219] Cell viability of PTEN-deficient U87 human glioma cells was also evaluated after treatment with control medium or medium containing variant 10, 13, 15, or 16. Seven days after treatment, cell viability was determined using a trypan blue exclusion assay and by direct visualization of cell morphology by light microscopy. All variants resulted in a decrease in cell viability compared to control-treated cells (FIG. 9).

[0220] Next, pairs of BRCA2-proficient and -deficient DLD1 colon cancer cells were treated with control medium or medium containing variant 10, 13, 15, or 16. After 7 days, cell viability was determined by trypan blue exclusion assay and by direct visualization of cell morphology by light microscopy. The variants were not toxic to BRCA2-proficient cells, but BRCA2-deficient cells were killed by the variants. These data indicate that the variants can selectively kill cancer cells with DNA repair defects. Furthermore, these data indicate that the variants will be able to discriminate between cancer cells with DNA repair defects and healthy cells and selectively kill cancer cells. Representative light microscopy images showing morphological changes in BRCA2-deficient cancer cells treated with the variants are shown in panel A of FIG. 10. Quantitative analysis of cell viability by trypan blue exclusion assay is shown in panel B of FIG. 10.

[0221] Example 3 - Co-administration of di-scFv with PARP inhibition in HDR-deficient cancer cells DLD-1 and MCF-7 cells were treated with control or di-scFv (SEQ ID NO: 41), and nuclear penetration was evaluated by protein L immunostaining of fixed cells. The di-scFv successfully penetrated the DLD-1 and MCF-7 cell nuclei (FIGS. 11 and 12).

[0222] Homology-directed repair (HDR)-deficient BRCA2-DLD1 cells and PTEN-U251 cells were treated with control, 5 nM olaparib, 10 μM di-scFv, or 10 μM di-scFv + 5 nM olaparib. Cell viability was determined by colony formation assay. Surprisingly, supra-additive cell death was observed in HDR-deficient cancer cells treated with di-scFv and a PARP inhibitor (FIG. 13).

[0223] Next, it was determined whether the combination of di-scFv and olaparib is simply universally cytotoxic regardless of the DNA repair status. To evaluate this possibility, HDR-proficient DLD1 cells were treated with the above regimen to confirm the selectivity of the combination therapy against HDR-deficient malignant cells. No effect on cell death was observed for di-scFv alone or in combination with a PARP inhibitor. These findings demonstrate that HDR-proficient cells remain resistant to the actions of both di-scFv and olaparib, even when used in combination.

[0224] Example 4 - Effect of di-scFv (SEQ ID NO: 41) on primary human glioblastoma (GBM) cells Primary human glioblastoma (GBM) cancer cells extracted from primary human GBM tumors from patients were treated with control or di-scFv (SEQ ID NO: 41), and the percentage of viable cells was evaluated by trypan blue staining. Five out of seven glioblastoma tumor explants treated with di-scFv (SEQ ID NO: 41) showed significant cancer cell death (Figure 14).

[0225] GBM cancer stem cells extracted from primary human GBM tumors from patients and grown as spheres were treated with control or di-scFv (SEQ ID NO: 41), and the effects of dose and incubation time on the reduction of sphere volume were evaluated by confocal microscopy of DX1-rhodamine cell permeation into GBM cells. Tumor spheres are recognized as a useful tool for preclinical studies because they retain tumor heterogeneity and more closely represent the original patient tumor. Treatment of human GBM cancer stem cells (CSCs) grown as tumor spheres with di-scFv (SEQ ID NO: 41) demonstrated cell permeation in GBM spheres and dose-dependently and time-dependently reduced sphere volume (Figure 15).

[0226] Example 5 - Evaluation of the effect of di-scFv (SEQ ID NO: 41) on human GBM cells in an orthotopic mouse model An orthotopic mouse model of GBM was generated by intracranial injection of GBM cells extracted from human GBM tumors. Once tumors had developed in the brain, the mice were treated by intravenous injection of either control or di-scFv variant 13 (SEQ ID NO: 41), and the effect of di-scFv on tumor volume reduction was evaluated by tumor extraction. Evaluation of brain sections showed that glioblastoma tumors in mice treated with di-scFv were more than 40% smaller than comparable tumors in control mice (Figure 16A). TUNEL staining also demonstrated an increased incidence of apoptosis in di-scFv-treated tumors (Figure 16B). The reduction in tumor size and increase in TUNEL staining observed in di-scFv-treated GBM tumors suggested that di-scFv variant 13 (SEQ ID NO: 41) successfully crossed the blood-brain barrier, localized in GBM tumors, and affected their growth. To confirm this, tumors and normal brains were examined for di-scFv by protein L immunostaining. As shown in Figure 16C, di-scFv was detected in the nuclei of GBM tumor cells but not in adjacent surrounding normal brain cells.

[0227] In addition, a group of seven mice was evaluated for survival effects, and mice treated with di-scFv showed a median survival of 87 days, more than 20% longer than controls (median 72 days). Mean survival data reflected these trends (83 days ± 3.2 days for di-scFv-treated mice and 71 days ± 1.2 days for controls) (Figure 16E). Statistical analysis showed a significant difference between the two groups, with a P value = 0.004. No toxicity or weight loss associated with di-scFv treatment was observed (Figure 16D).

[0228] Example 6 - Effect of PAT-DX1 on Focus Accumulation Pairs of BRCA2-proficient and -deficient DLD1 colon cancer cells and PTEN-proficient and -deficient U251 human glioma cells were treated with control medium or medium containing 10 μM of disulfide-stabilized scFv variant 13 (SEQ ID NO: 41), 5 nM olaparib, or a combination treatment. Phospho-53BP1 antibody staining was evaluated by CellProfiler to determine the average number of phospho-53BP1 foci per cell. Treatment with disulfide-stabilized scFv variant 13 (SEQ ID NO: 41) alone and in combination with olaparib increased the number of phospho-53BP1 foci in both BRCA2-deficient DLD1 and PTEN-deficient U251 cells, but not in proficient cells (Figure 17).

[0229] Those skilled in the art will recognize that numerous modifications and / or alterations can be made to the present disclosure without departing from the intended spirit and scope of the disclosure as broadly described. Accordingly, the present embodiments are illustrative in all respects and should not be construed as limiting.

[0230] All publications discussed above are hereby incorporated by reference in their entirety. Any discussion of documents, acts, materials, devices, articles, etc. included herein is for the purpose of providing context for the present disclosure only. None of these matters should be construed as an admission that any of these constitute prior art to the present disclosure or part of the common general knowledge in the relevant field as existing prior to the priority date of each claim of this application.

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Claims

1. A nuclear-permeable anti-DNA binding protein having an antigen-binding domain, wherein the antigen-binding domain binds to DNA and - A heavy chain variable region (V) having complementarity-determining region (CDR) 1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4 Ｈ ), and a light chain variable region (V) having CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8 Ｌ ), and - V comprising a sequence that is at least 98% identical to the sequence as shown in SEQ ID NO: 21 Ｈ , and V comprising a sequence that is at least 98% identical to the sequence as shown in SEQ ID NO: 27 Ｌ comprises, and the CDR is defined using the Kabat numbering system, the nuclear-permeable anti-DNA binding protein.

2. V comprising a sequence that is at least 99% identical to the sequence as shown in SEQ ID NO: 21 Ｈ and V comprising a sequence that is at least 99% identical to the sequence as shown in SEQ ID NO: 27 Ｌ The binding protein according to claim 1, comprising the same.

3. V comprising the array as shown in SEQ ID NO: 21 Ｈ and V comprising the array as shown in SEQ ID NO: 27 Ｌ The binding protein according to claim 1, comprising:

4. Said V Ｈ and V Ｌ The conjugate protein according to any one of claims 1 to 3, wherein are separated by a linker containing the sequence shown in SEQ ID NO:

30.

5. Said V Ｈ and V Ｌ The binding protein according to any one of claims 1 to 4, wherein is in a single polypeptide chain.

6. (i) single-chain Fv fragment (scFv), (ii) dimeric scFv (di-scFv), (iii) trimeric scFv (tri-scFv), (iv) Constant region of the antibody, Fc or heavy chain constant domain C Ｈ 2 and / or C Ｈ linked to any one of (i), (ii) or (iii) in C The binding protein according to claim 5, which is

7. The binding protein according to claim 6, which is scFv.

8. The binding protein according to claim 6, which is di-scFv.

9. The binding protein according to claim 8, wherein the scFv is separated by a linker containing the sequence shown in SEQ ID NO:

31.

10. Said V Ｈ and V Ｌ The binding protein according to any one of claims 1 to 3, wherein are in separate polypeptide chains.

11. The binding protein according to claim 10, which is an intact antibody.

12. The binding protein according to claim 1 or 2, which is a nuclear-permeable anti-DNA Fv fragment.

13. The binding protein according to claim 12, which is di-scFv.

14. The binding protein according to any one of claims 1 to 13, which is conjugated to another compound.

15. A nucleic acid encoding the binding protein defined in any one of claims 1 to 14.

16. A composition comprising the binding protein defined in any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

17. Use of the binding protein defined in any one of claims 1 to 14 or the composition according to claim 16 in the manufacture of a medicament for treating cancer.

18. The binding protein defined in any one of claims 1 to 14 or the composition according to claim 16 for use in the treatment of cancer.

19. The cancer is one or more of the following: - Homology-directed repair (HDR) deficient, - Resistant to PARP inhibition, - BRCA2 deficient, - PTEN deficient, The use according to claim 17.

20. The use according to claim 17, wherein the cancer is colon cancer, brain cancer, prostate cancer, ovarian cancer, breast cancer, endometrial cancer, melanoma, or pancreatic cancer.

21. The use according to claim 17, wherein the cancer is glioblastoma or triple-negative breast cancer.

22. The cancer is one or more of the following: - being homology-directed repair (HDR) deficient, - being resistant to PARP inhibition, - being BRCA2 deficient, - being PTEN deficient, The binding protein according to claim 18.

23. The binding protein according to claim 18, wherein the cancer is colon cancer, brain cancer, prostate cancer, ovarian cancer, breast cancer, endometrial cancer, melanoma, or pancreatic cancer.

24. The binding protein according to claim 18, wherein the cancer is glioblastoma or triple-negative breast cancer.

25. A nuclear-permeable anti-DNA binding protein having an antigen-binding domain, wherein the antigen-binding domain binds to DNA and - A heavy chain variable region (V) having a complementarity determining region (CDR) 1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 3, and a CDR3 as shown in SEQ ID NO: 4 Ｈ ), and a light chain variable region (V) having a CDR1 as shown in SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7, and a CDR3 as shown in SEQ ID NO: 8 Ｌ ), and V containing the array as shown in SEQ ID NO: 21 Ｈ and V containing the array as shown in SEQ ID NO: 27 Ｌ comprising the nuclear-permeable anti-DNA binding protein.

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