Anti-Factor IX Padova Antibodies

Abstract

The present invention provides binding constructs against Padova Factor IX, such as antibodies and antigen-binding fragments thereof. Related polypeptides, conjugates and kits are also provided. The present invention can be used in a method for detecting Padova Factor IX in a sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 337,118 filed on May 16, 2016 and No. 62 / 340,834 filed on May 24, 2016, the entire texts of both cases are hereby incorporated by reference.

[0003] Incorporation by reference of electronically submitted materials

[0004] The computer-readable nucleotide / amino acid sequence listing filed concurrently with this application is incorporated by reference in its entirety and is identified as follows: 28,804-byte ACII (Text) file named "50573_SeqListing.txt"; created on May 16, 2016. Background Art

[0005] Gene therapy has great development prospects as a treatment option for hemophilia in the future. In a clinical trial, a subject with severe hemophilia B was treated with an adeno-associated virus (AAV) vector encoding Padua Factor IX (FIX), which is a high-functional variant of FIX in which the Arg at position 338 of the mature peptide sequence (or at position 384 of the preproprotein sequence) undergoes a single amino acid substitution to become Leu. The specific detection of the transgenic product (Padua FIX) can be used to assess the success of factor replacement. However, the specific detection of Padua FIX in the treated patient is still a major challenge, because some patients have factor IX cross-reactive material (cross-reactive material, CRM). Some CRM positive (CRM+) patients express, for example, wild-type (WT) Factor IX, which cross-reacts with currently available Padua FIX binding agents, making it difficult to determine whether such CRM+ patients can express Padua FIX. Summary of the invention

[0006] Provided herein is a binding construct that specifically recognizes FIX of Padova (FIXp) and does not cross-react with wild-type FIX. In an exemplary embodiment, the binding construct is an antibody or antigen-binding fragment thereof that binds FIX of Padova containing the amino acid sequence SEQ ID NO: 1 and does not bind to the WT factor IX containing the amino acid sequence SEQ ID NO: 2. In an exemplary embodiment, the binding construct is a polypeptide comprising an amino acid sequence comprising each of SEQ ID NOs: 6-11, optionally wherein (i) one or more amino acids are present between each of SEQ ID NOs: 6-11, and / or (ii) the polypeptide optionally further comprises a FLAG tag comprising DYKDDDDK (SEQ ID NO: 12) and / or a hexahistidine tag comprising HHHHHH (SEQ ID NO: 13), optionally wherein the FLAG tag and / or the hexahistidine tag are located at the C-terminus of the polypeptide. In exemplary embodiments, the binding construct is a conjugate comprising an antigen binding fragment as described herein coupled to (i) a constant region of an immunoglobulin heavy chain, (ii) a constant region of an immunoglobulin light chain, or (iii) a constant region of an immunoglobulin heavy chain and a constant region of an immunoglobulin light chain. In exemplary embodiments, the binding construct is a conjugate comprising an antibody or antigen binding fragment as described herein connected or coupled to a heterologous portion. In exemplary aspects, the conjugate comprises an antibody or antigen binding fragment as described herein coupled to a polymer, carbohydrate, lipid, nucleic acid, oligonucleotide, amino acid, peptide, polypeptide, protein, or detection agent.

[0007] In addition, nucleic acids are provided, comprising nucleotide sequences encoding antibodies, antigen-binding fragments, polypeptides, conjugates or fragments thereof as described herein. Also provided are vectors comprising the nucleic acids and host cells comprising the nucleic acids or vectors.

[0008] Related kits are also provided herein. In exemplary embodiments, the kit includes an antibody, Fab, polypeptide, conjugate, nucleic acid, vector, host cell, or combination thereof as described herein, and optionally, instructions for use. In exemplary aspects, the kit also includes a solid support and optionally, the antibody, Fab, polypeptide, or conjugate is pre-coated on the solid support. In exemplary aspects, the kit also includes a secondary antibody that binds to the antibody, Fab, polypeptide, or conjugate provided in the kit.

[0009] The present invention also provides compositions comprising a mixture of an antibody or antigen binding fragment or polypeptide or conjugate as described herein and a biological sample, the biological sample being, for example, a biological sample obtained from a human. In exemplary aspects, the biological sample comprises human plasma or a dilution fraction thereof, and / or human tissue or cells thereof. In exemplary aspects, the biological sample comprises human plasma proteins, wherein at least one of the human plasma proteins is selected from the group consisting of factor IX, factor II, and factor X, and variants thereof. Optionally, the composition comprises a detection agent.

[0010] Such binding constructs provided herein can be used, for example, in such detection methods: they allow for the unambiguous or specific detection of Padova FIX in a sample, such as a clinical or preclinical sample comprising, for example, wild-type FIX. Thus, provided herein are methods for detecting Padova Factor IX comprising the amino acid sequence SEQ ID NO: 1 in a sample obtained from a subject. In an exemplary embodiment, the method comprises (i) contacting the sample with a binding construct (e.g., an antibody, antigen-binding fragment, polypeptide, or conjugate as described herein) to form a complex, such as an immune complex, comprising the FIXp and the binding construct; and (ii) detecting the complex in the sample.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 A schematic representation of the amino acid sequences of WT Factor IX and Padova FIX is presented.

[0013] Figure 2 Padova FIX (left panel) or WT Factor IX (right panel) with Ni coated with 5 μg / ml BC1 (blue bar closest to the Z-axis (i.e., to the left)), 1 μg / ml BC1 (red bar to the right of the blue bar), 0.2 μg / ml BC1 (green bar adjacent to the red bar), or 0.04 μg / ml BC1 are presented. 2+ Figure 3 is a graph of the binding signal of the plate.

[0014] Figure 3 Graphs showing the binding signals of BC1 (triangles), BC2 (diamonds), or BC3 (squares) coated on MaxiSorp plates to the indicated concentrations of Padova FIX are presented.

[0015] Figure 4 Presented are graphs of the binding signals of BC1 to the indicated concentrations of Padova FIX in a 5% human plasma solution containing 5 μg / ml WT Factor IX and without benzamidine.

[0016] Figure 5Presented are graphs of the binding signals of BC1 to the indicated concentrations of Padova FIX in 2% BSA / PBS (diamonds), 5% plasma solution containing 50 mM benzamidine (squares), 10% plasma solution containing 50 mM benzamidine (triangles), or 20% plasma solution containing 50 mM benzamidine (Xs).

[0017] Figure 6 Presented are graphs of binding signals of BC1 (squares), BC2 (triangles), BC4 (diamonds), or negative control (X) to the indicated concentrations of Padova FIX in a 20% (v / v) plasma solution containing WT Factor IX and 50 mM benzamidine.

[0018] Figure 7 Two graphs are presented showing the binding signals of BC1 (left graph) and BC4 (right graph) to the indicated concentrations of the following coagulation factors: Padova FIX (diamonds); Factor II (squares); and Factor X (triangles).

[0019] Figure 8 Two graphs showing the binding signals of BC5 (left graph) and BC6 (right graph) to the indicated concentrations (μg / ml) of WT Factor IX (diamonds) or Padova FIX (squares) are presented.

[0020] Fig. 9 A schematic diagram of the components of the ELISA described in Example 2 is presented.

[0021] Fig.10 A schematic diagram of the components of the ELISA described in Example 1 is presented.

[0022] Fig.11 Concentration-response curves of the Padova FIX ELISA performed using samples containing Padova FIX (circles) or reference human plasma samples (diamonds) are presented.

[0023] Fig.12 Presented is a graph of the calibration curve of the Padova FIX ELISA performed using six standards (Standards D1 to D6) with specified amounts of Padova FIX.

[0024] Fig.13 Dilution-response curves for the Padova FIX ELISA performed using buffer containing Padova FIX or normal plasma or plasma lacking FIX are presented.

[0025] Fig.14 Dilution-response curves for the Padova FIX ELISA performed using citrated monkey plasma samples are presented.

[0026] Fig.15Graphs are presented of coagulation activity and protein measurements in samples from subjects with FIX cross-reactive material (CRM+) following treatment with AAV2 / 8 viral vector expressing Padova FIX.

[0027] Fig.16 Graphs are presented of coagulation activity and protein measurements in samples from another subject with FIX cross-reactive material (CRM+) following treatment with AAV2 / 8 viral vector expressing Padova FIX.

[0028] Fig.17 Presented are graphs of coagulation activity and protein measurements in samples from subjects without FIX cross-reactive material (CRM+) following treatment with AAV2 / 8 viral vector expressing Padova FIX.

[0029] Fig.18 A graph showing the change in signal as a function of FIX concentration is presented, wherein the signal is generated by the Padova FIX-specific chromogenic activity assay.

[0030] Fig.19 A schematic diagram of the strategy used to generate Padova FIX-specific Fabs and the sequences of the panning and blocking peptides used are presented (L338 in Padova FIX is indicated in red and boxed, R338 in wild-type FIX is indicated in green and boxed).

[0031] Fig. 20 The schematic structure of the purified minibody (bivalent Fab) is presented.

[0032] Fig.21 Rendering Fig.21 A) X-ray structure of porcine FIXa and shows that Arg338 is located on the surface of the porcine FIXa heavy chain and ( Fig.21 B) Schematic representation of human Padova FIX and showing Leu338.

[0033] Fig. 22 The figures presented demonstrate that the purified Fab binds specifically to Padova FIX and not to wild-type FIX antigen. Fig. 22 Figures of ELISA performed by purified bivalent Fabs against wild-type and Padova antigens are presented. The bivalent Fabs obtained from different panning strategies were tested for their specificity against wild-type and Padova peptides and proteins. Results are expressed as fold increase relative to background ( Fig. 22 A), and ( Fig. 22 B) shows the scheme of the ELISA setup. The order of the bars is the same as in the legend (i.e., the Ab42 column is on the left). For details, see the method description in Example 5.

[0034] Fig.23The graph presented demonstrates that the purified bivalent Fab showed no cross-reactivity with wild-type FIX in 20% human plasma matrix. Fig.23 ELISA profiles of purified bivalent Fab in the presence of 20% plasma are presented. Fig.23 A) Purified bivalent Fab was coated and incubated with 20% human plasma containing 5 μg / mL wild-type FIX and the indicated concentrations of Padova FIX. Detection was performed with HRP-labeled polyclonal goat anti-FIX antibody (100 ng / ml). Fig.23 B) Scheme of ELISA setup. The order of each line is the same as in the legend.

[0035] Fig.24 The presented graphs demonstrate the surface plasmon resonance (SPR) analysis of the selected candidate Ab42. Fig.24 A) His-tagged miniantibody Ab42 with KD = 59 nM (ka: 4.3 × 10 4 1 / Ms; kd: 0.002531 1 / s) combined with Padova FIX, ( Fig.24 B) but not binding to FIX wt on the NTA-BIAcore sensor chip. The dashed line represents the original data, while the solid line indicates the fitted data. The order of each line is the same as the legend.

[0036] Fig.25 A graphical illustration of the immunoassay is presented.

[0037] Fig.26 The presented figures demonstrate the selectivity of the assay as represented by the concentration-response curves obtained for purified FIXp samples and fresh frozen reference plasma preparations with a normal FIX concentration of 5 μg / mL. Human plasma showed essentially no response.

[0038] Fig. 27 A 6-point calibration curve ranging from 0.85 to 27.1 ng FIXp / mL is presented with adequate linearity. Its accuracy is demonstrated by the correlation coefficient r, low relative total error (RTE), and the results of the back-fitting method.

[0039] Fig.28 A set of figures is presented demonstrating parallel studies performed in normal and FIX-deficient plasma. The slope of the dilution-response curve for plasma spiked with FIXp differs by <5% from that obtained for the buffer dilution series, indicating that the plasma matrix has no effect on the analytical performance.

[0040] Fig.29 The presented figure shows the Ca 2+ Effects on ELISA sensitivity.2+ The significant increase in sensitivity caused by the polyclonal detection antibody was most likely caused by the Ca-dependent EGF domain of FIX.

[0041] Fig.30 A set of graphs presented demonstrates the analysis of citrated plasma samples from the first patient treated with AAV2 / 8 viral vectors in a Phase 1 / 2 trial. The plasma sample obtained from the first patient was FIX cross-reactive material positive (CRM+).

[0042] Fig.31 A set of graphs are presented showing the analysis of citrated plasma samples from a second patient treated with AAV2 / 8 viral vectors in a Phase 1 / 2 trial. The plasma sample obtained from the second patient was FIX cross-reactive material positive (CRM+).

[0043] Fig.32 One set of figures presented demonstrates the analysis of citrated plasma samples from the third patient treated with AAV2 / 8 viral vectors in a Phase 1 / 2 trial. The plasma sample obtained from the third patient was not FIX cross-reactive material positive (CRM+).

[0044] Implementation

[0045] Provided herein is a binding construct for specific recognition Padova FIX and having minimal cross-reaction or no cross-reactivity with wild-type FIX. In exemplary aspects, the binding construct is bound to Padova FIX and is not bound to wild-type (WT) factors IX. In exemplary aspects, the binding construct is bound to Padova FIX (and is not bound to WT factors IX) in the presence of WT factors IX. In exemplary aspects, the binding construct is bound to Padova FIX under similar or identical conditions and is not bound to one or two of a WT factors IX and factor II and factor X, or any other sudden change or modified form thereof (except Padova FIX). In exemplary aspects, the binding construct is bound to Padova FIX and is neither bound to factor II nor bound to factor X. In exemplary aspects, the binding construct is bound to Padova FIX and is not bound to any one among WT factors IX, factor II and factor X. In exemplary aspects, the binding construct binds to Padova FIX and (does not bind to WT Factor IX, Factor II, and Factor X) in the presence of WT Factor IX, Factor II, and Factor X. In exemplary embodiments, the binding construct binds to an epitope of Padova FIX (SEQ ID NO: 1) even when in solution that includes WT Factor IX, Factor II, and Factor X at levels present in human plasma.

[0046] Epitope

[0047] As used herein, "epitope" means a region of Padova FIX combined with a binding construct or a region within Padova FIX. In some embodiments, the epitope is a linear epitope. As used herein, "linear epitope" refers to a region of Padova FIX combined with a binding construct or a region within Padova FIX, and the region is composed of adjacent amino acids in the amino acid sequence of Padova FIX. The amino acids of a linear epitope are adjacent to each other in the primary structure of Padova Factor IX. Therefore, a linear epitope is a fragment or a part of the amino acid sequence of the antigen (i.e., Padova FIX).

[0048] In other exemplary embodiments, the epitope is a conformational or structural epitope. "Conformational epitope" or "structural epitope" means an epitope consisting of amino acids that are closely adjacent to each other in position when Padova Factor IX is in its proper folded state. Different from a linear epitope, the amino acids of a conformational or structural epitope need not be adjacent to each other in the primary structure (i.e., amino acid sequence) of Padova FIX. A conformational or structural epitope need not consist of the adjacent amino acids in the amino acid sequence of the antigen (FIXp).

[0049] In an exemplary embodiment, the binding construct binds to an epitope of Padova FIX comprising the amino acid sequence SEQ ID NO: 1, wherein the epitope is a linear epitope within the amino acid sequence SEQ ID NO: 1. In an exemplary aspect, the linear epitope is within the amino acid sequence DRATCLLSTKFT (SEQ ID NO: 3). In an exemplary aspect, the linear epitope includes at least the LL in SEQ ID NO: 3. In an exemplary embodiment, the binding construct binds to a linear epitope of Padova FIX, even in the presence of WT factor IX, factor II and / or factor X. In an exemplary embodiment, the binding construct binds to an epitope of Padova FIX, even in the presence of 5 μg / mL WT factor IX. In an exemplary embodiment, the binding construct binds to an epitope of Padova FIX, even in the presence of 5 μg / mL WT factor IX and in a 20% human plasma matrix.

[0050] In exemplary aspects, the binding construct is not bound to the epi-position of WT FIX. In exemplary aspects, the binding construct is not bound to the epi-position in SEQ ID NO:2 or in DRATCLRSTKFT (SEQ ID NO:14) or in LVDRATCLRSTKFTIYNNMFCAGFH (SEQ ID NO:15). In exemplary aspects, under similar or identical conditions when the binding construct is bound to Padova FIX, the binding construct is not bound to the epi-position of WT FIX. In exemplary aspects, when in the solution of WT FIX containing normal plasma content, the binding construct is not bound to the epi-position of WT FIX. In exemplary aspects, when in the solution (such as buffer) containing 5 μg / mL WT FIX (such as human plasma matrix containing about 5 μg / mL WT FIX), the binding construct is not bound to the epi-position of WT FIX.

[0051] In an exemplary embodiment, the binding construct binds to an epitope of Padova FIX (SEQ ID NO: 1), wherein the epitope is a conformational epitope of the folded structure of the amino acid sequence LVDRATCLLSTKFTIYNNMFCAGFH (SEQ ID NO: 5). In an exemplary embodiment, the binding construct binds to an epitope of Padova FIX (SEQ ID NO: 1), wherein the epitope is a conformational epitope of the folded structure of the amino acid sequence LVDRATCLLSTKFTIYNNMFCAGFH (SEQ ID NO: 5), wherein the folded structure includes a disulfide bridge. In an exemplary embodiment, the binding construct binds to a conformational epitope of Padova FIX, even in the presence of WT factor IX, factor II and / or factor X. In an exemplary embodiment, the binding construct binds to an epitope of Padova FIX, even in the presence of 5 μg / mL WT factor IX.

[0052] Affinity and affinity

[0053] The binding constructs provided herein bind to Padova FIX in a non-covalent and reversible manner. In exemplary embodiments, the binding strength of the binding construct to Padova FIX can be described by its affinity, and affinity is a measure of the intensity of the interaction between the binding site of the binding construct and the epitope. In exemplary aspects, the binding constructs provided herein have high affinity to Padova FIX and therefore will bind to a larger amount of Padova FIX in a shorter period of time compared to low-affinity binding constructs. In exemplary aspects, the binding construct has an equilibrium association constant KA, and KA is at least 10 5 mol -1 , at least 10 6 mol -1, at least 10 7 mol -1 , at least 10 8 mol -1 , at least 10 9 mol -1 or at least 10 10 mol -1 . In exemplary aspects, the binding constructs provided herein show high affinity for the Padova FIX in human plasma. In exemplary aspects, in samples containing human plasma, the binding constructs are bound to the Padova Factor IX and are not bound to the WT Factor IX. In exemplary aspects, in samples containing at least or about 5% human plasma (e.g., 5% human plasma matrix), the binding constructs are bound to the Padova Factor IX and are not bound to the WT Factor IX. In exemplary aspects, in samples containing at least or about 10% human plasma (e.g., 10% human plasma matrix), the binding constructs are bound to the Padova Factor IX and are not bound to the WT Factor IX. In exemplary aspects, in samples containing at least or about 20% human plasma (e.g., 20% human plasma matrix), the binding constructs are bound to the Padova Factor IX and are not bound to the WT Factor IX. In exemplary aspects, in a sample containing at least or about 5% to about 40%, about 10% to about 30%, or about 15% to about 20% human plasma, the binding construct is bound to Padova Factor IX and is not bound to WT Factor IX. In exemplary aspects, the binding construct is bound to Padova Factor IX, even when there is a large amount of WT Factor IX in the sample. In exemplary aspects, in a sample containing a certain amount of human plasma (e.g., at least or about 5% human plasma, at least or about 10% human plasma, at least or about 20% human plasma) and at least or about 1 μg / mL WT Factor IX, or at least or about 2.5 μg / mL WT Factor IX, or at least or about 5 μg / mL WT Factor IX, or at least or about 10 μg / mL WT Factor IX, the binding construct is bound to Padova Factor IX and is not bound to WT Factor IX. In exemplary aspects, in samples containing other coagulation factors, the binding construct binds to Padova Factor IX and does not bind to WT Factor IX, and the other coagulation factors include but are not limited to Factor II, Factor V, Factor VI, Factor VII, Factor VIII, Factor X, Factor XI, Factor XII, and Factor XIII. In exemplary aspects, the binding construct binds to Padova Factor IX and does not bind to WT Factor IX and does not bind to Factor II or Factor X. In exemplary aspects, the binding construct binds to Padova Factor IX and does not bind to WT Factor IX and, in addition, does not bind to Factor II or Factor X.

[0054] In an exemplary embodiment, the strength of binding of the binding construct to Padova FIX can be described by its sensitivity. KD is the equilibrium dissociation constant between the binding construct and Padova FIX, i.e., k off / k on The ratio of KD to KA is inversely correlated. The KD value is related to the concentration of the binding construct (the amount of binding construct required for a particular experiment) and therefore the lower the KD value (the lower the concentration), the higher the affinity of the binding construct. In exemplary aspects, the binding strength of the binding construct to Padova FIX can be described by KD. In exemplary aspects, the KD of the binding construct provided herein for FIXp is about 1.0×10 -6 or smaller, about 1.0×10 -7 or smaller, about 1.0×10 -8 or smaller, about 1.0×10 -9 or smaller, about 1.0×10 -10 In exemplary aspects, the KD of the binding constructs provided herein is micromolar, nanomolar, picomolar, or femtomolar. In exemplary aspects, the KD of the binding constructs provided herein is about 10 -4 Up to 10 -6 , or 10 -7 Up to 10 -9 , or 10 -10 Up to 10 -12 , or 10 -13 Up to 10 -15 In the range of. In exemplary aspects, the KD of the binding construct provided herein is about 100nM or less. In certain aspects, the KD of the binding construct provided herein is about 20nM to about 100nM, about 25nM to about 95nM, about 30nM to about 90nM, about 35nM to about 85nM, about 40nM to about 80nM, about 45nM to about 75nM, about 50nM to about 70nM or about 55nM to about 65nM. In exemplary aspects, the KD of the binding construct is in the range of about 25nM to about 75nM. In exemplary aspects, the KD of the binding construct is in the range of about 50nM to about 60nM. In exemplary aspects, the KD of the binding construct is about 56nM.

[0055] Avidity is a measure of the overall strength of the antibody-antigen complex. It depends on three main parameters: the affinity of the binding construct to the epitope, the valence of the binding construct and Padova FIX, and the structural arrangement of the interacting parts. The higher the valence (number of antigen binding sites) of the binding construct, the higher the amount of antigen (Padova FIX) it can bind. In exemplary aspects, the binding construct has a stronger avidity for FIXp. In exemplary aspects, the binding construct is multivalent. In exemplary aspects, the binding construct is divalent.

[0056] structure

[0057] The binding constructs described herein can be engineered into one of numerous structures. In exemplary aspects, the binding constructs provided herein have the structure of an antibody or its antigen-binding fragment. In exemplary aspects, the binding constructs provided herein have a structure based on or derived from an antibody. In exemplary aspects, the binding constructs provided herein have the structure of a synthetic antibody mimetic, an engineered protein, or an aptamer, as described herein and in McEnaney et al., "Chemically Synthesized Molecules with the Targeting and Effector Functions of Antibodies," J. Am. Chem. Soc., 136(52):18034-18043 (2014); Binz and Pluckthun, "Engineered proteins as specific binding reagents," Curr Opin Biotechnol. 16(4):459-69 (2005); and Roque et al., "Antibodies and genetically engineered related molecules: production and purification." purification), "Biotechnol Prog.," 20(3):639-54 (2004).

[0058] Antibodies and antigen-binding fragments

[0059] In an exemplary embodiment, the binding construct is an antibody. The antibody can be any type of antibody known in the art, i.e., an immunoglobulin. In an exemplary embodiment, the antibody is an antibody belonging to a class or isotype IgA, IgD, IgE, IgG, or IgM. In an exemplary embodiment, the antibody described herein includes one or more α, δ, ε, γ, and / or μ heavy chains. In an exemplary embodiment, the antibody described herein includes one or more κ light chains. In exemplary aspects, the antibody is an IgG antibody and is optionally one of four human subclasses: IgG1, IgG2, IgG3, and IgG4.

[0060] Additionally, in some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

[0061] In some embodiments, the antibody is structurally similar to or derived from naturally occurring antibodies, such as antibodies isolated and / or purified from mammals, such as mice, rabbits, goats, horses, chickens, hamsters, humans, etc. In this regard, the antibody can be considered to be a mammalian antibody, such as a mouse antibody, a rabbit antibody, a goat antibody, a horse antibody, a chicken antibody, a hamster antibody, a human antibody, etc. In exemplary aspects, the antibody only includes the sequence of a mammalian antibody. Methods for making such antibodies are known in the art, some of which are further described in the chapters and sections entitled "Antibody Manufacturing Methods" herein. In exemplary aspects, the binding construct is a fully human antibody, or does not include the sequence of a non-human antibody.

[0062] In some embodiments, the antibody is a genetically engineered antibody and does not exist in nature. In exemplary embodiments, the antibody is a single-chain antibody, a humanized antibody, a chimeric antibody, a CDR-grafted antibody, a humaneered antibody, a bispecific antibody, a trispecific antibody, etc. Genetic engineering technology also provides the ability to prepare fully human antibodies from non-human sources.

[0063] In some aspects, the genetically engineered antibody is a single-chain antibody (SCA) specific for Padova FIX. Methods for preparing SCA are known in the art. See, for example, Davis et al., Nature Biotechnology 9: 165-169 (1991).

[0064] In some aspects, the antibody is a chimeric antibody. The term "chimeric antibody" is used herein to refer to an antibody containing a constant domain from one species and a variable domain from another species, or more generally, containing a continuous segment of an amino acid sequence from at least two species. In particular aspects, the chimeric antibody is bound to Padova FIX.

[0065] In some aspects, the antibody is a humanized antibody. When used with respect to an antibody, the term "humanization" refers to an antibody having at least CDR regions from a non-human source, which are engineered to have structures and immune functions more similar to real human antibodies rather than the original source antibodies. For example, humanization can involve transplanting CDRs from non-human antibodies, such as mouse antibodies, into human antibodies. Humanization can also involve selecting amino acid substitutions to make non-human sequences look more like human sequences.

[0066] The term "chimeric or humanized" used herein does not contain mutually exclusive intentions, but is meant to cover chimeric antibodies, humanized antibodies, and further humanized chimeric antibodies. Unless the context otherwise indicates, statements about chimeric antibodies (characteristics, uses, testing, etc. of chimeric antibodies) apply to humanized antibodies, and statements about humanized antibodies also apply to chimeric antibodies. Similarly, unless the context dictates otherwise, such statements should also be understood to apply to antibodies and antigen-binding fragments of such antibodies.

[0067] In some aspects, the antibody is humaneered TM ) antibodies. Humaneering technology is a proprietary method of KaloBios Pharmaceuticals, Inc. (South San Francisco, California) for converting non-human antibodies into engineered human antibodies. TM ) antibodies have high affinity and are highly similar to human germline antibody sequences. See, for example, Tomasevic et al., Growth Factors 32: 223-235 (2014).

[0068] In some aspects, the antibody is a CDR-grafted antibody specific for Padova FIX. Methods for preparing CDR-grafted antibodies are known in the art. See, for example, Lo, Benny, Antibody Engineering: Methods and Experimental Protocols ( Antibody Engineering:Methods andProtocols )》Volume 248 (2004), which is incorporated by reference in its entirety.

[0069] In exemplary embodiments, the antibody is engineered to be bispecific, trispecific or multispecific, and the antibody includes two or more different antigen binding regions. In some aspects, the antibody is a bispecific or trispecific antibody with specificity to Padova FIX. The method for preparing a bispecific or trispecific antibody is known in the art. See, for example, Marvin and Zhu, Acta Pharmacologica Sinica 26:649-658 (2005) and U.S. Patent 6,551,592. In exemplary aspects, the binding construct is a bispecific antigen binding construct with specificity to the first epitope of Padova FIX and the second epitope of Padova FIX. In exemplary embodiments, the antibody is a quadroma, a heterodimeric bispecific antibody, a bispecific antibody fusion, a bispecific antibody fragment, a bispecific T cell engager (bispecific T-cell engager, BiTE) or a multispecific antibody. In exemplary embodiments, the antibody is engineered to be divalent, trivalent or multivalent. See, e.g., Cuesta et al., “Multivalent antibodies: when design surpasses evolution,” Trends in Biotechnology 28, 355-362 (2010); Holliger et al., “Engineered antibody fragments and the rise of single domains,” Nature Biotechnology 23, 1126-1136 (2005); Chan et al., “Therapeutic antibodies for autoimmunity and inflammation,” Nat Rev Immunol 10, 301-S1 6 (2010); Byrne et al., “A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications.” In some embodiments, the antibody is in monomeric form, while in other embodiments, the antibody is coupled to one or more antibodies (eg, each of which recognizes the same epitope as the first antibody).Thus, in some aspects, the antibodies are in dimeric, polymeric, oligomeric or multimeric form.

[0070] In exemplary aspects, the binding construct is an antigen binding fragment of an antibody or includes an antigen binding fragment of an antibody.Antigen binding fragments (also referred to herein as "antigen binding moiety") can be any antigen binding fragment of an antibody described herein.The antigen binding fragment can be any part of an antibody having at least one antigen binding site, including but not limited to Fab, F(ab')2, monospecific or bispecific Fab2, trispecific Fab3, monovalent IgG, scFv, dsFv, scFv-Fc, bispecific bifunctional antibodies, trispecific trifunctional antibodies, mini antibodies, or fragments of IgNAR (e.g., V-NAR), or fragments of hcIgG (e.g., VhH), or double scFv, Fab expression library expressed fragments, etc. In exemplary aspects, the antigen binding fragment is a domain antibody, VhH domain, V-NAR domain, VH domain, VL domain, etc. However, the antibody fragments of the present disclosure are not limited to the antibody fragments of these exemplary types. In exemplary aspects, the binding construct includes a Fab fragment. In exemplary aspects, the binding construct comprises two Fab fragments. In exemplary aspects, the binding construct comprises two Fab fragments connected by a linker. In exemplary aspects, the binding construct comprises or is a minibody containing two Fab fragments. In exemplary aspects, the binding construct comprises or is a minibody containing two Fab fragments joined by a linker. Minibodies are known in the art. See, for example, Hu et al., Cancer Res 56:3055-3061 (1996). In exemplary aspects, the binding construct comprises or is a minibody containing two Fab fragments joined by a linker, optionally containing a minibody of an alkaline phosphatase domain.

[0071] Domain antibodies comprise the functional binding units of antibodies and may correspond to antibody heavy chains (V H ) or light chain (V L ). The molecular weight of a domain antibody can be about 13 kDa, or about one-tenth that of a complete antibody. Domain antibodies can be derived from complete antibodies, such as those described herein.

[0072] In some embodiments, the binding construct is a monomer or polymer, bispecific or trispecific, divalent or trivalent. In exemplary aspects, the binding construct provided herein is monospecific. In exemplary aspects, the binding construct provided herein is bispecific. In exemplary aspects, the binding construct provided herein is completely human. In exemplary aspects, the binding construct includes two Fab fragments and is divalent. In exemplary aspects, the binding construct is a homodimer formed by two Fab fragments of identical structure. Therefore, in exemplary aspects, the binding construct is divalent, but has monospecificity to Padova FIX. In exemplary aspects, the homodimer is dimerized by helix-turn-helix motif. In exemplary aspects, the binding construct is a homodimer formed by two Fab mini antibodies of identical structure. Therefore, in exemplary aspects, the binding construct is divalent, but has monospecificity to Padova FIX. In exemplary aspects, the homodimer formed by two Fab minibodies is dimerized via the alkaline phosphatase domain.

[0073] Antibody fragments containing antigen binding or idiotypes in antibody molecules can be produced by techniques known in the art. For example, such fragments include, but are not limited to, F(ab')2 fragments that can be produced by digesting antibody molecules with pepsin; Fab' fragments that can be produced by reducing the disulfide bridges of F(ab')2 fragments; and two Fab' fragments that can be produced by treating antibody molecules with papain and a reducing agent.

[0074] Conventional recombinant DNA techniques (see, e.g., Janeway et al., supra) can be used to produce single-chain variable region fragments (sFv) antibody fragments, which consist of truncated Fab fragments comprising an antibody heavy chain variable (V) domain connected to an antibody light chain V domain via a synthetic peptide. Similarly, disulfide-stabilized variable region fragments (dsFv) can be prepared by recombinant DNA techniques (see, e.g., Reiter et al., Protein Engineering, 7, 697-704 (1994)).

[0075] Recombinant antibody fragments, such as scFv, can also be engineered to assemble into stable multimeric oligomers with high binding affinity and specificity for different target antigens. Such bifunctional antibodies (dimers), trifunctional antibodies (trimers) or tetrafunctional antibodies (tetramers) are well known in the art, see, for example, Kortt et al., Biomol Eng. 2001, 18: 95-108, (2001) and Todorovska et al., J Immunol Methods 248: 47-66, (2001).

[0076] Bispecific antibodies (bscAbs) are molecules comprising two single-chain Fv fragments joined by a glycine-serine linker using recombinant methods. In an exemplary embodiment, the V light chains (V L ) and V heavy chain (V H ) domains were isolated using standard PCR methods. Then, in a two-step fusion PCR, the V L and V H cDNA is joined to form a single-chain fragment. Bispecific fusion proteins are prepared in a similar manner. Bispecific single-chain antibodies and bispecific fusion proteins are antibody substances included in the scope of the present disclosure. The teaching of exemplary bispecific antibodies is shown in U.S. Patent Application Publication No. 2005-0282233A1 and International Patent Application Publication No. WO 2005 / 087812, which are all incorporated herein by reference in their entirety.

[0077] In an exemplary embodiment, the binding construct is a biparatopic antibody or its biparatopic antigen binding fragment that can bind to two different non-overlapping epitopes on the same target antigen molecule. By simultaneously binding to the same cell surface target, the biparatopic antibody and its biparatopic antigen binding fragment may cause an enhancement in binding affinity, so that only cells that preferentially (strongly) bind to the expression target are bound, thereby fine-tuning the antibody selectivity. Existing data show that the biparatopic antibody or its biparatopic antigen binding fragment may even obtain new functions that the parent antibody (or antigen binding fragment) cannot achieve when used alone or in combination by simultaneously binding to two different epitopes on the same target molecule. In exemplary aspects, the binding construct provided herein is biparatopic for Padova FIX.

[0078] In exemplary embodiments, the Fab is engineered to be bispecific, trispecific or multispecific. In exemplary aspects, the Fab includes two or more different antigen binding regions. In some aspects, the Fab is a bispecific or trispecific antibody specific for Padova FIX and at least one other antigen. In exemplary aspects, the binding construct is a bispecific Fab specific for the first epitope of Padova FIX and the second epitope of Padova FIX. In exemplary embodiments, the Fab is engineered to be bivalent, trivalent or multivalent. In exemplary embodiments, the binding construct is a bivalent Fab fragment with monospecificity for Padova FIX. In some embodiments, the Fab is in monomeric form, and in other embodiments, the Fab is coupled to one or more Fabs (e.g., the same epitope of each of which recognizes the first Fab). Therefore, in some aspects, the Fab is dimerized, polymerized, oligomeric or multimeric. In exemplary aspects, the binding construct is a dimerized Fab fragment. In exemplary aspects, the binding construct is a fully human dimerization Fab fragment. In exemplary aspects, the binding construct is by helix-turn-helix motif dimerization. In exemplary embodiments, the Fab is engineered to be divalent, trivalent or multivalent. In exemplary embodiments, the binding construct is a dimerization bivalent Fab fragment with monospecificity to Padova FIX, wherein the binding construct is by helix-turn-helix motif dimerization.

[0079] In exemplary aspects, the binding construct, e.g., an antibody or antigen-binding fragment thereof, comprises the following amino acid sequence: SSYAIS (SEQ ID NO: 6); GIVPAFGTANYAQKFQG (SEQ ID NO: 7); SWGVISFAY (SEQ ID NO: 8); RASQDISSYLN (SEQ ID NO: 9); AASNLQS (SEQ ID NO: 10); and MQYDSLPFTF (SEQ ID NO: 11). In exemplary aspects, one or more amino acids are present between each of SEQ ID NOs: 6-11. In exemplary aspects, the binding construct, e.g., an antibody or antigen-binding fragment thereof, comprises the sequence of SEQ ID NO: 24 or SEQ ID NO: 25 or SEQ ID NOs: 24 and 25. In exemplary aspects, the binding construct, e.g., an antibody or antigen-binding fragment thereof, comprises the sequence of SEQ ID NO: 26 or SEQ ID NO: 27 or SEQ ID NOs: 26 and 27. In exemplary aspects, the amino acid sequence of the binding construct, such as an antibody or an antigen-binding fragment thereof, further comprises, for example, a sequence of a linker, an expression tag (e.g., a His tag, a FLAG tag, a myc tag, a fluorescent protein (e.g., a green fluorescent protein, a blue fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, a cyan fluorescent protein, an enhanced green fluorescent protein, etc.)). In exemplary aspects, the binding construct, such as an antibody or an antigen-binding fragment thereof, comprises a sequence of a His tag and / or a FLAG tag. In exemplary aspects, the FLAG tag comprises the sequence of SEQ ID NO: 12. In exemplary aspects, the His tag comprises the sequence of SEQ ID NO: 13. In exemplary aspects, the binding construct, such as an antibody or an antigen-binding fragment thereof, comprises a linker. In exemplary aspects, the binding construct, such as an antibody or its antigen-binding fragment, includes a synthetic double helix loop helix motif, such as Haylock et al., Int J. Oncol. 48 (2): 461-470 (2016) or Wang et al., Anal. Chem. 78: 997-1004 (2006) described in the motif. In exemplary aspects, the binding construct, such as an antibody or its antigen-binding fragment, includes a constant antibody domain. Such antibody domains are described in Hu et al., Cancer Res 56: 3055-3061 (1996) and McGregor et al., Mol Immuno 31: 219-226 (1994).In exemplary aspects, the binding construct, such as an antibody or an antigen-binding fragment thereof, comprises a bacterial alkaline phosphatase domain, such as the alkaline phosphatase domain described in Wang et al. (2006), supra. In exemplary aspects, the binding construct, such as an antibody or an antigen-binding fragment thereof, comprises the sequence of SEQ ID NO: 28 or SEQ ID NO: 27 or SEQ ID NO: 28 and 27.

[0080] Aptamer

[0081] In some embodiments, the binding construct is an antibody analog. In some aspects, the binding construct is an aptamer. Recent advances in the field of combinatorial science have identified short polymer sequences (such as oligonucleic acids or peptide molecules) with high affinity and specificity for a given target. For example, SELEX technology has been used to identify DNA and RNA aptamers with binding properties that can rival mammalian antibodies, and the field of immunology has produced and isolated antibodies or antibody fragments that bind to a large number of compounds, and phage display has been used to find new peptide sequences with extremely favorable binding properties. Based on the success of these molecular evolution techniques, molecules that bind to any target molecule can be produced with certainty. As in the following cases, the ring structure is usually involved in providing the desired binding properties: aptamers, which usually use hairpin loops produced by short regions without complementary base pairing; and antibodies of natural origin, which use the combination of annular hypervariable regions; and new phage display libraries using cyclic peptides, the results of which are displayed are improved compared to linear peptide phage display results. For more details about aptamers, see Gold, L., Singer, B., He, YY, Brody. E., "Aptamers As Therapeutic And Diagnostic Agents", J. Biotechnol. 74: 5-13 (2000). Related techniques for producing aptamers can be found in U.S. Pat. No. 6,699,843, which is incorporated herein by reference in its entirety.

[0082] Methods for producing antibodies or antigen-binding fragments

[0083] Suitable methods for preparing antibodies are known in the art. For example, standard hybridoma methods are described in the following books, such as Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), Immunobiology, 5th edition, Garland Publishing, New York, NY (New York, NY) (2001).

[0084] Briefly, polyclonal antibodies are prepared by immunizing animals with immunogens including polypeptides of the present invention and collecting antiserum from the immunized animals. Numerous animal species can be used to produce antiserum. In some aspects, the animal used to produce anti-antiserum is a non-human animal, including rabbits, mice, rats, hamsters, goats, sheep, pigs or horses. Since the blood volume of rabbits is relatively large, rabbits are a better choice for producing polyclonal antibodies. In an exemplary method for producing polyclonal antiserum with immunoreactivity to a selected Padova FIX epitope, 50 μg of Padova FIX antigen is emulsified in Freund's Complete Adjuvant for immunization of rabbits. At intervals of, for example, 21 days, 50 μg of epitopes are emulsified in Freund's incomplete adjuvant for additional immunization. After a certain time is given for antibody production, polyclonal antiserum can be obtained simply by bleeding the animal and preparing serum samples from whole blood.

[0085] The monoclonal antibodies used in the disclosed methods can be prepared using any technique capable of producing antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma techniques originally described by Koehler and Milstein (Nature 256: 495-497, 1975); human B cell hybridoma techniques (Kosbor et al., Immunology Today 4: 72, 1983; Cote et al., Proc Natl Acad Sci 80: 2026-2030, 1983); and EBV hybridoma techniques (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New York, NY, pp. 77-96, (1985).

[0086] Briefly, in an exemplary embodiment, to produce monoclonal antibodies, mice are regularly injected with recombinant Padova FIX (e.g., 10-20 μg emulsified in Freund's complete adjuvant), which is produced for recombinant Padova FIX. Mice are given PBS containing Padova FIX for final pre-fusion boosting immunity, and mice are killed after four days, and their spleens are taken out. Spleen is put into 10 ml serum-free RPMI 1640, and by grinding spleen between the frosted ends of two glass microscope slides immersed in serum-free RPMI 1640, a single cell suspension is formed, and the serum-free RPMI 1640 is supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units / ml penicillin and 100 μg / ml streptomycin (RPMI) (Gibco, Canada). The cell suspension was filtered through a sterile 70 mesh Nitex cell strainer (Becton Dickinson, Parsippany, NJ) and washed twice by centrifugation at 200 g for 5 minutes and resuspending the pellet in 20 ml serum-free RPMI. Splenocytes obtained from three untreated Balb / c mice were prepared in a similar manner and used as controls. NS-1 myeloma cells were maintained in logarithmic phase for three days before fusion in RPMI containing 11% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah), centrifuged at 200 g for 5 minutes, and the pellet was washed twice.

[0087] Splenocytes (1×10 8 ) and 2.0×10 7 NS-1 cells were combined and centrifuged, and the supernatant was aspirated. The cell mass was removed by tapping the test tube, and 1 ml of 37°C PEG 1500 (50% in 75 mM Hepes, pH 8.0) (Boehringer Mannheim) was added over a 1 minute period under stirring, followed by 7 ml of serum-free RPMI over a 7 minute period. 8 ml of RPMI was added and the cells were centrifuged at 200 g for 10 minutes. After discarding the supernatant, the pellet was resuspended in 200 ml of 15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco), 25 units / ml IL-6 (Boehringer Mannheim) and 1.5×10 6 The cells were then plated in RPMI at 100 splenocytes / mL and plated in 10 Corning flat-bottom 96-well tissue culture plates (Corning, Corning NY).

[0088] On the 2nd, 4th and 6th day after fusion, 100 μl of culture medium was removed from each well of the fusion plate and replaced with fresh culture medium. On the 8th day, the fusion was screened by ELISA to test the presence of mouse IgG bound to Padova FIX. The method is as follows: at 37°C, 100 ng of EGFR diluted in 25 mM Tris (pH 7.5) per well was coated with Immulon 4 plates (Dynatech, Cambridge, Mass.), and kept for 2 hours. Aspirate the coating solution and add 200 microliters / well blocking solution (0.5% fish skin gelatin (Sigma) diluted in CMF-PBS), and cultivate for 30 minutes at 37°C. Wash the plate three times with PBS (PBST) containing 0.05% Tween 20, and add 50 μl of culture supernatant. After incubation at 37°C for 30 minutes and washing as described above, 50 μl of horseradish peroxidase-conjugated goat anti-mouse IgG (fc) (Jackson ImmunoResearch, West Grove, Pa.) diluted 1:3500 in PBST was added. The plate was incubated as described above, washed four times with PBST and 100 μl of substrate was added, which consisted of 100 mM citrate (pH 4.5) containing 1 mg / ml o-phenylenediamine (Sigma) and 0.1 μl / ml 30% H2O2. After 5 minutes, 50 μl of 15% H2SO4 was added to stop the color reaction. A was read on a plate reader (Dynatech). 490 .

[0089] Selected fusion wells were cloned twice by dilution in 96-well plates and the number of colonies per well was scored visually after 5 days. Monoclonal antibodies produced by hybridomas were isotyped using the Isostrip system (Boehringer Mannheim, Indianapolis, Ind.).

[0090] When hybridoma technology is used, myeloma cell lines can be used. Such cell lines suitable for use in fusion procedures for producing hybridomas preferably do not produce antibodies, have high fusion efficiency, and have enzyme defects that prevent them from growing in certain selective media that only support the growth of the desired fused cells (hybridomas). For example, when the immunized animal is a mouse, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7 and S194 / 15XX0Bul can be used; for rats, R210.RCY3, Y3-Ag1.2.3, IR983F and 4B210 can be used; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 can all be used in combination with cell fusions.

[0091] Depending on the host species, various adjuvants can be used to increase the immune response. Such adjuvants include, but are not limited to, Freund's adjuvant; mineral gels such as aluminum hydroxide; and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Bacilli Calmette-Guerin (BCG) and Corynebacterium parvum are potentially useful adjuvants for human use.

[0092] As an alternative, other methods known in the art, such as the EBV hybridoma method (Haskard and Archer, J. Immunol. Methods, 74 (2), 361-67 (1984), and Roder et al., Methods Enzymol., 121, 140-67 (1986)), and phage vector expression systems (see, e.g., Huse et al., Science, 246, 1275-81 (1989)). In addition, methods for producing antibodies in non-human animals are described in, e.g., U.S. Patents 5,545,806, 5,569,825, and 5,714,352, and U.S. Patent Application Publication No. 2002 / 0197266A1.

[0093] As disclosed in Orlandi et al. (Proceedings of the National Academy of Sciences of the United States of America 86:3833-3837; 1989) and Winter and Milstein C (Nature 349:293-299, 1991), antibodies can also be produced by inducing lymphocyte populations in vivo or by screening recombinant immunoglobulin libraries or panels of highly specific binding agents. If the complete sequence of the antibody or antigen-binding fragment is known, methods for making recombinant proteins can be used. See, for example, "Protein production and purification", Nat Methods 5(2):135-146 (2008).

[0094] Phage display can also be used to produce antibodies of the present disclosure. In this regard, standard molecular biology and recombinant DNA techniques can be used to produce phage libraries encoding antigen-binding variable (V) domains of antibodies (see, for example, Sambrook et al. (ed.), "Molecular Cloning, A Laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory Press, New York (2001)). Select phages that specifically bind to the desired antigen encoding the variable region with the desired specificity, and reconstruct the complete or partial antibody to include the selected variable domains. The nucleic acid sequence encoding the reconstructed antibody is introduced into a suitable cell line, such as in the myeloma cells for producing hybridomas, so that the cells secrete antibodies with monoclonal antibody characteristics (see, for example, Janeway et al., aforementioned; Huse et al., aforementioned and U.S. Patent 6,265,150). Related methods are also described in U.S. Patent No. 5,403,484; U.S. Patent No. 5,571,698; U.S. Patent No. 5,837,500; U.S. Patent No. 5,702,892. These techniques are described in U.S. Patent No. 5,780,279; U.S. Patent No. 5,821,047; U.S. Patent No. 5,824,520; U.S. Patent No. 5,855,885; U.S. Patent No. 5,858,657; U.S. Patent No. 5,871,907; U.S. Patent No. 5,969,108; U.S. Patent No. 6,057,098; and U.S. Patent No. 6,225,447.

[0095] Antibodies can be produced by transgenic mice with specific heavy and light chain immunoglobulin genes.Such methods are known in the art and are described, for example, in the aforementioned U.S. Patent Nos. 5,545,806 and 5,569,825 and Janeway et al.

[0096] Methods for producing humanized antibodies are well known in the art and are described in detail in, for example, Janeway et al., supra; U.S. Pat. Nos. 5,225,539, 5,585,089, and 5,693,761; European Patent No. 0239400B1; and British Patent No. 2188638. Humanized antibodies can also be produced using antibody resurfacing techniques described in U.S. Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol., 235, 959-973 (1994). Preferred chimeric or humanized antibodies have human constant regions, while the variable region or at least one CDR of the antibody is derived from a non-human species. Methods for humanizing non-human antibodies are well known in the art. (See U.S. Patent Nos. 5,585,089 and 5,693,762.) In general, a humanized antibody has one or more amino acid residues introduced into its framework region from a non-human source. Humanization can be carried out, for example, using the methods described in Jones et al. (Nature 321:522-525, 1986), Riechmann et al. (Nature 332:323-327, 1988) and Verhoeyen et al. (Science 239:1534-1536, 1988), by replacing at least a portion of a rodent complementary determining region (CDR) with the corresponding region of an adult antibody. A variety of techniques for preparing engineered antibodies are described in, for example, Owens and Young, Journal of Immunological Methods, 168:149-165 (1994). Then, other changes can be introduced into the antibody framework to adjust affinity or immunogenicity.

[0097] Techniques developed for producing "chimeric antibodies" can be used, i.e., splicing mouse antibody genes to human antibody genes to obtain molecules with appropriate antigenic specificity and biological activity (Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Neuberger et al., Nature 312:604-608 (1984); and Takeda et al., Nature 314:452-454 (1985)). Alternatively, the techniques described for producing single-chain antibodies (U.S. Pat. No. 4,946,778) can be appropriately adjusted to produce EGFR or HSP90-specific single-chain antibodies.

[0098] Similarly, CDRs are separated using techniques known in the art to produce compositions comprising CDRs. Complementarity determining regions are characterized by six polypeptide loops, three loops each in the heavy or light chain variable region. The amino acid positions in CDRs are defined according to Kabat et al., "Sequences of Proteins of Immunological Interest", U.S. Department of Health and Human Services, (1983) (incorporated herein by reference). For example, the hypervariable regions of human antibodies are roughly limited to residues 28 to 35, 49-59 and residues 92-103 in the heavy and light chain variable regions (Janeway and Travers, "Immunobiology", 2nd edition, Garland Publishing, New York (1996)). Murine CDRs are also roughly at these amino acid residues. In the art, it is understood that CDR regions can be found within several amino acids of these predicted residues described above. Immunoglobulin variable regions are also composed of four "framework" regions (FR1-4) surrounding CDRs. The sequences of the framework regions of different light or heavy chains are highly conserved within a species and are also conserved between human and mouse sequences.

[0099] Compositions comprising one, two and / or three CDRs of a monoclonal antibody heavy chain variable region or light chain variable region are produced. The techniques for cloning and expressing nucleotide and polypeptide sequences in the art are well established (see, e.g., Sambrook et al., Molecular Cloning Laboratory Manual, 2nd edition, Cold Spring Harbor, New York (1989)). The amplified CDR sequences are connected to appropriate expression vectors. The vectors comprising one, two, three, four, five and / or six cloned CDRs optionally additionally contain polypeptide coding regions connected to the CDRs.

[0100] The framework regions (FR) of murine antibodies are humanized by substituting compatible human framework regions selected from a large database of human antibody variable sequences, containing more than 1,200 human V H Sequence and more than a thousand V L Sequences. The database of antibody sequences used for comparison was downloaded from Andrew CR Martin's KabatMan web page (http: / / www.rubic.rdg.ac.uk / abs / ). The Kabat method for identifying CDRs provides a means of delineating the approximate CDRs and framework regions from any human antibody and comparing the similarity with murine antibody sequences to determine the CDRs and FRs. The best matching human V H and V L Sequences were selected based on high overall framework match, similar CDR lengths, and classical and VH / V L The mouse CDRs are selected with minimal mismatch of contact residues. The human framework regions that are most similar to the mouse sequences are inserted between the mouse CDRs. Alternatively, the mouse framework regions can be modified by amino acid substitutions of all or part of the native framework regions that more closely resemble human antibody framework regions.

[0101] In addition, another useful technique for producing antibodies for use in the present disclosure can be a technique using a rational design approach. The goal of rational design is to make structural analogs (agonists, antagonists, inhibitors, peptide mimetics, binding partners, etc.) of biologically active polypeptides or compounds that interact with them. In one approach, a three-dimensional structure of an antibody or its epitope binding fragment is generated. This can be achieved by x-ray crystallography, computer modeling, or by a combination of the two methods. An alternative approach, "alanine scanning," involves randomly replacing residues throughout the molecule with alanine and determining the resulting effects on function.

[0102] Chemically constructed bispecific antibodies can be prepared by chemically cross-linking heterologous Fab or F(ab')2 fragments with chemical reagents such as the heterobifunctional reagent succinimidyl-3-(2-pyridyldithiol)-propionate (SPDP; Pierce Chemicals, Rockford, IL). Fab and F(ab')2 fragments can be obtained from intact antibodies by digestion with papain or pepsin, respectively (Karpovsky et al., J. Exp. Med. 160:1686-701 (1984); Titus et al., J. Immunol. 138:4018-22 (1987)).

[0103] Regardless of how the antibody is produced, methods for testing the ability of the antibody to bind to an epitope of Padova FIX are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, surface plasmon resonance, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266).

[0104] Peptides

[0105] Also provided herein is a polypeptide comprising an amino acid sequence comprising each of SEQ ID NOs: 6-11. The polypeptide binds to FIXp and does not bind to WT FIX, for example, the polypeptide binds only to FIXp, even in the presence of WT FIX, optionally, even in the presence of other coagulation factors such as factor II and factor X. In exemplary aspects, the polypeptide binds to FIXp in a sample containing at least 5%, at least 10%, or at least 20% human plasma. In exemplary aspects, one or more amino acids are present between each of SEQ ID NOs: 6-11. In exemplary aspects, the polypeptide comprises a sequence of SEQ ID NOs: 24 or 25 or a sequence of SEQ ID NOs: 24 and 25. In exemplary aspects, the polypeptide comprises a sequence of SEQ ID NOs: 26 or 27 or a sequence of SEQ ID NOs: 26 and 27. In exemplary aspects, the amino acid sequence of the polypeptide further includes, for example, a linker, an expression tag (e.g., a His tag, a FLAG tag, a myc tag, a fluorescent protein (e.g., a green fluorescent protein, a blue fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, a cyan fluorescent protein, an enhanced green fluorescent protein, etc.). In exemplary aspects, the polypeptide includes the sequence of a His tag and / or a FLAG tag. In exemplary aspects, the FLAG tag includes the sequence of SEQ ID NO: 12. In exemplary aspects, the His tag includes the sequence of SEQ ID NO: NO:13 sequence. In exemplary aspects, the polypeptide includes a linker. In exemplary aspects, the polypeptide includes a synthetic double helix loop helix motif sequence, such as Haylock et al., International Journal of Oncology 48 (2): 461-470 (2016) or Wang et al., Anal. Chem. 78: 997-1004 (2006) described in the sequence. In exemplary aspects, the polypeptide includes the sequence of a constant antibody domain. Such antibody domains are described in Hu et al., Cancer Research 56: 3055-3061 (1996) and McGregor et al., Molecular Immunology 31: 219-226 (1994). In exemplary aspects, the polypeptide includes a bacterial alkaline phosphatase domain, such as Wang et al. (2006), the alkaline phosphatase domain described in the foregoing. In exemplary aspects, the polypeptide includes SEQ ID NO:28 or SEQ ID The sequence of SEQ ID NO: 27 or the sequences of SEQ ID NO: 28 and 27.

[0106] Conjugate

[0107] The binding constructs described herein can be modified, for example, by glycosylation, amidation, carboxylation or phosphorylation, or by producing acid addition salts, amides, esters (particularly C-terminal esters) and N-acyl derivatives. The binding constructs can also be modified to produce derivatives by forming covalent or non-covalent complexes (i.e., conjugates) with other moieties. Covalently bound complexes can be prepared by connecting chemical moieties to the side chains of the amino acids constituting the binding construct, or to functional groups at the N-terminal or C-terminal ends.

[0108] In some embodiments, the binding constructs of the present disclosure are attached to, connected to, joined to, or coupled to a second portion (e.g., a heterologous portion) and the resulting product is a conjugate. Therefore, provided herein are conjugates comprising a binding construct described herein (covalently or non-covalently) coupled to a heterologous portion. As used herein, the term "heterologous portion" refers to any molecule (chemical or biochemical, naturally occurring or non-encoded molecule) different from the binding constructs of the present invention. Exemplary heterologous portions include, but are not limited to, polymers, carbohydrates, lipids, nucleic acids, oligonucleotides, DNA or RNA, amino acids, peptides, polypeptides, proteins, therapeutic agents (e.g., cytotoxic agents, cytokines), diagnostic agents, or detection agents.

[0109] In some embodiments, the binding construct is chemically modified with various heterologous moieties. In some embodiments, the chemical modification imparts other desired features as discussed herein. In some respects, chemical modification is in many different forms, such as heterologous peptides, polysaccharides, lipids, radioisotopes, non-standard amino acid residues and nucleic acids, metal chelators and various cytotoxic agents.

[0110] In some embodiments, the binding construct is fused to a heterologous peptide to impart various properties, such as increased solubility and / or stability and / or half-life, resistance to proteolytic cleavage, clearance modulation, targeting to specific cells or tissue types. In some embodiments, the binding construct is linked to the Fc domain of an IgG or other immunoglobulin. In some embodiments, the binding construct is fused to alkaline phosphatase (AP). Methods for preparing Fc or AP fusion constructs are found in International Patent Publication No. WO 02 / 060950. By fusing the binding construct to a protein domain having specific properties (e.g., half-life, bioavailability), these properties can be imparted to the binding constructs of the present invention.

[0111] The binding construct can be coupled to a detection agent (e.g., a detectable label or a reporter group), including but not limited to a radiolabel, a fluorescent label, an enzyme (e.g., an enzyme that catalyzes a heat or fluorescence reaction), a substrate, a solid matrix, or a carrier (e.g., biotin or avidin). In exemplary aspects, the fluorescent label includes a rhodamine dye, a fluorescein dye, and / or a cyanine dye. In exemplary embodiments, the fluorescent label includes a group of dyes, such as a rhodamine dye, TAMRA, and a fluorescein dye FAM. In another embodiment, the fluorescent label includes a group of fluorescent dyes formed by selecting two or more dyes from the group: Oregon Green 488, Flitorescein-EX, fluorescein isothiocyanate, rhodamine Red-X, Lissamine Rhodamine B, Calcein, fluorescein, rhodamine, one or more BODIPY dyes, Texas Red, Oregon Green 514, and one or more Alexa Fhiors. Representative BODIPY dyes include BODIPY FL, BODIPY R6G, BODIPYTMR, BODIPY 581 / 591, BODIPY TR, BODIPY 630 / 650, and BODIPY 650 / 665. Representative Alexa Fluors include Alexa Fluor 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750, and 790. In exemplary aspects, fluorescent labels include one or more of Oregon Green 488, fluorescein-EX, FITC, rhodamine Red-X, lissamine rhodamine B, calcein, fluorescein, rhodamine, BODIPYS and Texas Red, such as those disclosed in Molecular Probes Handbook, 11th edition (2010). In exemplary aspects, detectable labels are selected from radioisotopes, chromophores, fluorophores, fluorescent dyes, enzymes (e.g., horseradish peroxidase), linker molecules, or other moieties or compounds that emit detectable signals (e.g., radioactivity, fluorescence, colored signals) or emit detectable signals after the label is exposed to its substrate. A variety of detectable label / substrate pairs (e.g., horseradish peroxidase / diaminobenzidine, biotin / streptavidin, luciferase / fluorescein), methods for labeling antibodies, and methods for detecting antigens using labeled secondary antibodies are well known in the art.See, e.g., Harlow and Lane, eds., (Using Antibodies: A Laboratory Manual (1999) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0112] In some embodiments, the binding construct is directly joined to the heterologous part in the absence of a linker. In alternative aspects, the binding construct is indirectly connected to the heterologous part through one or more linkers. Whether directly joined together or indirectly joined together through a linker, the binding construct can be connected by a covalent bond (e.g., peptide, ester, amide or sulfhydryl bond) or a non-covalent bond (e.g., by hydrophobic interaction, hydrogen bond, van der Waals bond, electrostatic or ionic interaction) or a combination thereof. The binding construct of the present invention and the heterologous part can be connected by any means known in the art, including but not limited to any linker connection of the present invention.

[0113] The specific residues of the binding constructs described herein represent exemplary positions to which a heterologous moiety can be attached. For example, Cys, His, Lys and the N-terminal residue, Arg, Tyr, Asp, Glu, Ser, Thr, Pro represent positions to which a heterologous moiety can be attached. In some aspects, the residues (or portions thereof) are activated with one or more reagents and / or chemicals prior to attachment of the heterologous moiety.

[0114] Derivatization with bifunctional agents can be used to crosslink the binding construct to a water-insoluble carrier matrix. Such derivatization can also provide a linker capable of connecting adjacent binding elements in a binding construct, or connecting a binding element to a heterologous peptide, such as an Fc fragment. Common crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane; glutaraldehyde; N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate); and bifunctional maleimides, such as bis-N-maleimide-1,8-octane. Derivatization agents such as methyl-3-[(p-azidophenyl)dithio] propionimidate produce photoactivated intermediates capable of forming crosslinks in the presence of light. Alternatively, for protein immobilization, water-insoluble reactive matrices such as cyanogen bromide-activated carbohydrates and reactive substrates described in U.S. Pat. Nos. 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537, and 4,330,440, which are incorporated herein by reference, are employed.

[0115] In general, chemical derivatization can be carried out under any conditions suitable for reacting protein with a heterologous part, and the heterologous part is, for example, an activated polymer molecule. The method for preparing a chemical derivative of a polypeptide generally will include the following steps: (a) reacting the binding construct with a heterologous part, for example, an activated polymer molecule (such as a reactive ester or aldehyde derivative of a polymer molecule) under conditions where the binding construct is attached to one or more polymer molecules, and (b) obtaining a reaction product. Ideal reaction conditions will be determined based on known parameters and desired results. For example, the larger the ratio of polymer molecule: protein, the larger the amount of the attached polymer molecule. In some embodiments, the compound can have a single polymer molecule portion at the amino terminal. (See, for example, U.S. Patent No. 5,234,784).

[0116] The derivatized binding constructs disclosed herein may have additional activities, ie, enhanced or diminished biological activity, or other characteristics, such as increased or decreased half-life, compared to the underivatized molecule.

[0117] Conjugate: Fc fusion

[0118] For replaceable parts, such as the Fc region of human IgG, fusion can be directly fused to the binding construct of the present invention or fused by inserting sequences. For example, the human IgG hinge, CH2 and CH3 regions can be fused at the N-terminal or C-terminal end of the binding construct to attach the Fc region. The Fc-fusion construct thus obtained can be purified by protein A affinity column (Pierce, Rockford, Illinois). The half-life displayed in vivo by peptides and proteins fused to the Fc region can be substantially greater than that of unfused counterparts. Fusion with the Fc region can dimerize / multimerize the fusion polypeptide. The Fc region can be a naturally occurring Fc region, or an Fc region modified to obtain excellent characteristics, such as therapeutic or diagnostic quality, circulation time, and reduced aggregation. As described above, in some embodiments, the binding construct is coupled to an immunoglobulin or a portion thereof (e.g., variable region, CDR or Fc region), such as fusion. Known immunoglobulin (Ig) types include IgG, IgA, IgE, IgD or IgM. The Fc region is the C-terminal region of the Ig heavy chain, which is responsible for binding to Fc receptors to achieve activities such as recycling (extending half-life), antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC).

[0119] For example, according to some definitions, the human IgG heavy chain Fc region extends from Cys226 of the heavy chain to the C-terminus. The "hinge region" generally extends from Glu216 of human IgG1 to Pro230 (by aligning the cysteine ​​involved in the cysteine ​​bonding, the hinge region of other IgG isotypes can be compared with the IgG1 sequence). The Fc region of IgG contains two constant domains, CH2 and CH3. The CH2 domain of the human IgG Fc region generally extends from amino acid 231 to amino acid 341. The CH3 domain of the human IgG Fc region generally extends from amino acid 342 to 447. The amino acid numbering of the immunoglobulin or immunoglobulin fragment, or region mentioned is based on Kabat et al., 1991, Sequences of Immune-Related Proteins, U.S. Department of Public Health, Bethesda, Md. In related embodiments, the Fc region may include, in addition to CH1, one or more native or modified constant regions from an immunoglobulin heavy chain, such as the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4 regions of IgE.

[0120] Suitable heterologous parts include parts containing FcRn binding sites in immunoglobulin sequences. The rescue receptor FcRn is responsible for recycling immunoglobulins and returning them to the blood circulation. The region bound to the FcRn receptor in the IgG Fc part has been described based on X-ray crystallography (Burmeister et al., 1994, "Nature", 372:379). The main contact area of ​​Fc and FcRn is close to the junction of the CH2 domain and the CH3 domain. Fc-FcRn contacts are all within a single Ig heavy chain. The main contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311 and 314 of the CH2 domain, and amino acid residues 385-387, 428 and 433-436 of the CH3 domain.

[0121] The Fc region of the immunoglobulin can be amino acid modified. These variable Fc regions include at least one amino acid modification in the CH3 domain (residues 342-447) of the Fc region and / or at least one amino acid modification in the CH2 domain (residues 231-341) of the Fc region. Mutations that are believed to increase affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al., 2001, J. Biol. Chem. 276: 6591). Other mutations can reduce the binding of the Fc region to FcγRI, FcγRIIA, FcγRIIB, and / or FcγRIIIA without significantly reducing affinity for FcRn. For example, substitution of Asn at position 297 of the Fc region with Ala or another amino acid will remove a highly conserved N-glycosylation site and can reduce the immunogenicity of the Fc region, while extending its half-life and reducing binding to FcγRs (Routledge et al., 1995, Transplantation 60:847; Friend et al., 1999, Transplantation 68:1632; Shields et al., 1995, Journal of Biochemistry 276:6591). Amino acid modifications at positions 233-236 of IgG1 can reduce binding to FcγRs (Ward and Ghetie, 1995, Therapeutic Immunology 2:77; and Armour et al., 1999, Eur. J. Immunol. 29:2613). Some exemplary amino acid substitutions are described in US Pat. Nos. 7,355,008 and 7,381,408, each of which is incorporated herein by reference in its entirety.

[0122] Heterologous moieties: polymers, carbohydrates, and lipids

[0123] In exemplary embodiments, the heterologous moiety is a polymer. The polymer may be branched or unbranched. The polymer may have any molecular weight. In some embodiments, the polymer has an average molecular weight between about 2kDa to about 100kDa (the term "about" indicates that when preparing a water-soluble polymer, some molecules will be higher or lower than the specified molecular weight). In some aspects, the average molecular weight of the polymer is between about 5kDa and about 50kDa, between about 12kDa to about 40kDa, or between about 20kDa to about 35kDa.

[0124] In some embodiments, the polymer is modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, whereby the degree of polymerization can be controlled. In some embodiments, the polymer is water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. In some embodiments, when, for example, the composition is used for therapeutic purposes, the polymer is pharmaceutically acceptable. In addition, in some aspects, the polymer is a polymer mixture, such as a copolymer, a block copolymer.

[0125] In some embodiments, the polymer is selected from the group consisting of polyamides; polycarbonates; polyalkylenes and their derivatives, including polyalkylene glycols, polyoxyalkylenes, polyalkylene terephthalates; polymers of acrylates and methacrylates, including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate); polyvinyl polymers , including polyvinyl alcohol, polyvinyl ether, polyvinyl ester, polyvinyl halide, poly(vinyl acetate) and polyvinyl pyrrolidone; polyglycolide; polysiloxane; polyurethane and its copolymers; cellulose, including alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate and cellulose sulfate sodium salt; polypropylene; polyethylene, including poly(ethylene glycol), poly(ethylene oxide) and poly(ethylene terephthalate); and polystyrene.

[0126] In some aspects, the polymer is a biodegradable polymer, including synthetic biodegradable polymers (e.g., polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone)), and natural biodegradable polymers (e.g., alginate and other polysaccharides, including dextran and cellulose, collagen, its chemical derivatives (substitution, addition of chemical groups such as alkyl, alkylene, hydroxylation, oxidation, and other modifications commonly performed by those skilled in the art), albumin, and other hydrophilic proteins (e.g., zein and other prolamins and hydrophobic proteins), and any copolymers or mixtures thereof. Generally, these materials degrade in vivo by enzymatic hydrolysis or exposure to water, by surface or bulk erosion.

[0127] In some aspects, the polymer is a bioadhesive polymer such as a bioerodible hydrogel described in HS Sawhney, CP Pathak, and JA Hubbell, Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acid, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[0128] In some embodiments, the polymer is a water-soluble polymer or a hydrophilic polymer. Suitable water-soluble polymers are known in the art and include, for example, polyvinyl pyrrolidone, hydroxypropyl cellulose (HPC; Klucel), hydroxypropyl methylcellulose (HPMC; Methocel), nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose, hydroxypropyl pentylcellulose, methylcellulose, ethylcellulose (Ethocel), hydroxyethylcellulose, various alkyl celluloses and hydroxyalkyl celluloses, various cellulose ethers, cellulose acetate, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, vinyl acetate / butenoic acid copolymers, polyhydroxyalkyl methacrylates, hydroxymethyl methacrylate, methacrylic acid copolymers, polymethacrylic acid, polymethyl methacrylate, maleic anhydride / methyl vinyl ether copolymers, polyvinyl alcohol, sodium and calcium polyacrylates, polyacrylic acid, acidic carboxyl polymers, carboxypolymethylene, carboxyvinyl polymers, polyoxyethylene polyoxypropylene copolymers, polymethylvinyl ether-co-maleic anhydride, carboxymethylamide, potassium methacrylate divinylbenzene copolymers, polyoxyethylene glycol, polyethylene oxide, and derivatives, salts, and combinations thereof. In some aspects, water-soluble polymers or mixtures thereof include, but are not limited to, N-linked or O-linked carbohydrates, sugars, phosphates, carbohydrates; sugars; phosphates; polyethylene glycol (PEG) (including forms of PEG used to derivatize proteins, including mono (C1-C10) alkoxy polyethylene glycol or aryloxy polyethylene glycol); monomethoxy polyethylene glycol; dextran (such as, for example, low molecular weight dextran of about 6 kD), cellulose; cellulose; other carbohydrate polymers, poly (N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polyoxypropylene / ethylene oxide copolymer, polyoxyethylated polyols (such as glycerol) and polyvinyl alcohol. The present invention also encompasses bifunctional cross-linking molecules that can be used to prepare covalently attached polymers.

[0129] Particularly preferred water-soluble polymers used herein are polyethylene glycol (PEG). As used herein, polyethylene glycol is intended to encompass any of the PEG forms that can be used to derivatize other proteins, such as mono (C1-C10) alkoxy polyethylene glycol or aryloxy polyethylene glycol. PEG is a linear or branched neutral polyether, obtained with a wide range of molecular weights, and soluble in water and most organic solvents. When present in water, PEG effectively excludes other polymers or peptides primarily through its high dynamic chain mobility and hydrophobicity, thereby generating a water shell or hydrated sphere when attached to other proteins or polymer surfaces. PEG is nontoxic, non-immunogenic, and is approved for internal consumption by the Food and Drug Administration.

[0130] The method for preparing a PEGylated compound may comprise the following steps: (a) reacting the compound with polyethylene glycol (e.g., a reactive ester or aldehyde derivative of PEG) under conditions such that the compound is attached to one or more PEG groups, and (b) obtaining a reaction product. In general, the ideal reaction conditions for the acylation reaction will be determined based on known parameters and the desired results. For example, the greater the ratio of PEG:protein, the greater the percentage of the polyPEGylated product. In some embodiments, the binding construct will have a single PEG moiety at the N-terminus. See U.S. Patent No. 8,234,784, incorporated herein by reference.

[0131] In some embodiments, the heterologous moiety is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (e.g., starch, amylase, pullulan, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan).

[0132] In some embodiments, the heterologous moiety is a lipid. In some embodiments, the lipid is a fatty acid, an eicosanoid, a prostaglandin, a leukotriene, a thromboxane, an N-acylethanolamine, a glycerolipid (e.g., a monosubstituted glycerol, a disubstituted glycerol, a trisubstituted glycerol), a glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), a sphingolipid (e.g., sphingosine, ceramide), a sterol lipid (e.g., a steroid, cholesterol), a prenol lipid, a glycolipid or a polyketide, an oil, a wax, cholesterol, a sterol, a fat-soluble vitamin, a monoglyceride, a diglyceride, a triglyceride, a phospholipid.

[0133] Heterologous moiety: Therapeutic agent

[0134] In some embodiments, the heterologous portion is a therapeutic agent. The therapeutic agent can be any of the therapeutic agents known in the art. Examples of therapeutic agents encompassed herein include, but are not limited to, natural enzymes, proteins derived from natural sources, recombinant proteins, natural peptides, synthetic peptides, cyclic peptides, antibodies, receptor agonists, cytotoxic agents, immunoglobulins, beta-adrenergic blockers, calcium channel blockers, coronary vasodilators, cardiac glycosides, antiarrhythmics, cardiac sympathomimetics, angiotensin converting enzyme (ACE) inhibitors, diuretics, cardiotonic agents, cholesterol and triglyceride lowering agents, bile acid sequestrants, fibrates, 3-hydroxy-3-methylglutaryl (HMG) coenzyme A reductase inhibitors, nicotinic acid derivatives, antiadrenergics, alpha-adrenergic blockers, centrally acting antirenal Adrenergic agents, vasodilators, potassium sparing agents, thiazides and related agents, angiotensin II receptor antagonists, peripheral vasodilators, antiandrogens, estrogens, antibiotics, retinoids, insulin and analogs, alpha-glucosidase inhibitors, biguanides, meglitinides, sulfonylureas, thiazolidinediones, androgens, progestins, bone metabolism regulators, anterior pituitary hormones, hypothalamic hormones, posterior pituitary hormones, gonadotropins, gonadotropin-releasing hormone antagonists, ovulation induction drugs, selective estrogen receptor modulators, antithyroid agents, thyroid hormones, bulking agents forming agent), laxatives, antiperistaltic drugs, flora regulators, intestinal adsorbents, intestinal anti-infectives, anti-anoretics, anti-malnutrition drugs (anticachexic), anti-bulimics, appetite suppressants, anti-obesity agents, antacids, upper gastrointestinal agents, anticholinergics, aminosalicylic acid derivatives, biological response modifiers, corticosteroids, antispasmodics, 5-HT4 partial agonists, antihistamines, cannabinoids, dopamine antagonists, serotonin antagonists, cytoprotectants, histamine H2 receptor antagonists, mucosal protectants, proton pump inhibitors, Helicobacter pylori (H.pyl ori) eradication therapy, erythropoiesis stimulating agents, hematopoietic agents, anemic agents, heparin, antifibrinolytics, hemostatics, coagulation factors, adenosine diphosphate inhibitors, glycoprotein receptor inhibitors, fibrinogen-platelet binding inhibitors, thromboxane-A2 inhibitors, plasminogen activators, antithrombotic agents, glucocorticoids, mineralocorticoids, corticosteroids, selective immunosuppressants, antifungal agents, drugs involved in preventive therapy, AIDS-related infections, cytomegalovirus, non-nucleoside reverse transcriptase inhibitors, nucleoside analog reverse transcriptase inhibitors, protease inhibitors, anemia, Kaposi's sarcoma (Kaposi'ssarcoma), aminoglycosides, carbapenems, cephalosporins, glycopeptides, lincosamides, macrolides, oxazolidinones, penicillins, streptogramins, sulfonamides, trimethoprim and derivatives, tetracyclines, anthelmintics, amebicides, biguanides, cinchona alkaloids, folic acid antagonists, quinoline derivatives, Pneumocystis carinii carinii) therapy, hydrazides, imidazoles, triazoles, nitroimidazoles, cyclic amines, neuraminidase inhibitors, nucleosides, phosphate binders, cholinesterase inhibitors, adjunctive therapy, barbiturates and derivatives, benzodiazepines, gamma-aminobutyric acid derivatives, hydantoin derivatives, iminostilbene derivatives, succinimide derivatives, anticonvulsants, ergot alkaloids, antimigraine agents, biological response modifiers, carbamates, tricyclic derivatives, depolarizing agents, nondepolarizing agents, neuromuscular anesthetics, CNS stimulants, dopaminergic agents, monoamine oxidase inhibitors, COMT inhibitors, alkyl sulfonates, ethyleneimines, imidazotetrazines, nitrogen mustard analogs, nitrosoureas, platinum compounds , antimetabolites, purine analogs, pyrimidine analogs, urea derivatives, anthracyclines, actinomycins, camptothecin derivatives, epipodophyllotoxins, taxanes, vinca alkaloids and analogs, antiandrogens, antiestrogens, nonsteroidal aromatase inhibitors, protein kinase inhibitors, antineoplastic agents, azaspirodecanedione derivatives, anxiolytics, stimulants, monoamine reuptake inhibitors, selective serotonin reuptake inhibitors, antidepressants, benzisoxazole derivatives, butyrophenone derivatives, dibenzodiazepine derivatives, dibenzothiazepine derivatives, diphenylbutylpiperidine derivatives, phenothiazines, thienobenzodiazepine derivatives, thioxanthene derivatives, allergenic extracts (allergenic extracts), nonsteroidal agents, leukotriene receptor antagonists, xanthines, endothelin receptor antagonists, prostaglandins, pulmonary surfactants, mucolytics, antimitotics, uricosurics, xanthine oxidase inhibitors, phosphodiesterase inhibitors, methenamine salts, nitrofuran derivatives, quinolones, smooth muscle relaxants, parasympathomimetics, halogenated hydrocarbons, aminobenzoates, amides (e.g., lidocaine, articaine hydrochloride, bupivacaine hydrochloride,hydrochloride), antipyretics, hypnotics and sedatives, cyclopyrrolones, pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids, para-aminophenol derivatives, alcohol dehydrogenase inhibitors, heparin antagonists, adsorbents, emetics, opioid antagonists, cholinesterase reactivators, nicotine replacement therapy, vitamin A analogs and antagonists, vitamin B analogs and antagonists, vitamin C analogs and antagonists, vitamin D analogs and antagonists, vitamin E analogs and antagonists, vitamin K analogs and antagonists.

[0135] Conjugate: Detection Agent

[0136] In exemplary embodiments, the binding construct is coupled to a detector. In exemplary embodiments, the detector can emit a detectable (measurable) signal based on enzymatic activity, radioactivity, colorimetric activity and / or binding activity. In exemplary embodiments, the signal is radioactivity, colorimetric, colorimetric, fluorescence, chemiluminescence, enhanced chemiluminescence, direct fluorescence, time-resolved fluorescence, direct chemiluminescence, phosphorescence, enzyme or a signal based on the combination of micron particles or nanoparticles, streptavidin / avidin-biotin and protein A. In exemplary embodiments, the detector includes an enzyme, a radioisotope, a DNA reporter gene, a colorimetric or fluorescent reporter, or an electrochemiluminescent label. In exemplary aspects, the enzyme is horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase or beta-galactosidase. In exemplary aspects, the enzyme can cause chemiluminescence or light generation when exposed to certain reagents. In exemplary aspects, the radioisotope is I 125 In exemplary aspects, the DNA reporter gene is a DNA probe. In exemplary aspects, the fluorescent reporter is phycoerythrin (PE), such as B-PE, R-PE, or allophycocyanin (APC).

[0137] Conjugates: dimers and multimers

[0138] In some embodiments, the binding construct provided is in dimer or polymer form, wherein one or more binding constructs of the present invention are linked together. In some respects, the dimer is a homodimer comprising two binding constructs of the same type (such as same structure) linked together. In alternative aspects, the dimer is a heterodimer comprising two binding constructs of the present invention, wherein the structures of these two binding constructs are different from each other. In some respects, multimer is a homomultimer comprising more than a binding construct of the present invention and each binding construct belongs to the same type (such as same structure). In alternative aspects, multimer is a heteromultimer comprising more than a binding construct of the present invention and wherein the structures of at least two binding constructs in the heteromultimer are different from each other. In exemplary aspects, the binding construct comprises a dimer of two Fab fragments, such as a homodimer, wherein each Fab fragment is bonded to FIXp and is not bonded to WT FIX, for example, is bonded to FIXp, even in the presence of WT FIX or in a sample containing human plasma. In exemplary aspects, the homodimer including two Fab fragments is bivalent and monospecific for FIXp. In exemplary aspects, each Fab fragment in the homodimer includes the amino acid sequence of SEQ ID NO:6-11. In exemplary aspects, there are one or more amino acids between each of SEQ ID NO:6-11. In exemplary aspects, each Fab fragment in the homodimer includes SEQ ID NO:24 or SEQ ID NO:25 or SEQ ID NO:24 and 25 sequences. In exemplary aspects, each Fab fragment in the homodimer includes SEQ ID NO:26 or SEQ ID NO:27 or SEQ ID NO:26 and 27 sequences.

[0139] Two or more binding constructs can be linked together using standard linkers and procedures known to those skilled in the art. In certain embodiments, the linker connecting two (or more) binding constructs is a linker known in the art. In some embodiments, the linker is a disulfide bond. For example, each monomer in the dimer can include a sulfhydryl group and the sulfhydryl sulfur atom in each monomer participates in the formation of a disulfide bond. In some embodiments, the linker is a helix-turn-helix motif. In exemplary aspects, each monomer in the dimer is connected by a helix-turn-helix motif. In exemplary aspects, each monomer in the dimer is connected by an alkaline phosphatase domain.

[0140] In exemplary aspects, a homodimer including two Fab fragments includes a linker connecting the two Fab fragments. In exemplary aspects, the homodimer includes a synthetic double helix loop helix motif, such as Haylock et al., International Journal of Oncology 48 (2): 461-470 (2016) or Wang et al., Analytical Chemistry 78: 997-1004 (2006) described in the motif. In exemplary aspects, the homodimer includes a constant antibody domain. Such antibody domains are described in Hu et al., Cancer Research 56: 3055-3061 (1996) and McGregor et al., Molecular Immunology 31: 219-226 (1994). In exemplary aspects, the homodimer includes a bacterial alkaline phosphatase domain, such as Wang et al. (2006), the alkaline phosphatase domain described in the foregoing. In exemplary aspects, the homodimer comprises the sequence of SEQ ID NO: 28 or SEQ ID NO: 27 or SEQ ID NOs: 28 and 27.

[0141] Nucleic Acids

[0142] Also provided herein is nucleic acid, the nucleic acid includes a nucleotide sequence encoding any one of the binding constructs described herein (e.g., antibodies, Fabs, polypeptides, or conjugates). "Nucleic acid" as used herein includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally means a polymer of DNA or RNA, the DNA or RNA can be single-stranded or double-stranded, synthetic or obtained from natural sources (e.g., separation and / or purification), can contain natural, non-natural or altered nucleotides, and can contain natural, non-natural or altered internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, replacing the phosphodiester between each nucleotide in unmodified oligonucleotides. It is generally preferred that nucleic acid does not include any insertion, deletion, inversion, and / or substitution. However, in some cases, as discussed herein, nucleic acid includes one or more insertions, deletions, inversions, and / or substitutions that may be suitable.

[0143] Preferably, the nucleic acid of the present invention is a recombinant nucleic acid. The term "recombinant" as used herein refers to a molecule that is (i) constructed outside a living cell by joining a natural or synthetic nucleic acid segment to a nucleic acid molecule that can replicate in a living cell, or (ii) a molecule that is replicated by the molecule described in (i) above. For the purposes of this article, replication can be in vitro replication or in vivo replication.

[0144] Nucleic acids can be chemically synthesized and / or constructed by enzyme ligation based on procedures known in the art. See, for example, the aforementioned Sambrook et al., and the aforementioned Ausubel et al. For example, naturally occurring nucleotides or differently modified nucleotides (e.g., thiophosphate derivatives and acridine substituted nucleotides) can be used to chemically synthesize nucleic acids, and the differently modified nucleotides are designed to increase the biological stability of the molecule or increase the physical stability of the duplex formed during hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyl Q nucleoside, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substituted adenine, 7-methylguanine, 1-methylguan ...5-methylcytosine, N-substituted adenine, 7-methylguanine, 1-methylguanine, 5-methylcytosine, N-substituted adenine, 7-methylguanine, 1-methylguanine, 5-methylcytosine, N-substituted adenine, 7-methylguanine, 5-methylcytosine, N-substituted adenine, guanine, 5-methylaminomethyl uracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl Q nucleoside, 5'-methoxycarboxymethyl uracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, Q nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine. Alternatively, one or more nucleic acids disclosed herein can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0145] In some aspects, the nucleic acid encodes only a portion of the antibody, antigen binding fragment, polypeptide or conjugate. For example, when the conjugate includes a polymer that does not contain amino acids and is therefore not encoded by the nucleic acid, the nucleic acid encodes only the portion of the conjugate that can be encoded by the nucleic acid. In an exemplary embodiment, the nucleic acid includes a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence containing each of SEQ ID NOs: 6-11. In an exemplary aspect, the nucleic acid encodes a polypeptide comprising an amino acid sequence containing each of SEQ ID NOs: 6-11, wherein there are one or more amino acids between each of SEQ ID NOs: 6-11. In an exemplary aspect, the polypeptide encoded by the nucleic acid also includes an expression tag, such as a FLAG tag containing DYKDDDDK (SEQ ID NO: 12) and / or a hexahistidine tag containing HHHHHH (SEQ ID NO: 13).

[0146] The nucleic acids can be used, for example, in methods for the recombinant production of the binding constructs of the invention.

[0147] Recombinant expression vector

[0148] The nucleic acid of the present invention can be incorporated into a recombinant expression vector or "vector". In this regard, the present invention provides a recombinant expression vector or "vector" comprising any of the nucleic acid of the present invention. For the purposes of this article, the term "recombinant expression vector" or "vector" means a genetically modified oligonucleotide or polynucleotide construct, when the construct includes a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and the vector is in contact with a host cell under conditions sufficient to express mRNA, protein, polypeptide or peptide in the cell, the construct allows the host cell to express mRNA, protein, polypeptide or peptide. The vector of the present invention is not naturally occurring as a whole. However, parts of the vector may be naturally occurring. The recombinant expression vector of the present invention may include any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthesized or partially obtained from natural sources, and may contain natural, non-natural or altered nucleotides. The recombinant expression vector may include naturally occurring or non-naturally occurring internucleotide linkages, or these two types of linkages. Preferably, the altered nucleotides or non-naturally occurring internucleotide linkages do not hinder the transcription or replication of the vector.

[0149] The recombinant expression vector of the present invention can be any suitable recombinant expression vector, and can be used for transforming or transfecting any suitable host. Suitable carriers include those designed for breeding and amplification or for expression or both, such as plasmids and viruses. Carriers can be selected from the group consisting of pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, La Jolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, California). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. Examples of plant expression vectors include pBIO1, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, such as a retroviral vector.

[0150] The recombinant expression vector of the present invention can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., and Ausubel et al., above. Circular or linear expression vector constructs can be prepared to contain a replication system that functions in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, CoIE1, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0151] Ideally, the recombinant expression vector includes regulatory sequences specific for the type of host (e.g., bacteria, fungi, plants, or animals) into which it is to be introduced, such as transcription and translation start and stop codons, and whether the vector is DNA or RNA based, as appropriate.

[0152] The recombinant expression vector can include a natural or standardized promoter operably connected to the nucleotide sequence of the coded polypeptide (including its functional part and functional variant), or operably connected to the nucleotide sequence complementary or hybridized with the nucleotide sequence of the coded polypeptide. The selection of promoter, such as strong, weak, inducible, tissue-specific and development-specific, is within the common skills of practitioners. Similarly, the combination of nucleotide sequence and promoter is also within the skills of practitioners. Promoter can be a non-viral promoter or a viral promoter, such as cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter and the promoter found in the long terminal repeat sequence of murine stem cell virus.

[0153] The recombinant expression vector of the present invention can be designed for transient expression, stable expression or both. Similarly, the recombinant expression vector can be prepared for constitutive expression or inducible expression. In addition, the recombinant expression vector can be prepared to contain a suicide gene.

[0154] Host cells

[0155] The present invention also provides a host cell, which includes any one of the nucleic acid or recombinant expression vector described herein. The term "host cell" used herein refers to any type of cell that can contain and express the recombinant expression vector of the present invention. The host cell can be a eukaryotic cell, such as a plant, animal, fungus or algae cell, or can be a prokaryotic cell, such as a bacterial or protozoan cell. The host cell can be a cultured cell or a primary cell, i.e., a cell directly isolated from an organism, such as a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell grown in suspension. Suitable host cells are known in the art and include, for example, DH5α Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, such as a DH5α cell. For the purpose of producing a recombinant polypeptide, the host cell is preferably a mammalian cell, such as a CHO cell.

[0156] Reagent test kit

[0157] Provided herein is a kit comprising any one or more of the binding constructs disclosed herein. In an exemplary embodiment, the kit comprises an antibody or antigen binding fragment or polypeptide or conjugate or nucleic acid or vector or host cell as described herein, or any combination of the foregoing. In an exemplary aspect, the binding construct is provided in a kit in a predetermined amount or concentration. For example, the kit can be a detection kit for detecting Padova FIX in a sample, which includes a predetermined amount of binding constructs. In an exemplary embodiment, one or more of the binding constructs disclosed herein are provided in a kit in the form of an aqueous solution. In an exemplary aspect, the aqueous solution is provided to an end user on dry ice. In some aspects, the aqueous solution is provided to an end user separately from other components in the kit. In an exemplary embodiment, the binding construct of the present disclosure is provided in a kit in a lyophilized or other freeze-dried form. In an exemplary aspect, the binding construct of the present disclosure is provided in a kit in a frozen or cryopreserved form. In exemplary aspects, the concentration of the antibody or antigen-binding fragment or polypeptide or conjugate provided in the kit is about 1-10 μg / mL or about 1-5 μg / mL. In exemplary aspects, the concentration of the antibody or antigen-binding fragment or polypeptide or conjugate provided in the kit is about 1.5 μg / mL to about 2.0 μg / mL.

[0158] In exemplary aspects, the kit includes a solid support, and in exemplary aspects, the antibody or antigen binding fragment or polypeptide or conjugate is pre-coated on the solid support. In exemplary aspects, the kit includes a solid support selected from the group consisting of tubes, dishes, flasks, bags, plates (e.g., microtiter plates), membranes, filters, beads, fibers, probes, etc. In exemplary aspects, the solid support is made of a polymer. In exemplary aspects, the solid support is made of agarose, cellulose, dextran, polyacrylamide, latex, or controlled micropore glass. In exemplary aspects, the solid support is made of agarose. In exemplary aspects, the solid support is made of polyvinyl difluoride (PVDF), nitrocellulose, nylon 66, protran nitrocellulose, or paper. In exemplary aspects, the membrane is , , , , or In one embodiment, the solid support is a polymer bead, a microtiter plate, a membrane or a filter. In an exemplary aspect, the test kit includes a solid support pre-coated with a solution containing 100 ng or more, about 150 ng or more, about 200 ng or more, about 500 ng or more of an antibody or Fab or polypeptide or conjugate. In some aspects, the test kit includes a solid support pre-coated with a solution containing about 50 ng to about 550 ng, about 100 ng to about 500 ng, about 125 ng to about 400 ng, about 150 ng to about 350 ng, about 175 ng to about 300 ng or about 200 ng to about 250 ng of an antibody or Fab or polypeptide or conjugate. In certain aspects, the kit includes a solid support pre-coated with a solution containing about 100 ng to about 150 ng, about 150 ng to about 200 ng, about 200 ng to about 500 ng of an antibody or antigen-binding fragment or polypeptide or conjugate. In exemplary aspects, the kit includes a solid support containing a pre-equilibrated amount of an antibody or antigen-binding fragment or polypeptide or conjugate. In exemplary aspects, the kit includes a microtiter plate, wherein each well of the microtiter plate includes a solution, the solution including about 100 μL to about 500 μL of a solution containing about 1-10 μg / mL or about 1-5 μg / mL of an antibody or antigen-binding fragment or polypeptide or conjugate. In exemplary aspects, the kit includes a microtiter plate, wherein each well of the microtiter plate includes a solution, the solution including about 100 μL to about 500 μL of a solution containing about 2.5 μg / mL of an antibody or antigen-binding fragment or polypeptide or conjugate.

[0159] In exemplary aspects, the kit includes additional reagents, substrates, solvents, buffers, diluents, etc. used in the detection methods described herein. In exemplary aspects, any one or more of the additional components are provided in the kit in a predetermined amount, such as a necessary and suitable amount for the detection analysis. In exemplary aspects, the kit includes a secondary antibody that is bound to a Padova FIX binding antibody, an Fab, a polypeptide, or a conjugate. In exemplary aspects, the secondary antibody includes a detection agent. In exemplary embodiments, the detection agent includes an enzyme, a radioisotope, a DNA reporter gene, a colorimetric or fluorescent reporter, or an electrochemiluminescent label. The detection agent can be any of the detection agents described herein. In exemplary aspects, the secondary antibody of the Padova FIX binding antibody, an Fab, a polypeptide, or a conjugate is attached to a detection agent.

[0160] Composition

[0161] Provided herein are compositions comprising any one or more of the binding constructs disclosed herein. In exemplary aspects, the composition comprises a mixture of the binding construct as described herein and a detection agent. In exemplary aspects, the detection agent is any detection agent described herein. See the section entitled "Conjugate: Detection Agent".

[0162] In exemplary aspects, the composition includes a mixture of a binding construct as described herein and a biological sample obtained from a subject. In exemplary aspects, the biological sample is any biological sample described herein. See the section entitled "Sample". In exemplary aspects, the composition includes a mixture of an antibody or antigen binding fragment or polypeptide or conjugate as described herein and a biological sample including human plasma or its dilution fraction. In exemplary aspects, the composition includes a mixture of an antibody or antigen binding fragment or polypeptide or conjugate as described herein and a biological sample including human tissue or its cells. In exemplary aspects, the biological sample includes liver tissue. In exemplary aspects, the composition also includes a detection agent.

[0163] Also provided herein is a mixture of a composition comprising any one or more of the binding constructs disclosed herein and a sample (e.g., a biological sample) comprising human plasma proteins. In exemplary aspects, the composition comprises a mixture of an antibody or antigen binding fragment or polypeptide or conjugate as described herein and at least one human plasma protein, the human plasma protein being selected from the group consisting of factor IX, factor II, and factor X, and variants thereof. In exemplary aspects, the composition further comprises a detection agent.

[0164] Detection Methods

[0165] The binding construct provided herein can be used, for example, for a detection method that can clearly and specifically detect Padova FIX in a sample, wherein the sample is, for example, a clinical sample, wherein the clinical sample comprises, for example, Padova FIX and WT FIX. The binding construct can be used in any antibody-based analysis or technology or any immunoassay known in the art, such as, but not limited to, radioimmunoassay (RIA), magnetic immunoassay (MIA), immunocytochemistry (ICC) analysis, immunohistochemistry (IHC) analysis, immunofluorescence analysis, ELISA, EIA, ELISPOT, enzyme amplification immunoassay, radiobinding analysis, Western blotting, immunoprecipitation, dot blotting, flow cytometry, real-time immunoquantitative PCR, protein microarray, etc. See, for example, "Handbook of Immunoassays ( The Immunoassay Handbook) (4th edition); Theory and Applications of Ligand Binding, ELISA and Related Techniques, Wild, Elsevier Ltd. ed. (Oxford, UK) 2013; Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY) 2012; and Immunoassays: A Laboratory Manual, 4th edition, Cold Spring Harbor, NY 2013; Immunoassay )", Diamandis and Christopolous, Academic Press, 1996.

[0166] Therefore, provided herein are uses of binding constructs (e.g., antibodies or antigen binding fragments, polypeptides or conjugates), nucleic acids, vectors, host cells and / or kits described herein for detecting Padova Factor IX in a sample. In exemplary aspects, the sample is a biological sample obtained from a subject to which an expression vector containing a nucleic acid encoding Padova FIX has been administered. For example, in various embodiments, the subject suffers from a hemorrhagic disease and is undergoing Factor IX replacement therapy, which is optionally achieved by expression of a nucleic acid containing a nucleotide sequence encoding a heterologous Padova Factor IX.

[0167] Also provided herein is a method for detecting Padova Factor IX in a sample obtained from a subject. In an exemplary embodiment, the method comprises (i) contacting the sample with a binding construct (e.g., an antibody or Fab or polypeptide or conjugate) as described herein to form a complex (e.g., an immune complex) comprising Padova FIX and a binding construct (e.g., an antibody, Fab, polypeptide or conjugate), and (ii) detecting the complex.

[0168] In an exemplary embodiment, Padova FIX comprises the amino acid sequence of SEQ ID NO:1.

[0169] In an exemplary embodiment, detecting the complex includes detecting the signal of the detection agent. In an exemplary embodiment, the signal is based on enzyme activity, radioactivity, colorimetric activity and / or binding activity. In an exemplary embodiment, the signal is radioactivity, colorimetric, colorimetric, fluorescence, chemiluminescence, enhanced chemiluminescence, direct fluorescence, time-resolved fluorescence, direct chemiluminescence, phosphorescence, enzyme or based on the signal of the combination of micron particles or nanoparticles, streptavidin / avidin-biotin and protein A. In an exemplary embodiment, the detection agent includes enzyme, radioisotope, DNA reporter gene, colorimetric or fluorescent reporter, electrochemiluminescent label. In an exemplary embodiment, detecting the complex includes performing surface plasma resonance to detect the complex or measuring the change of resistance on the electrode (because Padova FIX is bound to an antibody, antigen binding fragment, polypeptide or conjugate). See González-Díaz et al., “Plasmonic Au / Co / Au nanosandwiches with Enhanced Magneto-Optical Activity,” Small 4(2):202-5 (2008); and Tsekenis (2008). “Label-less immunosensor assay for myelin basic protein based upon an ac impedance protocol.” Analytical Chemistry. Chemistry (2008) 80 (6): 2058-62. In exemplary aspects, the enzyme is horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase or beta-galactosidase. In exemplary aspects, the enzyme is exposed to an agent that causes it to chemiluminesce or generate light. In exemplary aspects, the radioisotope is I125. In exemplary aspects, the DNA reporter gene is a DNA probe.See, e.g., Rajkovic, "Immunoquantitative real-time PCR for detection and quantification of Staphylococcus aureus enterotoxin B in foods." Applied and Environmental Microbiology 72(10):6593-9 (2006); and Gofflot, "Immuno-quantitative polymerase chain reaction for detection and quantitation of prion protein." Journal of Immunoassay and Immunochemistry 25(3):241-58 (2004). In exemplary aspects, the fluorescent reporter is phycoerythrin (PE), e.g., B-PE, R-PE, or allophycocyanin (APC).

[0170] In an exemplary embodiment, the antibody or antigen binding fragment or polypeptide is coupled to a detection agent. In an exemplary embodiment, the conjugate includes a detection agent. In an exemplary embodiment, the antibody or antigen binding fragment or polypeptide is not coupled to a detection agent, or the conjugate does not include a detection agent. In such exemplary embodiments, the method includes contacting the sample with a secondary antibody containing a detection agent, wherein the secondary antibody is bound to the antibody or antigen binding fragment or polypeptide or conjugate. The secondary antibody can be any antibody of any isotype or species, as long as the secondary antibody will bind to an anti-Padova FIX antibody, its antigen binding fragment, polypeptide or conjugate.

[0171] In exemplary embodiments, the antibody or antigen binding fragment or polypeptide is coupled to a solid support. In exemplary embodiments, the conjugate comprises a solid support. For example, the solid support is selected from the group consisting of a tube, a dish, a flask, a bag, a plate (e.g., a microtiter plate), a membrane, a filter, a bead, a fiber, a probe, etc. In exemplary aspects, the solid support is made of a polymer. In exemplary aspects, the solid support is made of agarose, cellulose, dextran, polyacrylamide, latex, or controlled microporous glass. In exemplary aspects, the solid support is made of agarose. In exemplary aspects, the solid support is made of polyvinyl difluoride (PVDF), nitrocellulose, nylon 66, protran nitrocellulose, or paper. In exemplary aspects, the membrane is , , , , or Membrane (Sigma-Aldrich, St. Louis, MO). In exemplary aspects, the solid support is a polymeric bead, a magnetic or paramagnetic bead, a microtiter plate, a membrane, or a filter.

[0172] Subjects

[0173] In exemplary embodiments, the subject mentioned herein is a mammal, including but not limited to a mammal of the order Rodentia, such as mice and hamsters; and a mammal of the order Lagomorpha, such as rabbits; a mammal from the order Carnivora, including felines (cats) and canines (dogs); a mammal from the order Artiodactyla, including bovines (cows) and suids (pigs); or a mammal of the order Perissodactyla, including equines (horses). In some aspects, the mammal belongs to the order Primate, Ceboids or Simioids (monkeys) or to the order Anthropoids (humans and apes). In exemplary aspects, the mammal is a human. In exemplary aspects, the human subject is an adult, such as 18 years of age or older, or a teenager. In exemplary aspects, the subject has been administered an expression vector comprising a nucleotide sequence encoding the Padova FIX containing the amino acid sequence SEQ ID NO: 1. In exemplary aspects, the subject suffers from a bleeding disorder. In exemplary aspects, the subject suffers from a bleeding disorder in which the blood of the subject cannot properly coagulate. In exemplary aspects, the subject does not express factor IX, such as WT factor IX, or has a lower factor IX expression amount. In exemplary aspects, the subject has a mutation in a gene encoding factor IX. In exemplary aspects, the subject suffers from hemophilia, such as hemophilia B (also known as Christmas Disease). In exemplary aspects, the subject exhibits higher than normal coagulation activity. In exemplary aspects, the subject has naturally occurring Padova FIX, such as a gene mutation causing the expression of Padova FIX.

[0174] sample

[0175] In exemplary embodiments, the sample mentioned herein is a biological sample including one or more body fluids, such as human body fluids. In exemplary aspects, the sample includes body fluids, including but not limited to obtaining blood, plasma, serum, lymph, breast milk, saliva, mucus, semen, vaginal secretions, cell extracts, inflammatory fluids, cerebrospinal fluid, feces, vitreous humor or urine from a subject. In exemplary aspects, the sample includes blood, plasma or serum. In exemplary aspects, the sample is prepared by blood, plasma or serum. In exemplary aspects, the sample is a blood, plasma or serum fraction. In exemplary aspects, the sample is a blood sample, a plasma sample or a serum sample. In exemplary aspects, the sample includes blood or its fraction (e.g., plasma, serum). In exemplary aspects, the sample includes or is plasma.

[0176] In exemplary aspects, the sample is a human tissue sample. In exemplary aspects, the human tissue sample includes muscle tissue, epithelial tissue, connective tissue, or neural tissue. In exemplary aspects, the human tissue sample includes bone tissue. In exemplary aspects, the human tissue sample comprises heart tissue, spleen tissue, lymph node tissue, brain tissue, spinal cord tissue, nerve tissue, ear, nose or eye tissue, breast tissue, subcutaneous tissue, mammary tissue, bone marrow tissue, lymphatic tissue, nasopharyngeal tissue, laryngeal tissue, tracheal tissue, bronchial tissue, lung tissue, skin tissue, salivary gland tissue, tissue from tongue or mouth, oropharyngeal tissue, hypopharyngeal tissue, esophageal tissue, stomach tissue, small intestine tissue, appendix tissue, colon tissue, rectal tissue, anal tissue, liver tissue, bile duct tissue, pancreatic tissue, gallbladder tissue, kidney tissue, ureter tissue, bladder tissue, urethra tissue, uterus tissue, vaginal tissue, vulva tissue, ovarian tissue, placenta tissue, scrotal tissue, penis tissue, prostate tissue, testicular tissue, seminal vesicle tissue, pituitary tissue, pineal tissue, thyroid tissue, parathyroid tissue, adrenal tissue or pancreatic islet tissue. In exemplary aspects, the human tissue sample comprises liver tissue or spleen tissue or kidney tissue. In exemplary aspects, the human tissue sample includes liver tissue.

[0177] The following examples are provided only to illustrate the present invention and are not intended to limit the scope of the present invention in any way. Example

[0178] Example 1

[0179] This example demonstrates a method for preparing an antibody or antigen-binding fragment thereof of the present invention.

[0180] The candidate specific Padova FIX binding constructs were selected using phage display methods. The phage library was screened using a linear peptide comprising a single amino acid substitution at position 338, a structural peptide comprising a single amino acid substitution at position 338, or a full-length recombinant Padova FIX that captured a candidate. Three rounds of panning were performed with and without the wild-type FIX sequence competing, thus identifying several candidate binding constructs. BIACORE and ELISA experiments were performed to measure the specificity and affinity of the resulting candidate.

[0181] One candidate (named BC1) was obtained using a linear peptide containing the sequence DRATCLLSTKFT and two candidates (named BC2 and BC3) were obtained using a structural peptide containing the sequence LVDRATCLLSTKFTIYNNMFCAGFH. Yet another candidate (BC4) was obtained using peptides including linear or structural peptides alternating with Padova FIX.

[0182] Screening analysis shows that these candidates bind to Padova FIX. For BC2 and BC3, the binding signal (multiple relative to background) with wild-type FIX is less than 11, while the binding signal (multiple relative to background) with Padova FIX exceeds 71. For BC1, the binding signal (multiple relative to background) with wild-type FIX is 1, while the binding signal with Padova FIX is close to 200. In the second group of screening analysis, the binding signal of BC1, BC2 and BC3 with wild-type FIX is 1 or less, while the binding signals of BC1, BC2 and BC3 with Padova FIX are 70, 30 and 10 respectively. BC4 shows a binding signal with Padova FIX exceeding 36, while the signal with wild-type FIX is about 1.

[0183] In Ni 2+ The binding of BC1 to Padova FIX was tested by ELISA on plates where the coating concentration of BC1 and the concentration of Padova FIX were varied. The test concentrations of BC1 and Padova FIX were 0.04 μg / ml, 0.2 μg / ml, 1 μg / ml and 5 μg / ml. The binding signal of BC1 to Padova FIX was compared with the wt FIX control. At each test concentration, the binding of Padova FIX to BC1 was stronger than the binding of WT FIX to BC1 ( Figure 2 ).

[0184] The binding of BC1, BC2 and BC3 to Padova FIX in the presence of wild-type FIX was tested by ELISA ( Fig.10 ). MaxiSorp plates were coated with 5 μg / mL BC1, BC2, or BC3 and solutions containing different concentrations of Padova FIX and 5 μg / mL wild-type FIX was added to the coated plates. The Padova FIX concentrations tested were 0 μg / mL, 0.156 μg / mL, 0.313 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL. Figure 3 As shown in , BC1 exhibited a strong affinity for Padova FIX, even in the presence of WT Factor IX. In the presence of 5 μg / mL WT Factor IX, BC1 exhibited the highest sensitivity.

[0185] The binding of BC1 to various concentrations of Padova FIX (eg, Fig.10 ). Figure 4As shown in , the binding of BC1 to Padova FIX increased with increasing concentrations of Padova FIX, even in the presence of WT Factor IX and other plasma proteins. Padova FIX was varied from 25 ng / ml to 1000 ng / ml.

[0186] The binding of BC1 to Padova FIX was tested by ELISA in plasma solutions containing varying percentages of 50 mM benzamidine ( Fig.10 The solution is 5%, 10% or 20% (v / v) plasma. Figure 5 As shown, BC1 can detect Padova FIX in the range of 3.13 ng / ml to 200 ng / ml in 20% plasma solution.

[0187] The binding of BC1, BC2 and BC4 to Padova FIX was tested by ELISA in the presence of 20% human plasma containing 5 μg / mL WT Factor IX. The concentrations of Padova FIX used in this analysis were 0, 3.13, 6.25, 12.5, 25, 50, 100 and 200 ng / ml. Figure 6 As shown in , BC1 exhibited the highest sensitivity, while BC2 exhibited the lowest sensitivity and was unable to bind to Padova FIX in a 20% human plasma sample containing 5 μg / mL WT Factor IX. BC4 exhibited a lower binding capacity to Padova FIX than BC1 ( Figure 6 ). Like BC2, BC3 could not be used as a Padova FIX-specific detection antibody in samples containing 20% ​​human plasma (data not shown).

[0188] The binding of BC1 and BC4 to other coagulation factors was tested by ELISA. Specifically, the binding of BC1 or BC4 to Factor II and Factor X was tested. Figure 7 As shown in , BC1 has a high binding specificity for Padova FIX and shows no cross-reactivity against Factor II or Factor X, while in contrast, BC4 exhibits little cross-reactivity with Factor II.

[0189] BC1 has binding specificity for Padova FIX. In contrast, the other two binding constructs (BC5 and BC6) were prepared by phage display using structural peptides. Figure 8 As shown in , BC5 and BC6 do not have binding specificity for Padova FIX, as they were shown to bind significantly to WT FIX.

[0190] The KD value of BC1 was determined by Biacore surface plasmon resonance. The KD of BC1 was 56 nM. Compared with other candidates, BC1 showed the highest affinity for Padova FIX. This value is similar to (if not higher than) the KD (M) value of commercial antibodies. For example, the KD (M) value of commercially available anti-human wild-type FIX antibody is 3.11×10 -9 .

[0191] The sequences of the BC1 heavy and light chain CDRs were determined by PCR and sequence translation and are as follows:

[0192] Heavy chain CDR1 SSYAIS SEQ ID NO:6 Heavy chain CDR2 GIVPAFGTANYAQKFQG SEQ ID NO:7 Heavy chain CDR3 SWGVISFAY SEQ ID NO:8 Light chain CDR1 RASQDISSYLN SEQ ID NO:9 Light chain CDR2 AASNLQS SEQ ID NO:10 Light chain CDR3 MQYDSLPFTF SEQ ID NO:11

[0193] BC1 obtained with the linear peptide provided unique and specific binding to Padova FIX and this activity was confirmed. BC1 exhibited a detection limit of approximately 3 ng / mL plasma and showed no cross-reactivity with wild-type FIX, even at significantly higher (>5 μg / mL) concentrations.

[0194] These data demonstrate that a highly specific anti-Padova FIX antibody was generated. This antibody binds to Padova FIX in human plasma samples and does not bind to any of WT Factor IX, Factor II, and Factor X. These data demonstrate that this antibody can be used, for example, to conduct clinical assays to selectively distinguish wild-type FIX from Padova FIX antigen levels.

[0195] Example 2

[0196] This example demonstrates methods of using the antibodies or antigen-binding fragments thereof of the invention.

[0197] Using standard conditions, 2 μg / mL of the newly developed Fab fragment of Padova Factor IX (FlXp) specific binding antibody (BC1) was coated on a 96-well microtiter plate. Commercially available biotinylated polyclonal sheep anti-human FIX IgG and streptavidin peroxidase were used as the detection system. A schematic diagram of the assay components is shown in Fig. 9 middle.

[0198] Assay calibration was achieved by generating a six-point calibration curve with FIXp preparations, covering the FIXp concentration range from 27.1-0.85 ng / mL. Patient samples were diluted with HEPES / NaCl buffer containing 5 mg / mL bovine serum albumin, 10 mM benzamidine, 10 mM CaCl2, and 0.05% Tween 20.

[0199] Normal human plasma or purified human FIX did not show signals in FIXp-specific ELISA. An accurate calibration curve was obtained. Normal human plasma spiked with 1 / 10 dilution of FIXp showed acceptable recovery, and the dilution response curve was parallel to the dilution response curve of the buffer solution containing the analytical standard obtained. Importantly, the sample analysis of six patients treated with the expression vector encoding FIXp showed highly similar FIXp protein and FIX activity curves over time, and compared to CRM- patients, the samples of patients with cross-reactive substances positive (CRM+) did not show signal increase for FIXp protein, showing the specificity of the analysis.

[0200] The FIXp-specific ELISA also enables monitoring of treatment outcomes by measuring FIXp protein.

[0201] Example 3

[0202] This example demonstrates a selective ELISA for Padova FIX and the use of the ELISA for testing human plasma samples.

[0203] The BC1 preparation (0.94 mg / mL) was diluted 1 / 500 with 0.1 M NaHCO3-Na2CO3 (pH 9.5) and bound to the wells of Maxisorp F96 plates by incubating 100 μL per well overnight at 0 to +10°C. The dilution buffer (DB) used for diluting samples and reagents and for blocking the plates was 5 mg / mL biotin-free bovine serum albumin (BSA), 10 mM Ca 2+, 0.1M Hepes, 0.1M NaCl (pH 7.2) with 0.05% Tween 20 (Bio-Rad, EIA grade) and 10mM benzamidine. After coating, wash the plate with phosphate buffered saline containing 0.05% Tween 20 and block each well by incubating 60 minutes with 200 μL DB per well at room temperature (RT, 18 to 26 ° C). Terminate the blocking step by washing. Then, load the dilution of the standard / sample and prepare a continuous 1+1 dilution series directly on the plate. At room temperature, the dilution (100 microliters / well) is cultivated with the plate for 60 minutes. Then, wash the plate again and add the biotinylated polyclonal sheep anti-human FIX detection antibody (100 microliters / well; 1 / 500 operating dilution) prepared by F9-1030A (CoaChrom). After incubation at room temperature for 60 minutes, the plate was washed again, streptavidin peroxidase (DakoCytomation) was added (100 μl / well; 1 / 4,000 dilution) and incubated at room temperature for 30 minutes. After the final thorough washing procedure, the bound peroxidase activity was measured with the ready-to-use peroxidase substrate SureBlue, and the reaction was stopped with 3N sulfuric acid. The plate was then measured at 450 nm with an ELISA reader, subtracting the results obtained at 620 nm.

[0204] Fig.11 Shown are the concentration-response curves obtained for the purified human Padova FIX preparation and a fresh frozen control plasma preparation containing WT FIX at a concentration of 5 μg / mL.

[0205] The dose-response curves covering the concentration of recombinant human Padova FIX from 29 to 0.91 ng / mL obtained meet the recognized requirements for accuracy, precision and linearity, and are therefore considered suitable for extrapolation samples. Specifically, the correlation coefficient of the log-log regression curve is 0.9985, and the average accuracy is 101.4% and the precision is 7.0%. Accuracy and precision are calculated by the signals of the six concentrations in the calibration curve measured by back-fitting. In addition, these data demonstrate the absolute specificity of the method for Padova FIX: FIX wt containing 5 μg / mL normal plasma concentration and the human reference plasma measured using 1 / 10 minimum dilution does not cause any signal.

[0206] ELISA was performed as described above. A calibration curve was obtained using another recombinant human Padova FIX preparation with a protein concentration of 542.42 μg / ml. This preparation was clearly classified as a high-activity Padova FIX variant with a specific coagulation activity of 2310 IU / mg protein. The range of the serial dilution series was from 1 / 20,000 to 1 / 640,000, which determined that the FIX concentration range was from 27.1-0.85 ng / mL. Fig.12 The average calibration curve obtained by linear regression curve between the logarithm of the average signal corrected by blank and the FIX concentration of six analytical standards is shown. The inset shows that the back-fitted analytical calibrants D1 to D6 are consistent with their corresponding nominal concentrations. The calibration curve of Padova FIX shows good linearity in the concentration range from 27.1 to 0.85 ng / mL. This is shown by the average correlation coefficient r=0.9992 (range: 0.9986-0.9996) and confirmed by the back-fitted concentrations calculated for individual points in the calibration curve, which differ from the expected concentrations in the full range by less than 9% (range: 91.1% to 108.6%). These back-fitted data easily meet the requirements defined by the EMA guidelines for bioanalytical method validation, thereby identifying suitable calibration curves for ligand binding analysis. The relative total error (RTE) of the calibration curve is low. Specifically, the RTE is calculated by the average blank-corrected optical density (OD) of the back-fitted calibration curve standards. The resulting concentration is normalized by multiplying its dilution. The RTE can now be expressed as the absolute value of the difference between the nominal concentration of the analytical standard and the mean concentration determined by the back-fitting method plus two times the standard deviation of this mean concentration. Furthermore, the low RSD of the slope demonstrates that these curves can be obtained with the reproducibility required for analyses intended for use in a clinical setting.

[0207] ELISA was performed as described above. Fresh frozen normal human plasma and plasma deficient in FIX were spiked with Padova FIX. The starting dilution of the dilution series of both spiked plasma preparations was 1 / 10. Fig.13 A comparison of the dilution-response curves for these two plasma samples and samples obtained in buffer is shown. The slope of the dilution-response curve obtained for the plasma sample spiked with Padova FIX differs by less than 5% from the slope of the dilution-response curve obtained for the buffer dilution series. This shows that the plasma matrix has no effect on the analytical performance.

[0208] Due to the high sequence homology between human and monkey FIX, conventional polyclonal anti-human FIX antibodies also bind to cynomolgus monkey FIX (data on file). Therefore, the selectivity of the Padova FIX ELISA against the citrated monkey plasma matrix was also tested. Specifically, female and male citrated monkey plasma samples were spiked with Padova FIX and measured at a 1 / 10 dilution as the lowest starting dilution of the dilution series. Fig.14 A comparison of the dilution-response curves of these two plasma samples with samples obtained in buffer is shown.

[0209] The slope of the dilution-response curve obtained for the plasma sample spiked with Padova FIX differed by less than 4% from the slope of the dilution-response curve obtained for the buffer dilution series. This showed that the citrated monkey plasma matrix had no effect on the analytical performance. In addition, the recovery of the spiked Padova FIX concentration was 91.3% and 103.1% for female and male plasma samples, respectively.

[0210] In a Phase 1 / 2 clinical trial, Padova FIX was measured in citrated plasma samples from subjects with FIX cross-reactive material (CRM+) after treatment with AAV2 / 8 viral vectors expressing Padova FIX. Padova FIX ELISA was performed as described above using plasma samples from subjects. FIX coagulation activity and FIX antigen measurements were performed using plasma samples obtained from subjects using standard methods. Fig.15 Results of FIX activity and FIX protein measurements of plasma samples obtained from subjects in a Phase 1 / 2 clinical trial are shown, which consistently show FIX antigen concentrations close to 1U / mL, but FIX activity below the lower limit of quantitation. This data demonstrates that the subject exhibits CRM+ (i.e., FIX protein without coagulation activity) measurable with a conventional FIX ELISA. The Padova FIX ELISA results obtained in ng / mL were converted to activity and antigen units using the analytical standard specific activity (2310 IU / mg) for activity conversion and 5 μg / mL normal human FIX concentration for antigen conversion.

[0211] The Padova FIX ELISA data were similar to the FIX activity data, indicating that the measured FIX activity was dependent on the expression of Padova FIX. In contrast, the FIX antigen concentrations measured using the Padova FIX-specific ELISA were significantly lower than those obtained using the standard ELISA, demonstrating that the Padova FIX ELISA is able to distinguish CRM+ material from Padova FIX.

[0212] In a Phase 1 / 2 clinical trial, after treatment with an AAV2 / 8 viral vector expressing Padova FIX, Padova FIX was measured in a citrated plasma sample from another subject with FIX cross-reactive material (CRM+). Using a plasma sample from the other subject, the Padova FIX ELISA was performed as described above. FIX coagulation activity and FIX antigen measurements were performed using plasma samples obtained from the other subject using standard methods. Fig.16 The results of FIX activity and FIX protein measurements on plasma samples obtained from the other subject are shown, and these results always show a FIX antigen concentration close to 0.8U / mL, but the FIX activity is below the quantitative limit. This data proves that the subject shows CRM+ (i.e., FIX protein without coagulation activity) that can be measured with conventional FIX ELISA. The Padova FIX ELISA results obtained in ng / mL are converted to activity and antigen units using the analytical standard specific activity (2310IU / mg) for activity conversion and 5μg / mL normal human FIX concentration for antigen conversion.

[0213] The Padova FIX ELISA data were similar to the FIX activity data, indicating that the measured FIX activity was dependent on the expression of Padova FIX. In contrast, the FIX antigen concentrations measured using the Padova FIX-specific ELISA were significantly lower than those obtained using the standard ELISA, showing that the Padova FIX ELISA was able to distinguish CRM+ material from Padova FIX.

[0214] In a Phase 1 / 2 clinical trial, Padova FIX was measured in citrated plasma samples from subjects without FIX CRM+ after treatment with AAV2 / 8 viral vectors expressing Padova FIX. The Padova FIX ELISA was performed as described above using plasma samples from subjects without FIX CRM+. FIX coagulation activity and FIX antigen measurements were performed using plasma samples obtained from subjects without FIX CRM+ using standard methods. Fig.17 Results of FIX activity and FIX protein measurements of plasma samples obtained from subjects who consistently showed no CRM+ are shown. Padova FIX ELISA results obtained in ng / mL were converted to activity and antigen units using the analytical standard specific activity (2310 IU / mg) for activity conversion and 5 μg / mL normal human FIX concentration for antigen conversion. Padova FIX ELISA data were similar to FIX activity and FIX protein data, indicating that the measured FIX activity depends on the expression of Padova FIX.

[0215] Example 4

[0216] This article also uses BC1 to develop a FIX colorimetric activity assay. Padova FIX is selectively purified from the sample matrix by binding to the plate-bound BC1. Unbound sample components, including human wild-type FIX, are removed by thorough washing, and then the colorimetric factor IX test 221806 (Hyphen Biomed) is performed on each well of the microplate. The sample is diluted with the dilution buffer described above. Fig.18 The dose-response curves obtained for the Padova FIX and normal reference plasma preparations are shown.

[0217] Padova FIX showed a linear concentration-response curve, whereas normal reference plasma concentrations containing FIX at a concentration of approximately 1 U / mL did not show any measurable signal. These data demonstrate the feasibility of the method, describe the Padova FIX specific activity assay, and determine its specificity.

[0218] Example 5

[0219] The following describes the isolation of antibodies that discriminate between wild-type FIX and the Padova FIX variant.

[0220] Padova FIX is a highly functional variant (FIXR338L) of the naturally occurring wild-type FIX with a single amino acid exchange. The usefulness of Padova FIX in hemophilia B gene therapy has been shown in preclinical models and is currently being studied in clinical 1 / 2 phase programs. The assessment of the success of the therapy depends largely on the determination of the Padova FIX transgenic expression amount, but the determination of the Padova FIX transgenic expression amount is hindered by the lack of antibodies that distinguish wild-type FIX from Padova FIX. The antibody that specifically recognizes Padova FIX but does not cross-react with wild-type FIX enables the development of an analytical method for clearly detecting the Padova FIX in clinical samples. Phage display methods are used to select specific Padova FIX conjugates. Linear peptides and structural peptides containing single amino acid substitutions at position 338, and full-length recombinant Padova FIX are used to screen phage libraries. Three rounds of panning performed with and without the competition with wild-type FIX sequences are used to identify several conjugates. BIACORE (surface plasmon resonance) and ELISA experiments were performed to determine the specificity and affinity of the resulting antibodies. Various antibodies were initially identified by different phage display panning approaches. One antibody generated by the linear peptide approach exhibited unique and specific binding to Padova FIX. The selected antibody had a detection limit of approximately 3 ng / mL plasma and showed no cross-reactivity with wild-type FIX, even at significantly higher (>50 μg / mL) concentrations. The highly specific anti-Padova FIX antibodies described above can be used to perform clinical assays to selectively distinguish wild-type FIX from Padova FIX antigen levels.

[0221] introduction:

[0222] Padova FIX is a naturally occurring, highly functional variant of wild-type (wt) FIX with a single amino acid exchange (FIXR338L). This gain-of-function mutation results in an 8- to 10-fold increase in specific coagulant activity compared to normal FIX. In vitro, the specific coagulant activity of recombinant FIX-R338L was 5- to 10-fold higher than that of recombinant wild-type FIX [1]. The usefulness of Padova FIX in gene therapy for hemophilia B has been demonstrated in preclinical models and is currently being studied in a Phase 1 / 2 clinical trial. Assessment of the success of such therapy relies heavily on the determination of the expression of the Padova FIX transgene, which is hampered by the lack of antibodies that distinguish wild-type FIX from Padova FIX.

[0223] The aim of this study was to generate antibodies that specifically bind to Padova FIX but do not cross-react with wild-type FIX to allow the detection of Padova FIX in clinical samples in the presence of wild-type FIX.

[0224] The following describes the methods performed during this study.

[0225] Generation of anti-Padova FIX-specific antibodies: based on HuCAL Library and Four different methods were used to isolate antibodies specific for Padova FIX ( Fig.19 ). Two of the approaches were performed with peptides containing a single amino acid substitution at position 338 in solid or liquid phase (bead) assays. The third and fourth strategies contained full-length active Padova FIX protein. Three rounds of panning were performed for each strategy, including appropriate negative controls (wild-type peptide or wild-type FIX). Fabs of unique positive clones were prepared and tested for specific binding to Padova FIX antigen. For ELISA, antigen [5 μg / mL] was coated and incubated with Fab fragments [2 μg / mL], followed by detection with anti-Fab AP conjugates.

[0226] Plasma ELISA: Fab was coated on MaxiSorp ELISA plates [5 μg / mL] and incubated with 20% human plasma diluted in PBS buffer plus 50 mM benzamidine. Plasma was spiked with 5 μg / mL rFIX wt and increasing concentrations of Padova rFIX (produced in-house in HEK293). Detection was performed using HRP-labeled polyclonal goat anti-FIX antibody (100 ng / mL).

[0227] Biacore: At 25°C, use Biacore TMAll experiments were performed using a GE Healthcare 200 instrument and a nickel-coated biosensor chip (NTA-Chip). The instrument was prepped three times with HBS-EP operating buffer and flow cell 1 (FC1) was used as a reference flow cell, which was not modified and lacked Fab ligands. Flow cell 2 (FC2) was used to fix about 500RU of Padova FIX-specific Ab42. Ligand concentration ranged from 100 to 6.25nM.

[0228] The following describes the results of this study.

[0229] Structural analysis of porcine wt FIX revealed a single Padova modification (R338L) located on the surface of the protein[2]( Fig.21 ), and thus is a suitable epitope for generating highly specific antibodies. The purified bivalent Fab was tested for antigen specificity by ELISA ( Fig. 22 ) and cross-reactivity with wild-type FIX in 20% human plasma ( Fig.23 ). In a 20% plasma matrix containing 5 μg / mL spiked wild-type FIX, only two of the four isolated bivalent Fabs (Ab42 and Ab76) bound specifically to Padova FIX. Only Ab42 did not show any cross-reactivity against human FII and human FX (data not shown).

[0230] Based on these data, it can be concluded that high specificity anti-Padova FIX miniantibody Ab42 has been produced using phage display technology. The selected candidate shows no cross-reactivity with wild-type FIX or other common blood factor proteins such as FII or FX. In human plasma matrix (20%) and in the presence of wild-type FIX at common concentrations, effective binding with Padova FIX is shown. Miniantibody Ab42 is currently used to carry out Padova FIX analysis (see below) in the plasma obtained from patients treated during clinical 1 / 2 phase trials.

[0231] In the studies described above, the following references were cited: (1) Simioni et al., X-Linked Thrombophilia with a Mutant Factor IX (Factor IX Padua), N Engl J Med 2009;361:1671-5; and (2) Brandstetter et al., X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B. Proc Natl Acad Sci U S A 1995 Oct 10;92(21):9796-800.

[0232] The following describes the development and application of the Padova FIX-specific immunoassay for monitoring gene therapy for hemophilia B.

[0233] Gene therapy holds great promise as a future treatment option for hemophilia. In a clinical phase 1 / 2 trial, AAV2 / 8 viral vectors were used to express Padova FIX (FIXp), a highly functional variant of FIX with a single amino acid exchange (R338L), in subjects with severe hemophilia B. Specific detection of the transgene product is key to assessing the success of therapy but remains challenging in patients with FIX cross-reactive material (CRM+). A FIXp-specific ELISA was developed and applied to measure FIXp expressed in the plasma of hemophilia B patients following treatment with AAV2 / 8 viral vectors expressing Padova FIX. 2 μg / mL of the Fab fragment of the newly developed FIXp-specific binding antibody was coated onto a 96-well microplate using standard conditions. Biotinylated polyclonal sheep anti-human FIX IgG and streptavidin peroxidase were used as the detection system. By generating a six-point calibration curve with a FIXp preparation, covering the FIXp concentration range from 27.1-0.85ng / mL, analytical calibration was achieved. The patient's sample was diluted with HEPES / NaCl buffer containing 5mg / mL bovine serum albumin, 10mM benzamidine, 10mM CaCl2 and 0.05% Tween20. Normal human plasma or purified human FIX did not show a signal in the FIXp-specific ELISA. An accurate calibration curve was obtained. Normal human plasma spiked with 1 / 10 dilution of FIXp showed an acceptable recovery rate, and the dilution response curve was parallel to the dilution response curve of the buffer containing the analytical standard obtained. Importantly, the samples of six patients treated with AAV encoding FIXp showed extremely similar FIXp protein and FIX activity curves over time, and compared to CRM- patients, the samples of CRM+ patients did not show an increase in signal for FIXp protein, indicating the specificity of the analysis. FIXp-specific ELISA can also monitor treatment outcomes by measuring FIXp protein. This represents the first set of data showing the feasibility of this approach.

[0234] introduction:

[0235] Gene therapy holds great promise as a future treatment option for hemophilia[1]. In a phase 1 / 2 trial, an AAV2 / 8 viral vector was used to express FIX of Padova (FIXp)[2], a highly functional variant of FIX with a single amino acid exchange (R338L), in subjects with severe hemophilia B. Specific detection of the transgene product is critical to assessing the success of therapy but remains challenging in patients with FIX cross-reactive material (CRM+).

[0236] Fig.25Describe the principle of the method (analysis) described in this study.Anti-Padova FIX Fab is coated on each well of microplate and selectively captures the Padova FIX in the sample.After removing unbound sample compounds with washing steps, the bound Padova FIX is detected using internal biotinylated polyclonal sheep anti-FIX IgG and streptavidin peroxidase.The HRP activity of the binding is measured using the ready-to-use HRP substrate SureBlue.

[0237] The following describes the methods performed during this study.

[0238] ELISA procedure: Fab preparation Ab42 (0.94 mg / mL) was diluted 1 / 500 with 0.1 M NaHCO3-Na2CO3 (pH 9.5) and bound to each well of a Maxisorp F96 plate by incubating 100 μL per well overnight at 0 to +10°C. The dilution buffer (DB) used for dilution of samples and reagents and for blocking the plate contained 0.1 M HEPES, 0.1 M NaCl (pH 7.2), 5 mg / mL biotin-free bovine serum albumin (BSA), 10 mM Ca 2+ , 0.05% Tween 20 (Bio-Rad, EIA grade) and 10mM benzamidine. After coating, wash the plate with phosphate buffered saline containing 0.05% Tween 20. Then, block the wells by incubating them with 200μL DB per well for 60 minutes at room temperature (RT). Terminate the blocking step by washing. Next, load the dilutions of the standard extreme / sample and prepare a continuous 1+1 dilution series directly on the plate. At room temperature, incubate the dilutions (100 microliters / well) for 60 minutes. Wash the plate again and add the biotinylated polyclonal sheep anti-human FIX detection antibody prepared by F9-1030A (A-Coa) (100 microliters / well; 11500 dilution). After incubation at room temperature for 60 minutes, wash the plate again, and add streptavidin peroxidase (DakoCytomation) (100 microliters / well; 1 / 4,000 dilution) and incubate at room temperature for 30 minutes. After the final washing procedure, the peroxidase activity of the binding is measured with the ready-to-use peroxidase substrate SureBlue (KPL), and the reaction is stopped with 3N sulfuric acid. The plate is then measured at 450nm, and the result obtained at 620nm is subtracted. A calibration curve is constructed with purified recombinant Padova FIX (FIXp) preparation, and the display protein concentration is 542.4 μg / mL. The specific coagulation activity of 2,310IU / mg protein clearly shows that this preparation is classified as a highly active Padova FIX variant. The serial dilution series ranges from 1 / 20,000 to 1 / 640,000 and determines that the FIX concentration range is from 27.1 to 0.85ng / mL.

[0239] The results of this study are described below and in the accompanying figures. FIX coagulation activity and FIX antigen measurements were performed using established standard methods; in addition, specific measurements of Padova FIX protein in citrated plasma samples were performed using ELISA. The results of the Padova FIX ELISA obtained in ng / mL were converted to activity and antigen units. Specifically, the activity units were calculated using the specific activity of the recombinant purified Padova FIX preparation of 2,310 IU FIX / mg applied as an analytical standard for the FIXp ELISA, while the conversion for antigen plasma units was based on a normal FIX plasma concentration of 5 μg / mL. Figure 26-29 Assay selectivity, calibration curves, parallel studies, and the effect of calcium on ELISA sensitivity in normal and FIX-deficient plasma are presented.

[0240] Plasma samples were obtained from patients treated with AAV2 / 8 viral vectors. Figure 30-32 The activity and expression of Padova FIX in samples obtained from three patients are shown. Plasma samples obtained from subject 05-001 consistently show FIX antigen concentrations close to 1U / mL, but FIX activity is below the lower limit of quantification. This data discloses the presence of FIX cross-reactive substances (CRM+) that can be detected with conventional FIX ELISA, i.e., the presence of FIX proteins without coagulation activity. The FIXp ELISA data are similar to the FIX activity data, indicating that the measured FIX activity depends on the expression of Padova FIX. In contrast, the FIX antigen concentration measured using the Padova FIX-specific ELISA is significantly lower than the antigen concentration obtained using the standard ELISA, indicating that the Padova FIX ELISA can distinguish CRM+ substances from Padova FIX. Similar data are seen in samples from subject 10-005, but at lower CRM+ levels; while the samples from subject 03-004 do not contain CRM+, making the time-concentration curves of the two ELISA systems parallel.

[0241] From these data it can be concluded that the Padova FIX-specific ELISA based on the capture of FIXp using a highly specific Fab fragment can monitor treatment outcomes by measuring FIXp. These data are the first set of data to demonstrate the feasibility of this approach.

[0242] In the studies described above, the following references were cited: [1] Simioni P et al. (2009): NEJM 361, 1671-1675, X-Linked thrombophilia with a Mutant Factor IX (Factor IX Padua); [2] Crudele JM et al. (2015), Blood 125: 1553-1561, AAV liver expression of FIX-Paduaprevents and eradicates FIX inhibitor without increasing thrombogenicity inhemophilia B dogs and mice.

[0243] The embodiments of this specification can also be described as follows:

[0244] 1. An antibody or antigen-binding fragment thereof that binds to Padova Factor IX having the amino acid sequence of SEQ ID NO: 1 and does not bind to wild-type (WT) Factor IX having the amino acid sequence of (SEQ ID NO: 2).

[0245] 2. The antibody or antigen-binding fragment of embodiment 1, which binds to the epitope of SEQ ID NO: 1, wherein the epitope is a linear epitope within the amino acid sequence DRATCLLSTKFT (SEQ ID NO: 3).

[0246] 3. The antibody or antigen-binding fragment of embodiment 1, which binds to the epitope of SEQ ID NO: 1, wherein the epitope is a conformational epitope of the folded structure of the amino acid sequence LVDRATCLLSTKFTIYNNMFCAGFH (SEQ ID NO: 5).

[0247] 4. The antibody or antigen-binding fragment of embodiment 1 or 3, wherein the folded structure comprises a disulfide bridge.

[0248] 5. The antibody or antigen-binding fragment of any of the preceding embodiments, which does not bind to the amino acid sequence DRATCLRSTKFT (SEQ ID NO: 14) or LVDRATCLRSTKFTIYNNMFCAGFH (SEQ ID NO: 15).

[0249] 6. The antibody or antigen-binding fragment of any of the preceding embodiments, wherein the antibody or antigen-binding fragment has a K of about 100 nM or less. D Binds to the Padova Factor IX.

[0250] 7. The antibody or antigen-binding fragment of any of the preceding embodiments, wherein the antibody or antigen-binding fragment has a K in the range of about 25 to about 75 nM. D Binds to the Padova Factor IX.

[0251] 8. The antibody or antigen-binding fragment of any of the preceding embodiments, wherein the antibody or antigen-binding fragment has a K in the range of about 50 nM to about 60 nM. D Binds to the Padova Factor IX.

[0252] 9. The antibody or antigen-binding fragment of any of the preceding embodiments, wherein the antibody or antigen-binding fragment has a K in the range of about 20 nM to about 100 nM, about 25 nM to about 95 nM, about 30 nM to about 90 nM, about 35 nM to about 85 nM, about 40 nM to about 80 nM, about 45 nM to about 75 nM, about 50 nM to about 70 nM, or about 55 nM to about 65 nM. D Binds to the Padova Factor IX.

[0253] 10. An antibody or antigen-binding fragment as described in any of the preceding embodiments, wherein the antibody or antigen-binding fragment binds to the Padova Factor IX in a sample containing human plasma and does not bind to WT Factor IX, optionally, wherein the sample comprises at least or about 5%, at least or about 10%, or at least or about 20% human plasma and the sample comprises at least or about 5 μg / mL wild-type Factor IX.

[0254] 11. The antibody or antigen-binding fragment of any of the preceding embodiments, wherein the antibody or antigen-binding fragment does not bind to a Factor II polypeptide or a Factor X polypeptide.

[0255] 12. The antibody or antigen-binding fragment of any of the preceding embodiments, wherein the antibody or antigen-binding fragment binds neither to Factor II nor to Factor X.

[0256] 13. The antibody or antigen-binding fragment of any of the preceding embodiments, which is a Fab or Fab2' antibody fragment.

[0257] 14. The antibody or antigen-binding fragment of any of the preceding embodiments, which is monospecific.

[0258] 15. The antibody or antigen-binding fragment of any of the preceding embodiments, which is fully human.

[0259] 16. The antibody or antigen-binding fragment of any of the preceding embodiments, which is bivalent.

[0260] 17. The antibody or antigen-binding fragment of any of the preceding embodiments, which is bivalent but monospecific for Padova FIX.

[0261] 18. The antibody or antigen-binding fragment of any of the preceding embodiments, comprising a dimerized Fab fragment or a dimerized Fab minibody.

[0262] 19. The antibody or antigen-binding fragment of any of the preceding embodiments, which is a dimerized Fab fragment connected by a linker sequence.

[0263] 20. An antibody or antigen-binding fragment as described in any of the preceding embodiments, which comprises (i) the following amino acid sequence: SSYAIS (SEQ ID NO:6); GIVPAFGTANYAQKFQG (SEQ ID NO:7); SWGVISFAY (SEQ ID NO:8); RASQDISSYLN (SEQ ID NO:9); AASNLQS (SEQ ID NO:10); and MQYDSLPFTF (SEQ ID NO:11), or (ii) the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:25 or SEQ ID NOs:24 and 25, or (iii) the amino acid sequence of SEQ ID NO:26 or SEQ ID NO:27 or SEQ ID NOs:26 and 27.

[0264] 21. A polypeptide comprising an amino acid sequence comprising each of SEQ ID NOs: 6-11, optionally wherein (i) one or more amino acids are present between each of SEQ ID NOs: 6-11, and / or (ii) the polypeptide optionally further comprises a FLAG tag comprising DYKDDDDK (SEQ ID NO: 12) and / or a hexahistidine tag comprising HHHHHH (SEQ ID NO: 13).

[0265] 22. A polypeptide as described in embodiment 21, wherein the FLAG tag and / or the hexahistidine tag is located at the C-terminus of the polypeptide.

[0266] 23. A conjugate comprising the antibody or antigen-binding fragment or polypeptide of any of the preceding embodiments conjugated to a heterologous moiety.

[0267] 24. The conjugate of embodiment 23, wherein the heterologous portion can be selected from the group consisting of a polymer, a carbohydrate, a lipid, a nucleic acid, an oligonucleotide, an amino acid, a peptide, a polypeptide, a protein, and a detection agent.

[0268] 25. The conjugate of embodiment 23 or 24, wherein the antibody or antigen-binding fragment or polypeptide is coupled to agarose, cellulose, dextran, polyacrylamide, latex or controlled pore glass.

[0269] 26. The conjugate of any one of embodiments 23 to 25, wherein the antibody or antigen-binding fragment or polypeptide is conjugated to a fluorophore, a chromophore, a radioisotope, an enzyme label or biotin.

[0270] 27. A conjugate as described in any one of embodiments 23 to 26, comprising a homodimer of the polypeptide described in embodiment 19.

[0271] 28. The conjugate of embodiment 27, wherein the polypeptides in the dimer are connected by a helix-turn-helix structure.

[0272] 29. A nucleic acid comprising a nucleotide sequence encoding the antibody, antigen-binding fragment, polypeptide, conjugate or fragment thereof as described in any one of the preceding embodiments.

[0273] 30. A vector comprising the nucleic acid described in embodiment 29.

[0274] 31. A host cell comprising the nucleic acid of embodiment 29 or the vector of embodiment 30.

[0275] 32. A kit comprising the antibody or antigen-binding fragment of any one of embodiments 1 to 20, the polypeptide of embodiment 21 or 22, the conjugate of any one of embodiments 23 to 28, the nucleic acid of embodiment 29, the vector of embodiment 30 and / or the host cell of embodiment 31.

[0276] 33. The kit of embodiment 32 further comprises a secondary antibody that binds to the antibody, antigen-binding fragment, polypeptide or conjugate.

[0277] 34. The kit of embodiment 32 or 33 further comprises a solid carrier.

[0278] 35. A kit as described in any one of embodiments 32 to 34, wherein the antibody, antigen-binding fragment, polypeptide or conjugate is pre-coated on a solid support.

[0279] 36. A kit as described in embodiment 34 or 35, wherein the solid support is a polymer bead, a microtiter plate, a membrane or a filter.

[0280] 37. The kit of embodiment 35 or 36, comprising a solid support pre-coated with a solution containing about 100 ng or more, about 150 ng or more, about 200 ng or more, about 500 ng or more of the antigen-binding fragment.

[0281] 38. A kit as described in any one of embodiments 32 to 37, wherein the kit further comprises instructions for use.

[0282] 39. A composition comprising a mixture of an antibody or antigen-binding fragment thereof as described in any one of embodiments 1 to 20 and a biological sample obtained from a human, wherein the biological sample comprises human plasma or a diluted fraction thereof, and / or human tissue or cells thereof, wherein optionally, the composition comprises a detection agent.

[0283] 40. A composition comprising a mixture of the antibody or antigen-binding fragment thereof of any one of embodiments 1 to 20 and a biological sample containing human plasma proteins obtained from a human, wherein at least one of the human plasma proteins is selected from the group consisting of factor IX, a variant of factor IX, factor II, a variant of factor II, factor X, and a variant of factor X.

[0284] 41. The composition of embodiment 40, wherein the composition comprises a detection agent.

[0285] 42. Use of the antibody or antigen-binding fragment of any one of embodiments 1 to 20, the polypeptide of embodiment 21 or 22, the conjugate of any one of embodiments 23 to 28, the nucleic acid of embodiment 29, the vector of embodiment 30, the host cell of embodiment 31 and / or the kit of any one of embodiments 32 to 38 for detecting Padova Factor IX in a sample.

[0286] 43. A method for detecting Padova Factor IX in a sample obtained from a subject, comprising (i) contacting the sample with an antibody or antigen-binding fragment as described in any one of embodiments 1 to 20, a polypeptide as described in embodiment 21 or 22, or a conjugate as described in any one of embodiments 23 to 28 to form a complex comprising the Padova Factor IX and the antibody, antigen-binding fragment, polypeptide or conjugate, and (ii) detecting the complex in the sample.

[0287] 44. A method as described in embodiment 43, wherein the antibody or antigen-binding fragment or polypeptide is conjugated to a detection agent and / or a solid support, or wherein the conjugate includes a detection agent.

[0288] 45. The method of embodiment 43 or 44, comprising contacting the sample with a secondary antibody comprising a detection agent, wherein the secondary antibody binds to the antibody or antigen-binding fragment or polypeptide or conjugate.

[0289] 46. ​​A method as described in any one of embodiments 43 to 45, wherein detecting the complex includes detecting a signal of the detection agent.

[0290] 47. A method as described in embodiment 46, wherein the signal is enzymatic activity, binding activity and / or color development activity.

[0291] 48. A method as described in any one of embodiments 43 to 47, wherein the sample is a blood sample, a serum sample or a plasma sample.

[0292] 49. The method of any one of embodiments 43 to 48, wherein the subject has been treated with a vector comprising a nucleotide sequence encoding Padova Factor IX.

[0293] 50. A binding construct as described herein.

[0294] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0295] Unless otherwise indicated herein or clearly contradicted by context, the use of the terms "a", "an", "the" and similar referents in the context of describing the present disclosure (especially in the context of the appended claims) should be construed to cover both the singular and the plural. Unless otherwise indicated, the terms "comprising", "having", "including", and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise noted.

[0296] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value and endpoint of the range, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0297] Unless otherwise indicated herein or otherwise clearly contradictory to the context, all methods described herein can be performed in any suitable order. Unless otherwise required, any and all embodiments used, or exemplary language provided herein (e.g., "such as") are intended only to better illustrate the present disclosure and are not intended to limit the scope of the present disclosure. Any language in this specification should not be interpreted as indicating that any unrequired element is necessary to practice the present disclosure.

[0298] Preferred embodiments of the present disclosure are described herein, including the best mode of implementing the present invention known to the inventor. Those of ordinary skill in the art will readily understand the variations of those preferred embodiments after reading the foregoing description. The inventor expects that a skilled person will adopt these variations when appropriate, and the inventor expects that the present disclosure will be practiced in a manner different from the manner specifically described herein. Therefore, the present disclosure includes all modifications and equivalents of the subject matter described in the appended claims as allowed by applicable law. In addition, unless otherwise indicated herein or otherwise clearly contradictory to the context, the present disclosure encompasses any combination of the above-mentioned elements with all possible variations thereof.

Claims

1. An antibody or antigen-binding fragment thereof that binds to Padova Factor IX comprising the amino acid sequence of SEQ ID NO: 1 and does not bind to wild-type Factor IX, wherein the antibody or antigen-binding fragment thereof comprises the following amino acid sequences: the heavy chain CDR1 of SSYAIS (SEQ ID NO: 6); the heavy chain CDR2 of GIVPAFGTANYAQKFQG (SEQ ID NO: 7); the heavy chain CDR3 of SWGVISFAY (SEQ ID NO: 8); the light chain CDR1 of RASQDISSYLN (SEQ ID NO: 9); the light chain CDR2 of AASNLQS (SEQ ID NO: 10); and the light chain CDR3 of MQYDSLPFTF (SEQ ID NO: 11).

2. The antibody or antigen-binding fragment of claim 1, which does not bind to the amino acid sequence DRATCLRSTKFT (SEQ ID NO: 14).

3. The antibody or antigen-binding fragment of claim 1, which does not bind to the amino acid sequence LVDRATCLRSTKFTIYNNMFCAGFH (SEQ ID NO: 15).

4. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment has a K of 100 nM or less. D Binds to the Padova Factor IX.

5. The antibody or antigen-binding fragment of claim 4, wherein the antibody or antigen-binding fragment has a K in the range of 25 to 75 nM. D Binds to the Padova Factor IX.

6. The antibody or antigen-binding fragment of claim 5, wherein the antibody or antigen-binding fragment has a K in the range of 50 nM to 60 nM. D Binds to the Padova Factor IX.

7. The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment binds to the Padova Factor IX in a sample containing human plasma and does not bind to wild-type Factor IX.

8. The antibody or antigen-binding fragment of claim 7, wherein the sample comprises at least 5% human plasma and the sample comprises at least 5 μg / mL wild-type Factor IX.

9. The antibody or antigen-binding fragment of claim 7, wherein the sample comprises at least 10% human plasma and the sample comprises at least 5 μg / mL wild-type Factor IX.

10. The antibody or antigen-binding fragment of claim 7, wherein the sample comprises at least 20% human plasma and the sample comprises at least 5 μg / mL wild-type Factor IX.

11. The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment does not bind to at least one of a factor II polypeptide or a factor X polypeptide.

12. The antibody or antigen-binding fragment of any one of claims 1 to 6, which is a Fab or Fab2' antibody fragment.

13. The antibody or antigen-binding fragment of any one of claims 1-6, which is monospecific.

14. The antibody or antigen-binding fragment of any one of claims 1-6, which is fully human.

15. The antibody or antigen-binding fragment of any one of claims 1-6, which is bivalent.

16. The antibody or antigen-binding fragment of claim 15, comprising a dimerized Fab fragment or a dimerized Fab minibody. The antibody or antigen-binding fragment according to claim 16 , which is a dimerized Fab fragment connected by a linker.

18. The antibody or antigen-binding fragment of any one of claims 1 to 6, comprising (i) the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25, or the amino acid sequences of SEQ ID NOs: 24 and 25; or (ii) the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27, or the amino acid sequences of SEQ ID NOs: 26 and 27.

19. A conjugate, wherein the conjugate is the antibody or antigen-binding fragment according to any one of claims 1 to 6 conjugated to a heterologous moiety, wherein The heterologous part is a detection agent, a solid support, a His tag and / or a FLAG tag.

20. A nucleic acid comprising a nucleotide sequence encoding the polypeptide portion of the antibody, antigen-binding fragment, or conjugate according to any one of claims 1 to 6. A vector comprising the nucleic acid according to claim 20 .

22. A host cell comprising the nucleic acid according to claim 20 or a vector comprising the nucleic acid.

23. A kit comprising (i) the antibody or antigen-binding fragment according to any one of claims 1 to 6, (ii) the conjugate according to claim 19, (iii) a nucleic acid comprising a nucleotide sequence encoding the polypeptide portion of the antibody, antigen-binding fragment of (i) or the conjugate of (ii), (iv) a vector comprising the nucleic acid of (iii), and / or (v) A host cell comprising the nucleic acid of (iii) or the vector of (iv). The kit according to claim 23 , further comprising instructions for use.

25. The kit of claim 23, further comprising a secondary antibody that binds to the antibody or antigen-binding fragment, or the conjugate.

26. The kit of claim 23, further comprising a solid support.

27. The kit of claim 26, wherein the antibody, antigen-binding fragment or conjugate is pre-coated on the solid support.

28. The kit of claim 26, wherein the solid support is a polymer bead, a microtiter plate, a membrane, or a filter.

29. The kit of claim 27, comprising a solid support pre-coated with a solution containing 100 ng or more of the antigen-binding fragment.

30. The kit of claim 27, comprising a solid support pre-coated with a solution containing 150 ng or more of the antigen-binding fragment.

31. The kit of claim 27, comprising a solid support pre-coated with a solution containing 200 ng or more of the antigen-binding fragment.

32. The kit of claim 27, comprising a solid support pre-coated with a solution containing 500 ng or more of the antigen-binding fragment.

33. A composition comprising a mixture of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and a biological sample obtained from a human, the biological sample comprising human plasma or a diluted fraction thereof, and / or human tissue or cells thereof.

34. The composition of claim 33, wherein the composition comprises a detection agent.

35. A composition comprising a mixture of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and a biological sample containing human plasma proteins obtained from a human, wherein at least one of the human plasma proteins is selected from the group consisting of coagulation factor IX or a variant thereof, coagulation factor II, and coagulation factor X.

36. The composition of claim 35, wherein the composition comprises a detection agent.

37. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 6, the conjugate according to claim 19, the nucleic acid according to claim 20, the vector according to claim 21, and / or the host cell according to claim 22 in the preparation of a kit for detecting Padova Factor IX in a sample.

38. Use of an antibody or Fab according to any one of claims 1 to 6 or a conjugate according to claim 19 in the preparation of a kit for detecting Padova Factor IX in a sample obtained from a subject, wherein the detection comprises (i) contacting the sample with the antibody or Fab or conjugate to form a complex comprising the Padova Factor IX and the antibody, Fab or conjugate, and (ii) detecting the complex in the sample.

39. The use of claim 38, wherein the detecting comprises contacting the sample with a secondary antibody comprising a detection agent, wherein the secondary antibody binds to the antibody or antigen-binding fragment or conjugate.

40. The use of claim 38, wherein detecting the complex comprises detecting a signal of the detection agent.

41. The use according to claim 40, wherein the signal is enzymatic activity, binding activity and / or colorimetric activity.

42. The use according to claim 38, wherein the sample is a blood sample, a serum sample or a plasma sample.

43. The use according to claim 38, wherein the subject has been treated with a vector comprising a nucleotide sequence encoding Padova Factor IX.

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