Antibodies and assays for determining clotting factor viii activity

Abstract

The present disclosure provides anti-ELNN antibodies, as well as methods for assaying the level of Clotting Factor VIII (FVIII) activity level of certain chimeric proteins. Methods provided include methods for determining the level of FVIII activity in the blood or plasma of a human subject who has Willebrand disease (VWD) and to whom an ELNN- and / or VWF fragment-containing chimeric protein such as efanesoctocog alfa has been administered.

Description

ANTIBODIES AND ASSAYS FOR DETERMINING CLOTTING FACTOR VIII ACTIVITY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No.63 / 611,692 filed December 18, 2023, which is incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The content of the electronically submitted sequence listing in XML file (Name: 748429_SA9-493-1_ST26.xml; Size: 84,582 bytes; Date of Creation: December 18, 2023) is incorporated herein by reference in its entirety. BACKGROUND OF THE DISCLOSURE

[0002] Von Willebrand disease is characterized by the qualitative and quantitative deficiency in VWF. There are multiple subtypes of VWD (type 1, 2A, 2B, 2M, 2N, and 3). Type 2N and type 3 are rare recessive disorders resulting from the mutation in the FVIII binding region of VWF in type 2N and a complete lack of or very low levels of VWF in type 3. FVIII levels are substantially reduced in patients with type 2N and 3 VWD.

[0003] Inherited VWD is phenotypically heterogeneous and classified into 3 types. Type 1 and 3 reflect a partial or complete quantitative deficiency of VWF, respectively, while type 2 VWD reflects qualitative defects in VWF. Type 1 VWD is characterized by a mild to moderate reduction in plasma levels of VWF and is the most common (70-80%) form of VWD. The bleeding phenotype of type 1 VWD patients is generally milder than in type 2 and type 3 VWD. In type 2 VWD, 4 subtypes are recognized representing different qualitative VWF defects. Type 3 VWD is characterized by undetectable levels of VWF in plasma and very low plasma levels of FVIII (Mannucci PM. Blood.2001;97(7):1915-9). The VWF gene, which largely determines VWF levels in circulation, is located at chromosome 12. A large variety of mutations or deletions in the VWF gene are associated with VWD (de Jong A. and Eikenboom J. Throm res.2017;159:65-75).

[0004] Similar to patients with hemophilia A who have FVIII deficiency, arthropathy caused by joint bleeds has occasionally been described in patients with moderate and severe VWD. This is often associated with joint pain, lower health-related quality of life, and significantly more radiological and self-reported joint damage. This has led to the use of prophylaxis to prevent joint bleeds in some VWD patients experiencing this complication.SUMMARY OF THE DISCLOSURE

[0005] The present disclosure provides, inter alia, anti-ELNN antibodies, as well as methods for assaying the level of Clotting Factor VIII (FVIII) activity level of certain chimeric proteins. Methods provided include methods for determining the level of FVIII activity in the blood or plasma of a human subject who has Willebrand disease (VWD) and to whom an ELNN- and / or VWF fragment- containing chimeric protein such as efanesoctocog alfa has been administered.

[0006] Certain aspects of the present disclosure are directed to a method of determining the level of exogenous Clotting Factor VIII (FVIII) activity in the blood or plasma of a human subject who has von Willebrand disease (VWD) and to whom a chimeric protein has been administered, wherein the chimeric protein comprises a FVIII protein and an ELNN polypeptide, the method comprising (i) separating the chimeric protein from a biological sample from the subject with an anti-ELNN antibody or antigen-binding fragment thereof to obtain a captured chimeric protein sample, wherein the biological sample is a blood sample or a plasma sample; (ii) assaying the level of FVIII activity in the captured chimeric protein with a chromogenic assay to obtain an assayed FVIII activity value; and (iii) reporting the assayed FVIII activity value or an approximated value thereof without correcting the assayed FVIII activity value or approximated value thereof.

[0007] In some embodiments, the chimeric protein was administered to the subject less than one week before the sample was taken from the subject. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a plasma sample. In some embodiments, correcting the assayed FVIII activity value or approximated value thereof comprises reducing the value. In some embodiments, correcting the assayed FVIII activity value or approximated value thereof comprises reducing the value as if an overestimation of the FVIII activity needs to be accounted for. In some embodiments, an activated partial thromboplastin time (aPTT)-based one stage clotting assay is not used to assess the level of FVIII activity.

[0008] In some embodiments, the anti-ELNN antibody or antigen-binding fragment thereof comprises three light chain complementarity determining region (LCDR) sequences, wherein LCDR1, LCDR2, and LCDR3 comprises SEQ ID NOs: 38, 39 and 40, respectively, and wherein the anti-ELNN antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining region (HCDR) sequences, wherein HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 32, 33 and 34, respectively. In some embodiments, the anti-ELNN antibody or an antigen binding fragment thereof comprises a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:32 and a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:37. In some embodiments, the anti-ELNN antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising three heavy-chain CDRs, and a light chain variable region comprising three light-chain CDRs, wherein said three heavy-chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:32 (HCVR), and said three light-chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:37 (LCVR).

[0009] In some embodiments, the anti-ELNN antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 32 and a light chain variable region (LCVR) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 37. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 32 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 31 and a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 36. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 36.

[0010] In some embodiments, the antibody comprises two heavy chains comprising the amino acid sequence of SEQ ID NO:31, and two light chains comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody comprises two heavy chains consisting of the amino acid sequence of SEQ ID NO:31, and two light chains consisting of the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody or antigen binding fragment thereof binds to an ELNN polypeptide and comprises (i) a heavy chain variable region which is at least 98% identical to SEQ ID NO: 32 and (ii) a light chain variable region which is at least 98% identical to SEQ ID NO: 37.

[0011] In some embodiments, the anti-ELNN antibody or antigen binding portion thereof comprises (i) a light chain variable domain comprising an amino acid sequence at least 90% identical to a light chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs 38, 39 and 40, and (ii) a heavy chain variable domain comprising an amino acid sequence at least 90% identical to a heavy chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs: 32, 33, and 34.

[0012] In some embodiments, the ELNN polypeptide comprises at least three instances of the sequence: SATPE (SEQ ID NO: 28). In some embodiments, the ELNN polypeptide comprises at least 3 instances of the sequence: ESATPE (SEQ ID NO: 29). In some embodiments, the ELNN polypeptide comprises at least 4 instances of the following sequence: SATPE (SEQ ID NO: 28).In some embodiments, the ELNN polypeptide comprises at least 5 instances of the following sequence: SATPE (SEQ ID NO: 28). In some embodiments, the ELNN polypeptide comprises at least 6 instances of the following sequence: SATPE (SEQ ID NO: 28). In some embodiments, each of the at least 3, at least 4, at least 5, or at least 6 instances of SATPE (SEQ ID NO: 28) are as part of the following sequence: ESATPE (SEQ ID NO: 29). In some embodiments, each instance of SATPE (SEQ ID NO: 28) and / or ESATPE (SEQ ID NO: 29) in the sequence of the ELNN polypeptide is followed by at least 3 amino acids at the C-terminal end thereof. In some embodiments, the ELNN polypeptide comprises the sequence of SEQ ID NO: 14, 24, 25, 26, and / or 27. In some embodiments, wherein the anti-ELNN antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:37.

[0013] In some embodiments, the anti-ELNN antibody or binding portion thereof comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:31, wherein the anti-ELNN antibody or binding portion thereof comprises a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:36. In some embodiments, the anti-ELNN antibody or binding portion thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:31, and wherein the anti-ELNN antibody or binding portion thereof a light chain comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the anti- ELNN antibody or binding portion thereof comprises two heavy chains comprising the amino acid sequence of SEQ ID NO:31, and wherein the anti-ELNN antibody or binding portion thereof comprises two light chains comprising the amino acid sequence of SEQ ID NO:36.

[0014] Certain aspects of the present disclosure are directed to a method of determining the level of exogenous Clotting Factor VIII (FVIII) activity in the blood or plasma of a human subject who has von Willebrand disease (VWD) and to whom a chimeric protein has been administered, wherein the chimeric protein comprises a FVIII protein and an ELNN polypeptide, the method comprising (i) separating the chimeric protein from a biological sample from the subject with an anti-ELNN antibody or antigen-binding fragment thereof to obtain a captured chimeric protein sample, wherein the biological sample is a blood sample or a plasma sample; (ii) assaying the level of FVIII activity in the captured chimeric protein with a chromogenic assay to obtain an assayed FVIII activity value; and (iii) reporting the assayed FVIII activity value or an approximated value thereof without correcting the assayed FVIII activity value or approximated value thereof, wherein the chimeric protein further comprises a von Willebrand Factor (VWF) fragment.

[0015] Certain aspects of the present disclosure are directed to a method for determining the level of Clotting Factor VIII (FVIII) activity in the blood or plasma of a human subject who has vonWillebrand disease (VWD) and to whom a chimeric protein has been administered, wherein the chimeric protein comprises (a) a FVIII protein and (b) an ELNN polypeptide and / or a von Willebrand Factor (VWF) fragment, the method comprising (i) assaying the level of FVIII activity in the chimeric protein with an activated partial thromboplastin time (aPTT)-based one stage clotting assay to obtain an assayed FVIII activity value, wherein the aPTT-based one stage clotting assay does; (ii)reducing the assayed FVIII activity value to obtain an adjusted FVIII activity value; and (iii) reporting the adjusted FVIII activity value or an approximated value thereof.

[0016] In some embodiments, the chimeric protein comprises a FVIII protein and an ELNN polypeptide. In some embodiments, the ELNN polypeptide is inserted into the FVIII protein of the chimeric protein. In some embodiments, the FVIII activity value or the approximated value thereof is reported to the subject, a caregiver of the subject, or a medical professional. In some embodiments, the subject has a body mass index of ≥30 kg / m2. In some embodiments, the subject is a female. In some embodiments, the subject is a male. In some embodiments, the subject is a human.

[0017] In some embodiments, the VWD is Type 2N VWD. In some embodiments, the VWD is Type 3 VWD.

[0018] Certain aspects of the present disclosure are directed to a method of monitoring the level of FVIII in the blood or plasma of a human subject who is receiving treatment for Willebrand disease (VWD), the method comprising performing a method for determining the level of FVIII activity disclosed herein at least once per year, e.g., at least once every 6 months or at least once per month. In some embodiments, the level of FVIII activity is determined at least once per year. In some embodiments, the level of FVIII activity is determined at least once every 9 months. In some embodiments, the level of FVIII activity is determined at least once every 6 months. In some embodiments, the level of FVIII activity is determined at least once per month.

[0019] In some embodiments, multiple doses of the chimeric protein are administered to the subject at a dosing interval. In some embodiments, at least one of the multiple doses is from about 10 IU / kg to about 30 IU / kg and the dosing interval is at least about 7 days. In some embodiments, at least one of the multiple doses is from about 25 IU / kg and the dosing interval is at least about 7 days.

[0020] Certain aspects of the present disclosure are directed to a method for determining the level of Clotting Factor VIII (FVIII) activity from a chimeric protein in a biological sample, wherein the chimeric protein comprises a FVIII protein and an ELNN polypeptide, the method comprising (i) separating the chimeric protein from the biological sample from the subject with an anti-ELNN antibody to obtain a captured chimeric protein sample, wherein the biological sample is a bloodsample or a plasma sample; and (ii) assaying the level of FVIII activity in the captured chimeric protein with a chromogenic assay to obtain an assayed FVIII activity value.

[0021] In some embodiments, the chimeric protein is in a pharmaceutical composition comprising sucrose, histidine, arginine, calcium chloride, and polysorbate 80. In some embodiments, the chimeric protein is in a pharmaceutical composition comprising: (a) about 5% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride; and (e) about 0.05% polysorbate 80, and a pH of about 7.0. In some embodiments, the pharmaceutical composition is lyophilized. In some embodiments, the lyophilized pharmaceutical composition is reconstituted with sterile water prior to administering to the subject.

[0022] In some embodiments, the chimeric protein comprises a VWF fragment. In some embodiments, the VWF fragment comprises a D’D3 fragment of VWF. In some embodiments, the VWF fragment consists of a D’D3 fragment of VWF. In some embodiments, the D’D3 fragment of VWF is mutated such that it cannot form disulfide binds with another D’D3 fragment of VWF. In some embodiments, the D' domain of the D’D3 fragment of VWF comprises the sequence of SEQ ID NO: 21. In some embodiments, the D3 domain of the D’D3 fragment of VWF comprises the sequence of SEQ ID NO: 23. In some embodiments, wherein the FVIII protein comprises at least a partial deletion of the wild-type FVIII B domain. In some embodiments, the chimeric protein comprises an ELNN polypeptide comprising the sequence of SEQ ID NO: 14. In some embodiments, the chimeric protein comprises an ELNN polypeptide comprising the sequence of SEQ ID NO: 24.

[0023] In some embodiments, the chimeric protein comprises a first polypeptide which comprises a FVIII protein and a first immunoglobulin (“Ig”) constant region or a portion thereof, and a second polypeptide which comprises a VWF fragment and a second Ig constant region or a portion thereof. In some embodiments, the chimeric protein comprises (i) a first polypeptide comprising a FVIII protein, an ELNN polypeptide inserted within and replacing a portion of the B domain, and a first Fc region; and (ii) a second polypeptide comprising a VWF fragment, an additional ELNN polypeptide, an a2 linker, and a second Fc region. In some embodiments, the chimeric protein is a FVIII-ELNN-Fc / D’D3-ELNN-Fc heterodimer. In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 1 and the second polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 2, and wherein the first polypeptide chain and the second polypeptide chain are covalently linked by two disulfide bonds between the Ig constant region domains of the first and second polypeptide chains.

[0024] In some embodiments, the chimeric protein is efanesoctocog alfa.

[0025] Certain aspects of the present disclosure are directed to an antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, wherein the antibody or antigen-binding fragment thereof comprises three light chain complementarity determining region (CDR) sequences of SEQ ID NOs: 38, 39 and 40, and three heavy chain complementarity determining region (CDR) sequences of SEQ ID NOs: 32, 33 and 34, and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0026] Certain aspects of the present disclosure are directed to an antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, wherein the antibody or antigen-binding fragment thereof comprises three light chain complementarity determining region (CDR) sequences of SEQ ID NOs: 38, 39 and 40, and three heavy chain complementarity determining region (CDR) sequences of SEQ ID NOs: 32, 33 and 34, and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: ESATPE (SEQ ID NO: 29).

[0027] Certain aspects of the present disclosure are directed to an antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, comprising three heavy-chain CDRs of a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:32 and three light-chain CDRs of a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:37 and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0028] Certain aspects of the present disclosure are directed to an antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, comprising three heavy-chain CDRs of a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:32 and three light-chain CDRs of a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:37 and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: ESATPE (SEQ ID NO: 29).

[0029] Certain aspects of the present disclosure are directed to an antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, comprising a heavy chain variable region comprising three heavy-chain CDRs, and a light chain variable region comprising three light-chain CDRs, wherein said three heavy-chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:32 (HCVR), and said three light-chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:37 (LCVR), and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: ESATPE (SEQ ID NO: 29). In some embodiments, the antibody or antigen- binding fragment thereof comprises a heavy chain variable region (HCVR) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:32 and a light chain variable region (LCVR) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:37. Insome embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:31 and a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:36. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:31 and a light chain comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody comprises two heavy chains comprising the amino acid sequence of SEQ ID NO:31, and two light chains comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody comprises two heavy chains consisting of the amino acid sequence of SEQ ID NO:31, and two light chains consisting of the amino acid sequence of SEQ ID NO:36.

[0030] Certain aspects of the present disclosure are directed to a monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen binding fragment thereof binds to an ELNN polypeptide and comprises (i) a heavy chain variable region which is at least 98% identical to SEQ ID NO: 32 and (ii) a light chain variable region which is at least 98% identical to SEQ ID NO: 37, and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0031] Certain aspects of the present disclosure are directed to a monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen binding fragment thereof binds to an ELNN polypeptide and comprises (i) a heavy chain variable region which is at least 98% identical to SEQ ID NO: 32 and (ii) a light chain variable region which is at least 98% identical to SEQ ID NO: 37, and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 29).

[0032] Certain aspects of the present disclosure are directed to an antigen binding protein comprising (i) a light chain variable domain comprising an amino acid sequence at least 90% identical to a light chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs 38, 39 and 40, and (ii) a heavy chain variable domain comprising an amino acid sequence at least 90% identical to a heavy chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs: 32, 33, and 34, and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28). In some embodiments, the ELNN polypeptide comprises the sequence: ESATPE (SEQ ID NO: 28). In some embodiments, at least 3 instances of the sequence SATPE (SEQ ID NO: 28) are ESATPE (SEQ ID NO: 29). In some embodiments, each of the instances of the sequence SATPE (SEQ ID NO: 28) is ESATPE (SEQ ID NO: 29). In someembodiments, the ELNN polypeptide comprises at least 4 instances of the following sequence: SATPE (SEQ ID NO: 28). In some embodiments, the ELNN polypeptide comprises at least 5 instances of the following sequence: SATPE (SEQ ID NO: 28). In some embodiments, the ELNN polypeptide comprises at least 6 instances of the following sequence: SATPE (SEQ ID NO: 28). In some embodiments, each of the at least 3, at least 4, at least 5, or at least 6 instances of SATPE (SEQ ID NO: 28) are part of the following sequence: ESATPE (SEQ ID NO: 29). In some embodiments, each instance of SATPE (SEQ ID NO: 28) and / or ESATPE (SEQ ID NO: 29) in the sequence of the ELNN polypeptide is followed by at least 3 amino acids at the C-terminal end thereof. In some embodiments, the ELNN polypeptide comprises the sequence of SEQ ID NO: 14, 24, 25, 26, and / or 27.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:37.

[0034] Certain aspects of the present disclosure are directed to an antibody or antigen-binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:31 and a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:36.

[0035] Certain aspects of the present disclosure are directed to an antibody or antigen-binding fragment comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:31 and a light chain comprising the amino acid sequence of SEQ ID NO:36.

[0036] Certain aspects of the present disclosure are directed to an antibody comprising two heavy chains comprising the amino acid sequence of SEQ ID NO:31, and two light chains comprising the amino acid sequence of SEQ ID NO:36.

[0037] In some embodiments, the antibody, antigen-binding fragment, or antigen binding protein is isolated antibody, antigen-binding fragment, or antigen binding protein.

[0038] Certain aspects of the present disclosure are directed to a polynucleotide or set of polynucleotides encoding any of the antibodies, antigen-binding fragments, or antigen binding proteins disclosed herein.

[0039] Certain aspects of the present disclosure are directed to a vector comprising the polynucleotide or set of polynucleotides encoding any of the antibodies, antigen-binding fragments, or antigen binding proteins disclosed herein.

[0040] Certain aspects of the present disclosure are directed to a host cell comprising a vector comprising the polynucleotide or set of polynucleotides encoding any of the antibodies, antigen-binding fragments, or antigen binding proteins disclosed herein. In some embodiments, the host cell is a CHO cell or a HEK293 cell. BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

[0041] FIG. 1 is a schematic representation of a rFVIIIFc-VWF-ELNN heterodimer. Abbreviations are FVIII: factor VIII; VWF: von Willebrand Factor; A1, A2, A3, C1, C2: domains of FVIII; D'D3: domains of VWF; ELNN: extended length polypeptides; Fc: Fc region of immunoglobulin constant region.

[0042] FIG.2 is a graphical representation of the design of the clinical study. Abbreviations are DDAVP: Desmopressin acetate (1-deamino-8-D-arginine vasopressin); FVIII: Factor VIII; IV: Intravenous; PK: Pharmacokinetics; VWF: von Willebrand factor.

[0043] FIG. 3 is a graphical representation of the mean and individual participant baseline- corrected FVIII activity-time profiles quantified by the (A) OSA and (B) CCS. Abbreviations are CCS: capture chromogenic assay; FVIII, factor VIII; h, hour; OSA, one-stage assay. Mean values are displayed with black filled circles. Participants with Type 2N VWD (Participants 2 and 3) are displayed with filled triangles and Xs, respectively. Participants with Type 3 VWD (Participants 1, 4, 5, and 6) displayed with open circles, open stars, open diamonds, and open squares, respectively.

[0044] FIG. 4 is a graphical display showing binding of the 4D9G3 antibody to 183 unique peptides. The x-axis indicates peptide number, and the y-axis represents ELISA signal intensity.

[0045] FIG. 5 is a graphical display showing binding of the 4D9G3 antibody to 183 unique peptides.43 peptides containing the ESATPE epitope showed significant binding (black bars), 5 peptides contain only SATPE showed reduced binding (dark gray bars), and the remaining 135 peptides, which did not contain the epitope, showed very low binding (light gray bars). The x-axis indicates peptide number, while the y-axis represents ELISA signal intensity.

[0046] FIG. 6 is a graphical representation of the binding of the 43 peptides found to be significant binders. All peptides with equal or less than 2 aa on the C-terminus of the epitope showed inferior signal. On the other hand, if the epitope is on the very N-terminus, binding to the 4D9G3 antibody was not affected. DETAILED DESCRIPTION

[0047] In the absence of exogenous VWF, the half-life of recombinant FVIII has been shown to be very short in certain VWD patients (VWD type 2N and 3) patients compared to hemophilia A patients, suggesting that regular FVIII products that depend on VWF cannot be readily used inpatients with severe VWD disease, even when the goal is that of correcting the FVIII defect only (e.g., for prophylaxis against bleeds or perioperative management). Efanesoctocog alfa temporarily replaces the missing FVIII needed for effective hemostasis in patients with a deficiency of FVIII. Efanesoctocog alfa is the first FVIII therapy that is engineered to circulate independently from VWF, thereby uncoupling FVIII from VWF clearance and extending half-life.

[0048] The present disclosure provides, inter alia, methods for assaying, determining, and monitoring the level of administered FVIII (e.g., FVIII of a chimeric protein such as efanesoctocog alfa) in patients who have VWD. In some embodiments, these methods comprise use of a chimeric protein such as efanesoctocog alfa.

[0049] Also provided are anti-ELNN antibodies and fragments thereof and methods using such antibodies and fragments. Non-limiting examples of such methods include binding ELNN polypeptides, capturing ELNN polypeptides (e.g., isolating ELNN polypeptide-containing polypeptides from a solution such as a sample), and labeling ELNN polypeptides (e.g., with anti- ELNN antibodies or fragments thereof that are conjugated to a fluorophore or other detectable molecule). I. Definitions

[0050] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence," is understood to represent one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0051] Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0052] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In some embodiments, the term indicates deviation from the indicated numerical value by ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%. In some embodiments, “about”indicates deviation from the indicated numerical value by ±10%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±2%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.9%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.8%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.7%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.6%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.05%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.01%.

[0053] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related.

[0055] Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0056] In some embodiments, a polynucleotide is an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). Depending on context, a "polynucleotide" may be any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a larger polynucleotide, such as a genome or vector. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which is not in its natural milieu (e.g., it is not within a wild-type cell or virus). For example, a recombinant polynucleotide encoding a Factor VIIIprotein contained in a vector is considered isolated for the purposes of the present disclosure. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in asolution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides ofthe present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid can include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

[0057] Certain proteins secreted by mammalian cells are associated with a secretory signal peptide which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that signal peptides are generally fused to the N-terminus of the polypeptide and are cleaved from the complete or full-length polypeptide to produce a secreted or mature form of the polypeptide. In some embodiments, a native signal peptide or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. It will be understood that, in instances where a polypeptide sequence is disclosed with a signal peptide, the form of the polypeptide sequence without the signal peptide is also disclosed.

[0058] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, but does not refer to any particular amino acid length. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with any of these terms, depending on context. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

[0059] An "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can simply be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are consideredisolated for the purpose of the disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0060] Also included in the present disclosure are fragments or variants of polypeptides, and any combination thereof. The term "fragment" or "variant" when referring to polypeptide binding domains or binding molecules of the present disclosure include any polypeptides that retain at least one or more of the properties (e.g., FcRn binding affinity for an FcRn binding domain or Fc variant, coagulation activity for an FVIII variant, or FVIII binding activity for the VWF fragment) of the reference polypeptide. Fragments of polypeptides include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein, but do not include the naturally occurring full-length polypeptide (or mature polypeptide). Variants of polypeptide binding domains or binding molecules of the present disclosure include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can be naturally or non-naturally occurring. Non- naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions, or additions.

[0061] The term "VWF fragment" includes any VWF fragment that interacts with FVIII and retains at least one or more properties that are normally provided to FVIII by full-length VWF, e.g., preventing or reducing premature activation to FVIIIa, preventing or reducing premature proteolysis, preventing or reducing clearance, preventing or reducing association with phospholipid membranes that could lead to premature clearance, preventing or reducing binding to FVIII clearance receptors that can bind naked FVIII but not VWF-bound FVIII, and / or stabilizing FVIII (such as FVIII heavy chain and light chain interactions). A VWF fragment referred to herein is a VWF polypeptide that is less than the full-length VWF protein, wherein the VWF fragment retains the ability to interact with and / or bind to FVIII. In some embodiments, a VWF fragment is a fragment (which may be mutated) of full-length VWF that binds to a FVIII polypeptide such that the FVIII polypeptide has reduced binding to, or does not bind, full length VWF (e.g., endogenous VWF in a subject). In some embodiments, the VWF fragment is a human VWF fragment, or a mutant VWF fragment. In some embodiments, the VWF fragment comprises the D’ and D3 domains of VWF. In some embodiments, the VWF fragment is a D’D3 fragment that is mutated to substitute one or two cysteines to alanines such that the fragment is not part of a dimer.

[0062] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In some embodiments, a string of amino acids can be conservatively replaced with a structurally similar string that differs in order and / or composition of side chain family members.

[0063] As known in the art, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. Similarly, "sequence identity" between two polynucleotides is determined by comparing the nucleotide sequence of one polynucleotide to the sequence of a second polynucleotide. The terms “% identical”, “% identity” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids (as applicable) which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. For example, the optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol.48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment. When discussed herein, whether any particularpolypeptide is, e.g., at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide, can be determined using methods and computer programs / software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, the parameters are set such that the percentage of identity is calculated over the full-length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

[0064] An amino acid corresponding to an amino acid in a VWF sequence or a FVIII sequence may be identified by alignment to maximize the identity or similarity between a first VWF or FVIII sequence and a second VWF or FVIII sequence. The number used to identify a corresponding amino acid in a second VWF or FVIII sequence is based on the number used to identify the corresponding amino acid in the first VWF or FVIII sequence.

[0065] As used herein, the term "insertion site" refers to a position in a FVIII polypeptide, or fragment, variant, or derivative thereof, which is immediately downstream of the position at which a half-life extending moiety or heterologous moiety can be inserted. An "insertion site" is specified as a number, the number being the number of the amino acid in mature native (wild type) human FVIII (SEQ ID NO: 8) to which the insertion site corresponds, which is immediately C-terminal to the position of the insertion. For example, the phrase "comprises an ELNN polypeptide at an insertion site which corresponds to amino acid 1656 of SEQ ID NO: 8" indicates that the heterologous moiety is inserted immediately after the amino acid residue corresponding to amino acid residue 1656 of SEQ ID NO: 8 (without requiring amino acid residue 1657 of SEQ ID NO: 8 to be present).

[0066] The terms "inserted," "is inserted," "inserted into" or grammatically related terms, as used herein with respect to insertions of ELNN polypeptide into FVIII refers to the position of an ELNN polypeptide in a chimeric protein relative to the analogous position in native mature human FVIII (SEQ ID NO: 8). As used herein the terms refer to the characteristics of the recombinant FVIII polypeptide relative to native mature human FVIII, and do not indicate, imply or infer any methods or process by which the chimeric protein was made. For example, in reference to a chimeric protein provided herein, the phrase "an ELNN polypeptide is inserted immediately downstream of residue 745 of the FVIII polypeptide" means that the chimeric protein comprises an ELNNpolypeptide immediately downstream of an amino acid residue which corresponds to amino acid residue 745 in native mature human FVIII, e.g., bounded by amino acids corresponding to amino acid residues 745 and 746 of native mature human FVIII (without requiring the presence of an amino acid residue corresponding to 746 of native mature human FVIII), and does not connote an order or method of production for which the chimeric protein was constructed.

[0067] As used herein, the terms “ELNN polypeptide” and “ELNN” are synonymous, and refer to extended length polypeptides comprising non-naturally occurring, substantially non-repetitive sequences (e.g., polypeptide motifs) that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions. Such extended length polypeptides include unstructured hydrophilic polypeptides comprising repeating motifs of 6 natural amino acids (G, A, P, E, S, and / or T). In some embodiments, an ELNN polypeptide comprises multiple motifs of 6 natural amino acids (G, A, P, E, S, T), wherein the motifs are the same or comprise a combination of different motifs. ELNN polypeptides can confer certain desirable pharmacokinetic, physicochemical and pharmaceutical properties when linked to a VWF fragment or a FVIII sequence of the disclosure to create a chimeric protein. Such desirable properties include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics. ELNN polypeptides are known in the art, and non-limiting descriptions relating to and examples of ELNN polypeptides referred to as XTEN polypeptides are available in Schellenberger et al., (2009) Nat Biotechnol 27(12):1186-90; Brandl et al., (2020) Journal of Controlled Release 327:186-197; and Radon et al., (2021) Advanced Functional Materials 31, 2101633 (pages 1-33), the entire contents of each of which are incorporated herein by reference.

[0068] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature. The amino acid sequences which normally exist in separate proteins can be brought together in the fusion polypeptide, or the amino acid sequences which normally exist in the same protein can be placed in a new arrangement in the fusion polypeptide, e.g., fusion of a Factor VIII domain of the disclosure with an immunoglobulin Fc domain. A fusion protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. A chimeric protein can further comprise a second amino acid sequence associated with the first amino acid sequence by a covalent, non-peptide bond or a non-covalent bond.

[0069] With respect to amino acid and nucleotide sequences, the term "linked" as used herein refers to a first amino acid sequence or nucleotide sequence covalently or non-covalently joined to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid ornucleotide sequence can be directly joined or juxtaposed to the second amino acid or nucleotide sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence. Depending on context, the term "linked" means not only a fusion of a first amino acid sequence to a second amino acid sequence at the C-terminus or the N-terminus, but also includes insertion of the whole first amino acid sequence (or the second amino acid sequence) at an insertion site of the second amino acid sequence (or the first amino acid sequence, respectively). In some embodiments, the first amino acid sequence can be linked to a second amino acid sequence by a peptide bond or a linker. In some embodiments, a first nucleotide sequence can be linked to a second nucleotide sequence by a phosphodiester bond or a linker. In some embodiments, the linker can be a peptide or a polypeptide (for polypeptide chains) or a nucleotide or a nucleotide chain (for nucleotide chains) or any chemical moiety (for both polypeptide and polynucleotide chains). The term "linked" may also be indicated by a hyphen (-).

[0070] With respect to two polypeptides, the term "associated with" refers to one or more covalent or non-covalent bonds formed between a first polypeptide and a second polypeptide. In some embodiments, the term "associated with" means a covalent, non-peptide bond or a non- covalent bond. This association can be indicated by a colon, i.e., (:). In some embodiments, it means a covalent bond other than a peptide bond. For example, the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a thiol group on a second cysteine residue. In many naturally occurring IgG molecules, the CH1 and CL regions are associated by a disulfide bond and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system). Examples of covalent bonds include, but are not limited to, a peptide bond, a metal bond, a hydrogen bond, a disulfide bond, a sigma bond, a pi bond, a delta bond, a glycosidic bond, an agnostic bond, a bent bond, a dipolar bond, a Pi backbond, a double bond, a triple bond, a quadruple bond, a quintuple bond, a sextuple bond, conjugation, hyperconjugation, aromaticity, hapticity, or antibonding. Non-limiting examples of non-covalent bond include an ionic bond (e.g., cation-pi bond or salt bond), a metal bond, a hydrogen bond (e.g., dihydrogen bond, dihydrogen complex, low-barrier hydrogen bond, or symmetric hydrogen bond), van der Walls force, London dispersion force, a mechanical bond, a halogen bond, aurophilicity, intercalation, stacking, entropic force, or chemical polarity. In some embodiments, the one or more covalent bonds between the first amino acid chain and the second amino acid chain is two disulfide bonds. In some embodiments, the one or more covalent bonds between the first amino acid chain and the second amino acid chain is two disulfide bonds between a first Fc portion on the first amino acid chain and a second Fc portion on the second amino acid chain, wherein the two disulfide bonds occur in the hinge region of the two Fc portions.

[0071] In some embodiments, a polypeptide has an enzymatic cleavage site cleaved by an enzyme that is activated during the clotting cascade, such that cleavage of such sites occurs at the site of clot formation. Exemplary such sites include, e.g., those recognized by thrombin, Factor XIa or Factor Xa. Other enzymatic cleavage sites are known in the art and described in elsewhere herein. In constructs that include more than one processing or cleavage site, it will be understood that such sites can be the same or different.

[0072] The composition, e.g., the chimeric protein, can be administered to a subject using methods known in the art. In some embodiments, the administration is intravenous. In some embodiments, the administration is subcutaneous. In some embodiments, the administration is self-administration. In some embodiments, a parent administers the chimeric protein to a child. In some embodiments, the chimeric protein is administered to a subject by a healthcare practitioner, such as a medical doctor, a medic, or a nurse.

[0073] In some embodiments, a single dose is administered to a subject. In some embodiments, multiple doses are administered to a subject. A single dose can be administered all at once, e.g., as a bolus, or over a period of time, e.g., via an intravenous infusion.

[0074] In some embodiments, the composition, e.g., the chimeric protein, is delivered via a slow push IV injection of 8 ±2 minutes. In some embodiments, the composition is delivered at a rate of administration determined by the subject’s comfort level. In some embodiments, the composition is delivered via a slow push IV injection at a rate of administration determined by the subject’s comfort level and according to the following vial injection rate recommendations: For subjects weighing ≤55 kg, the minimum injection duration per vial is 2 minutes per vial; for subjects weighing >55 kg, the minimum injection duration per vial is 1 minute per vial.

[0075] As used herein, the term "dosing interval" refers to the amount of time that elapses between when a first dose of a composition and a subsequent dose of the same composition are administered to a subject. A dosing interval can refer to the time that elapses between multiple doses. For example, a dosing interval can be written as once a week, once every two weeks, etc.

[0076] A “pharmaceutical label” is a display of written matter upon a container or printed medium that comprises statements about the administration, efficacy, and / or safety of a therapeutic agent such as a chimeric protein or a pharmaceutical composition comprising a chimeric protein.

[0077] aPTT tests measure the efficacy of both the "intrinsic" (also referred to the contact activation pathway) and the common coagulation pathways. This test is commonly used to measure clotting activity of commercially available recombinant clotting factors, e.g., FVIII.. In one embodiment, aPTT is tested using an assay where FVIII activity is measured using the Dade®Actin®FSL Activated PTT Reagent (Siemens Health Care Diagnostics) on a BCS®XP analyzer (Siemens Healthcare Diagnostics).

[0078] The aPTT assay may also be used for assessing the potency of a chimeric protein prior to administration to a patient or subject (Hubbard AR, et al. J Thromb Haemost 11: 988–9 (2013)). The aPTT assay may further be used in conjunction with any of the assays described herein, either prior to administration or following administration to a patient or subject.

[0079] Aspects relating to the use of aPTT assays in connection with efanesoctocog alfa and hemophilia A (including with respect to different reagents) are described in Pipe et al., “A global comparative field study to evaluate the factor VIII activity of efanesoctocog alfa by one-stage clotting and chromogenic substrate assays at clinical haemostasis laboratories”, Haemophilia 2023;1–10; DOI:10.1111 / hae.14831, the entire contents of which are incorporated herein by reference.

[0080] The chromogenic assay mechanism is based on the principles of the blood coagulation cascade, where activated FVIII accelerates the conversion of Factor X into Factor Xa in the presence of activated Factor IX, phospholipids and calcium ions. The Factor Xa activity is assessed by hydrolysis of a p-nitroanilide (pNA) substrate specific to Factor Xa. The initial rate of release of p-nitroaniline measured at 405 nM is directly proportional to the Factor Xa activity and thus to the FVIII activity in the sample. In some embodiments, the chromogenic assay is the BIOPHEN FVIII:C assay (Hyphen Biomed, Neurville sur Oise, France)

[0081] The chromogenic assay may also be used for assessing the potency of a chimeric protein prior to administration to a patient or subject. (Hubbard AR, et al. J Thromb Haemost 11: 988–9 (2013)). The chromogenic assay may further be used in conjunction with any of the assays described herein, either prior to administration or following administration to a patient or subject.

[0082] In some embodiments, the chromogenic assay is a capture chromogenic assay that uses an antibody (such as an anti-ELNN antibody) to separate exogenous FVIII (e.g., in the context of a chimeric protein, such as FVIII of efanesoctocog alfa) from endogenous FVIII in a sample (e.g., plasma), before assessing the level of the exogenous FVIII activity using a chromogenic assay. A non-limiting example of a capture chromogenic assay is described in Example 1. In some embodiments, the exogenous FVIII (such as a chimeric protein comprising FVIII, e.g., efanesoctocog alfa) is used as a standard (e.g., a calibration standard) for a capture chromogenic assay.

[0083] As used herein, a “captured chimeric protein sample” is a sample derived from a biological sample (such as blood or plasma) from a subject in which the ratio of chimeric protein to endogenous FVIII protein is greater than the ratio of chimeric protein to endogenous FVIIIprotein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured chimeric protein sample is less than 10% the amount of the endogenous FVIII protein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured chimeric protein sample is less than 5% the amount of the endogenous FVIII protein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured chimeric protein sample is less than 1% the amount of the endogenous FVIII protein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured chimeric protein sample is less than 0.5% the amount of the endogenous FVIII protein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured chimeric protein sample is less than 0.1% the amount of the endogenous FVIII protein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured chimeric protein sample is less than 0.01% the amount of the endogenous FVIII protein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured chimeric protein sample is undetectable by a Western Blot assay. In some embodiments, the biological sample is blood that has been obtained from a subject who has type 2N or type 3 VWD. In some embodiments, the biological sample is plasma that has been obtained from a subject who has type 2N or type 3 VWD.

[0084] Consistently, a “captured efanesoctocog alfa sample” is a sample derived from a biological sample (such as blood or plasma) from a subject in which the ratio of efanesoctocog alfa protein to endogenous FVIII protein is greater than the ratio efanesoctocog alfa protein to endogenous FVIII protein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured efanesoctocog alfa sample is less than 10% the amount of the endogenous FVIII protein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured efanesoctocog alfa sample is less than 5% the amount of the endogenous FVIII protein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured efanesoctocog alfa sample is less than 1% the amount of the endogenous FVIII protein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured efanesoctocog alfa sample is less than 0.5% the amount of the endogenous FVIII protein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured efanesoctocog alfa sample is less than 0.1% the amount of the endogenous FVIII protein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured efanesoctocog alfa sample is less than 0.01% the amount of the endogenous FVIII protein in the biological sample. In some embodiments, the amount of endogenous FVIII protein in the captured efanesoctocog alfa sample is undetectable by a Western Blot assay. In some embodiments, the biological sample is blood that has beenobtained from a subject who has type 2N or type 3 VWD. In some embodiments, the biological sample is plasma that has been obtained from a subject who has type 2N or type 3 VWD.

[0085] As used herein, an “approximated value” of a FVIII activity value (such as an assayed FVIII activity value or an adjusted FVIII activity value) is a value derived by approximating (e.g., rounding) the FVIII activity value (e.g., to the nearest integer (up or down), the nearest half of an integer, the nearest tenth of an integer, or the nearest hundredth of an integer), so long as the approximated value does not deviate from the FVIII activity value by more than 5 or ±20%. In some embodiments, the approximated value does not deviate from the FVIII activity value by more than 5. In some embodiments, the approximated value does not deviate from the FVIII activity value by more than 1. In some embodiments, the approximated value does not deviate from the FVIII activity value by more than ±20%. In some embodiments, the approximated value does not deviate from the FVIII activity value by more than ±15%. In some embodiments, the approximated value does not deviate from the FVIII activity value by more than ±10%. In some embodiments, the approximated value does not deviate from the FVIII activity value by more than ±5%. In some embodiments, the approximated value does not deviate from the FVIII activity value by more than ±5%. Unlimiting illustrative examples include the following: (i) the FVIII activity value is 14.455 IU / dL and the approximated value is 14.46 (e.g., approximated to the nearest hundredth with the number 5 being rounded up to 6), (ii) the FVIII activity value is 14.455 IU / dL and the approximated value is 14.5 (e.g., rounded to the nearest tenth with the number 4 being rounded up to 5); (iii) the FVIII activity value is 14.456 IU / dL and the approximated value is 14 (e.g., rounded to the nearest integer); (iv) the FVIII activity value is 14.456 IU / dL and the approximated value is 15 (e.g., rounded to the nearest 5thinteger). II. Chimeric Proteins

[0086] In an aspect, the present disclosure is directed to detecting levels of a chimeric protein. In some embodiments, the chimeric protein comprises a FVIII protein and an ELNN polypeptide. In some embodiments, the ELNN polypeptide is inserted into the FVIII protein of the chimeric protein. In some embodiments, the chimeric protein was administered to the subject less than one week before a sample was taken from the subject. In some embodiments, the chimeric protein further comprises a von Willebrand Factor (VWF) fragment. In some embodiments, a chimeric protein comprises a first polypeptide which comprises a Factor VIII (“FVIII”) protein or a portion thereof and a first immunoglobulin (“Ig”) constant region or a portion thereof, and a second polypeptide which comprises a von Willebrand Factor (“VWF”) fragment and a second Ig constant region or a portion thereof. In some embodiments, the chimeric protein comprises (i) a first polypeptide comprising a FVIII polypeptide, an ELNN polypeptide inserted within the B domain ofthe FVIII protein (e.g. replacing a portion of the B domain, such as the majority of the B domain), and a first Fc region; and (ii) a second polypeptide comprising a VWF fragment, a second ELNN polypeptide sequence, an a2 linker, and a second Fc region. In some embodiments, the chimeric protein disclosed herein is a FVIII-ELNN-Fc / D’D3-ELNN-Fc heterodimer. A graphical representation of an rFVIIIFc-VWF-ELNN heterodimer is shown in FIG.1.

[0087] In some embodiments, the chimeric protein is efanesoctocog alfa. Efanesoctocog alfa, also known as “BIVV001”, “Efa”, and “rFVIIIFc-VWF-XTEN”, is described in Chhabra et al. Blood 2020; 135(17): 1484-1496, Konkle et al., N Engl J Med 2020; 383:1018-1027, and the International Nonproprietary Names for Pharmaceutical Substances (INN) WHO Drug Information, 2019, Vol.33, No.4, p.828-30, the entire contents of each of which are hereby incorporated by reference in their entireties. Efanesoctocog alfa is an exemplary FVIII-ELNN-Fc / D’D3-ELNN-Fc heterodimer.

[0088] In some embodiments, the chimeric protein is a FVIII-ELNN-Fc / D’D3-ELNN-Fc heterodimer comprising (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the chimeric protein comprises (i) a first polypeptide and (ii) a second polypeptide that are covalently linked via one or more disulfide bonds (e.g., two disulfide bonds). In some embodiments, the chimeric protein comprises a FVIII polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the chimeric protein comprises a VWF fragment encoded by the nucleic acid sequence of SEQ ID NO: 6.

[0089] In some embodiments, the chimeric protein is efanesoctocog alfa. III. Methods of Assaying, Determining, and Monitoring FVIII Levels

[0090] In an aspect, the present disclosure is directed to a method for determining the level of Clotting Factor VIII (FVIII) activity in the blood or plasma of a human subject who has von Willebrand disease (VWD) and to whom a chimeric protein has been administered, wherein the chimeric protein comprises (a) a FVIII protein and (b) an ELNN polypeptide and / or a von Willebrand Factor (VWF) fragment, the method comprising: (i) assaying the level of FVIII activity in the captured chimeric protein with an activated partial thromboplastin time (aPTT)-based one stage clotting assay to obtain an assayed FVIII activity value; (ii) reducing the assayed FVIII activity value to obtain an adjusted FVIII activity value; and (iii) reporting the adjusted FVIII activity value or an approximated value thereof. In some embodiments, the FVIII activity in the blood is determined. In some embodiments, the FVIII activity in the plasma is determined. In some embodiments, the aPTT-based one stage clotting assay does not use a reagent comprising ellagicacid. In some embodiments, the aPTT-based one stage clotting assay uses a reagent comprising ellagic acid. In some embodiments, the aPTT-based one stage clotting assay uses cephalin. In some embodiments, the aPTT-based one stage clotting assay uses ellagic acid and cephalin. In some embodiments, the cephalin is brain cephalin. In some embodiments, the cephalin is rabbit brain cephalin. In some embodiments, the aPTT-based one stage clotting assay does not use a reagent comprising both ellagic acid and phosphatides. In some embodiments, the aPTT-based one stage clotting assay does not use a reagent comprising both ellagic acid and phospholipids comprising phosphatides but not phosphatidylserine. In some embodiments, the aPTT-based one stage clotting assay does not use the reagent Actin®FS.

[0091] In some embodiments, the assayed FVIII activity value is reduced by about 25-50%. In some embodiments, the assayed FVIII activity value is reduced by about 30-55%. In some embodiments, the assayed FVIII activity value is reduced by about 35-60%. In some embodiments, the assayed FVIII activity value is reduced by about 40-60%. In some embodiments, the assayed FVIII activity value is reduced by about 45-75%. In some embodiments, the assayed FVIII activity value is reduced by about 50-75%. In some embodiments, the assayed FVIII activity value is reduced by about 75%. In some embodiments, the assayed FVIII activity value is reduced by about 70%. In some embodiments, the assayed FVIII activity value is reduced by about 65%. In some embodiments, the assayed FVIII activity value is reduced by about 60%. In some embodiments, the assayed FVIII activity value is reduced by about 55%. In some embodiments, the assayed FVIII activity value is reduced by about 50%. In some embodiments, the assayed FVIII activity value is reduced by about 45%. In some embodiments, the assayed FVIII activity value is reduced by about 40%. In some embodiments, the assayed FVIII activity value is reduced by about 35%. In some embodiments, the assayed FVIII activity value is reduced by about 30%. In some embodiments, the assayed FVIII activity value is reduced by at least 25%.

[0092] In some embodiments, detecting the level of exogenous Clotting Factor VIII (FVIII) activity in the blood or plasma of a human subject who has von Willebrand disease (VWD) and to whom a chimeric protein has been administered, wherein the chimeric protein comprises a FVIII protein and an ELNN polypeptide, comprises: (i) separating the chimeric protein from a biological sample from the subject with an anti-ELNN antibody or an antigen-binding fragment thereof to obtain a captured chimeric protein sample, wherein the biological sample is a blood sample or a plasma sample; (ii) assaying the level of FVIII activity in the captured chimeric protein with a chromogenic assay to obtain an assayed FVIII activity value; and (iii) reporting the assayed FVIII activity value or an approximated value thereof without correcting the assayed FVIII activity value or approximated value thereof. In some embodiments, the chimeric protein is used as a standard (e.g., a calibration standard) for the chromogenic assay. In some embodiments, correcting theassayed FVIII activity value or approximated value thereof comprises reducing the value. In some embodiments, correcting the assayed FVIII activity value or approximated value thereof comprises reducing the value as if an overestimation of the FVIII activity needs to be accounted for. In some embodiments, an activated partial thromboplastin time (aPTT)-based one stage clotting assay is not used to assess the level of FVIII activity. In some embodiments, the chimeric protein is separated from the biological sample with an anti-ELNN antibody. In some embodiments, the chimeric protein is separated from the biological sample with an antigen-binding fragment of an anti-ELNN antibody. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a plasma sample.

[0093] In one aspect, the present disclosure is directed to a method for determining the level of Clotting Factor VIII (FVIII) activity in the blood or plasma of a human subject who has von Willebrand disease (VWD) and to whom a chimeric protein has been administered, wherein the chimeric protein comprises (a) a FVIII protein and (b) an ELNN polypeptide and / or a von Willebrand Factor (VWF) fragment, the method comprising: (i) assaying the level of FVIII activity in the captured chimeric protein with an activated partial thromboplastin time (aPTT)-based one stage clotting assay to obtain an assayed FVIII activity value; (ii) reducing the assayed FVIII activity value to obtain an adjusted FVIII activity value; and (iii) reporting the adjusted FVIII activity value or an approximated value thereof. In some embodiments, FVIII activity value or the approximated value thereof is reported to the subject, a caregiver of the subject, or a medical professional. In some embodiments, the aPTT-based one stage clotting assay does not use a reagent comprising ellagic acid. In some embodiments, the aPTT-based one stage clotting assay uses a reagent comprising ellagic acid. In some embodiments, the aPTT-based one stage clotting assay uses cephalin. In some embodiments, the aPTT-based one stage clotting assay uses ellagic acid and cephalin. In some embodiments, the cephalin is brain cephalin. In some embodiments, the cephalin is rabbit brain cephalin. In some embodiments, the aPTT-based one stage clotting assay does not use a reagent comprising both ellagic acid and phosphatides. In some embodiments, the aPTT-based one stage clotting assay does not use a reagent comprising both ellagic acid and phospholipids comprising phosphatides but not phosphatidylserine. In some embodiments, the aPTT-based one stage clotting assay does not use the reagent Actin®FS.

[0094] In some embodiments, the assayed FVIII activity value is reduced by about 25-50%. In some embodiments, the assayed FVIII activity value is reduced by about 50%. In some embodiments, the assayed FVIII activity value is reduced by at least 25%.

[0095] In some embodiments, the subject has a body mass index of ≥30 kg / m2. In some embodiments, the subject is a female. In some embodiments, the subject is a male. In some embodiments, the subject is human.

[0096] In one aspect, the present disclosure is directed to a method of monitoring the level of FVIII in the blood or plasma of a human subject who is receiving treatment for Willebrand disease (VWD), the method comprising performing any of the methods disclosed herein at least once per year, e.g., at least once every 6 months or at least once per month. In some embodiments, the level of FVIII activity is determined at least once per year. In some embodiments, the level of FVIII activity is determined at least once every 9 months. In some embodiments, the level of FVIII activity is determined at least once every 6 months. In some embodiments, the level of FVIII activity is determined at least once per month. In some embodiments, the methods disclosed herein can be performed twice a month, three times a month, 4 times a month, one time a week, twice a week, three times a week, four times a week, five times a week, six times a week, or daily. In some embodiments, the methods disclosed herein can be performed once every two months, once every three months, once every four months, once every five months, once every six months or longer. In some embodiments, multiple doses of the chimeric protein are administered to the subject at a dosing interval. In some embodiments, the dosing interval is one day, two days, three days, four days, five days, six days, or 7 days. In some embodiments, the dosing interval is 7 days or longer.

[0097] In some embodiments, the subject has VWD, wherein the VWD is Type 2N VWD. In some embodiments, the subject has VWD, wherein the VWD is Type 3 VWD.

[0098] In some embodiments, multiple doses of the chimeric protein are administered. In some embodiments, each of the multiple doses is between about 10IU / kg and 100 IU / kg. In some embodiments, each of the multiple doses is 25 IU / kg. In some embodiments, each of the multiple doses is 50 IU / kg. IV. Anti-ELNN Antibodies

[0099] In an aspect, the present disclosure is directed to an anti-ELNN antibody or antigen- binding fragment thereof. In some embodiments, the anti-ELNN antibody or antigen-binding fragment thereof comprises three light chain complementarity determining region (CDR) sequences of SEQ ID NOs: 38, 39 and 40, and three heavy chain complementarity determining region (CDR) sequences of SEQ ID NOs: 32, 33 and 34. In some embodiments, the anti-ELNN antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, comprising a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:32 and threelight-chain CDRs of a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:37, wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28). In an aspect, the present disclosure is directed to an anti- ELNN antibody or antigen-binding fragment thereof. In some embodiments, the anti-ELNN antibody or antigen-binding fragment thereof comprises three light chain complementarity determining region (CDR) sequences of SEQ ID NOs: 38, 39 and 40, and three heavy chain complementarity determining region (CDR) sequences of SEQ ID NOs: 32, 33 and 34. In some embodiments, the anti-ELNN antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, comprising a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:32 and three light-chain CDRs of a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:37, wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: ESATPE (SEQ ID NO: 29). In some embodiments, the anti- ELNN antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, comprising a heavy chain variable region comprising three heavy-chain CDRs, and a light chain variable region comprising three light-chain CDRs, wherein said three heavy-chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:32 (HCVR), and said three light-chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:37 (LCVR), and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28). In some embodiments, the anti-ELNN antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, comprising a heavy chain variable region comprising three heavy-chain CDRs, and a light chain variable region comprising three light-chain CDRs, wherein said three heavy-chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:32 (HCVR), and said three light-chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:37 (LCVR), and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: ESATPE (SEQ ID NO: 29).

[0100] In some embodiments, the CDRs are identified by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition and / or the contact definition.

[0101] In some embodiments, the anti-ELNN antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:32 and a light chain variable region (LCVR) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:37. In some embodiments, the anti-ELNN antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the anti- ELNN antibody comprises a heavy chain comprising an amino acid sequence that is at least 95%identical to SEQ ID NO:31 and a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:36.

[0102] In some embodiments, the anti-ELNN antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:31 and a light chain comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the anti-ELNN antibody comprises two heavy chains comprising the amino acid sequence of SEQ ID NO:31, and two light chains comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the anti-ELNN antibody comprises two heavy chains consisting of the amino acid sequence of SEQ ID NO:31, and two light chains consisting of the amino acid sequence of SEQ ID NO:36. In some embodiments, the anti-ELNN antibody or antigen binding fragment thereof binds to an ELNN polypeptide and comprises (i) a heavy chain variable region which is at least 98% identical to SEQ ID NO: 32 and (ii) a light chain variable region which is at least 98% identical to SEQ ID NO: 37, and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28). In some embodiments, the anti-ELNN antibody comprises two heavy chains consisting of the amino acid sequence of SEQ ID NO:31, and two light chains consisting of the amino acid sequence of SEQ ID NO:36. In some embodiments, the anti-ELNN antibody or antigen binding fragment thereof binds to an ELNN polypeptide and comprises (i) a heavy chain variable region which is at least 98% identical to SEQ ID NO: 32 and (ii) a light chain variable region which is at least 98% identical to SEQ ID NO: 37, and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: ESATPE (SEQ ID NO: 29).

[0103] In some embodiments, the anti-ELNN antibody or antigen binding portion thereof comprises (i) a light chain variable domain comprising an amino acid sequence at least 90% identical to a light chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs 38, 39 and 40, and (ii) a heavy chain variable domain comprising an amino acid sequence at least 90% identical to a heavy chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs: 32, 33, and 34, wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28). In some embodiments, the anti-ELNN antibody or antigen binding portion thereof comprises (i) a light chain variable domain comprising an amino acid sequence at least 90% identical to a light chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs 38, 39 and 40, and (ii) a heavy chain variable domain comprising an amino acid sequence at least 90% identical to a heavy chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs: 32, 33, and 34, wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: ESATPE (SEQ ID NO: 29).

[0104] In some embodiments, the ELNN polypeptide comprises the sequence: SATPE (SEQ ID NO: 28). In some embodiments, the ELNN polypeptide comprises the sequence: ESATPE (SEQ ID NO: 29). In some embodiments, the ELNN polypeptide comprises at least 2 instances of the following sequence: SATPE (SEQ ID NO: 28). In some embodiments, the ELNN polypeptide comprises at least 5 instances of the following sequence: SATPE (SEQ ID NO: 28). In some embodiments, the ELNN polypeptide comprises at least 6 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0105] In some embodiments, the ELNN polypeptide comprises at least 3 instances, at least 4 instances, at least 5 instances, or at least 6 instances of the following sequence: SATPE (SEQ ID NO: 28), wherein each of the at least 3, at least 4, at least 5, or at least 6 instances of SATPE (SEQ ID NO: 28) are part of the following sequence: ESATPE (SEQ ID NO: 29). In some embodiments, each instance of SATPE (SEQ ID NO: 28) and / or ESATPE (SEQ ID NO: 29) in the sequence of the ELNN polypeptide is followed by at least 3 amino acids at the C-terminal end thereof.

[0106] In some embodiments, the ELNN polypeptide comprises the sequence of SEQ ID NO: 14, 24, 25, 26, and / or 27. In some embodiments, the ELNN polypeptide comprises a von Willebrand Factor fragment.

[0107] In some embodiments, the anti-ELNN antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the anti-ELNN antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 31 and a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 36. In some embodiments, the anti-ELNN antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the anti-ELNN antibody comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 31, and two light chains comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the anti-ELNN antibody, antigen-binding fragment, or antigen binding protein, which is an isolated antibody, antigen-binding fragment, or antigen binding protein.

[0108] In some embodiments, the anti-ELNN antibody is in a mouse IgG1 backbone. In some embodiments, the anti-ELNN antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 42, and a light chain variable region comprising an amino acid sequence that is at least 95%identical to SEQ ID NO: 47. In some embodiments, the anti-ELNN antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence that is identical to SEQ ID NO: 42, and a light chain variable region comprising an amino acid sequence that is identical to SEQ ID NO: 47. In some embodiments, the anti-ELNN antibody or antigen- binding fragment comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 41, and a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 46. In some embodiments, the anti-ELNN antibody or antigen- binding fragment comprises a heavy chain comprising an amino acid sequence that is identical to SEQ ID NO: 41, and a light chain comprising an amino acid sequence that is identical to SEQ ID NO: 46. In some embodiments, the anti-ELNN antibody comprises two heavy chains comprising amino acid sequences at least 95% identical to SEQ ID NO: 41, and two light chains comprising amino acid sequences at least 95% identical to SEQ ID NO: 46. In some embodiments, the anti- ELNN antibody comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 41, and two light chains comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the anti-ELNN antibody comprises a heavy chain, wherein the heavy chain comprises three complementary determining regions (CDR), wherein HCDR1 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 43, wherein HCDR2 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 44, wherein HCDR3 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 45. In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 43, HCDR2 comprises the amino acid sequence of SEQ ID NO: 44, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the anti-ELNN antibody comprises a light chain, wherein the light chain comprises three complementary determining regions (CDR), wherein LCDR1 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 48, wherein LCDR2 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 49, wherein LCDR3 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 50. In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 48, LCDR2 comprises the amino acid sequence of SEQ ID NO: 49, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 50.

[0109] In some embodiments, the anti-ELNN antibody is in a mouse IgG2a backbone. In some embodiments, the anti-ELNN antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 53, and a light chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 57. In some embodiments, the anti-ELNN antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence that is identical to SEQ ID NO: 53, and a light chain comprising an amino acid sequence that is identicalto SEQ ID NO: 57. In some embodiments, the anti-ELNN antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 51, and a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 52. In some embodiments, the anti-ELNN antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence that is identical to SEQ ID NO: 51, and a light chain comprising an amino acid sequence that is identical to SEQ ID NO: 52. In some embodiments, the anti-ELNN antibody comprises two heavy chains comprising amino acid sequences at least 95% identical to SEQ ID NO: 51, and two light chains comprising amino acid sequences at least 95% identical to SEQ ID NO: 52. In some embodiments, the anti-ELNN antibody comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 51, and two light chains comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the anti-ELNN antibody comprises a heavy chain, wherein the heavy chain comprises three complementary determining regions (CDR), wherein HCDR1 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 54, wherein HCDR2 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 55, wherein HCDR3 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 56. In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 54, HCDR2 comprises the amino acid sequence of SEQ ID NO: 55, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, the anti-ELNN antibody comprises a light chain, wherein the light chain comprises three complementary determining regions (CDR), wherein LCDR1 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 58, wherein LCDR2 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 59 (KVS), wherein LCDR3 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 60. In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 58, LCDR2 comprises the amino acid sequence of SEQ ID NO: 59 (KVS), and LCDR3 comprises the amino acid sequence of SEQ ID NO: 60.

[0110] In some embodiments, an anti-ELNN antibody binds to ELNN polypeptides that comprise at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28). In some embodiments, an anti-ELNN antibody binds to ELNN polypeptides that comprise at least 3 instances of the following sequence: ESATPE (SEQ ID NO: 29). Non-limiting examples of such ELNNs include ELNNs comprising the sequence of SEQ ID NO: 14, 24, 25, 26, and / or 27. In some embodiments, 2 or more of the instances are separated by one or more amino acids within the ELNN polypeptide’s sequence. In some embodiments, all of the instances are separated by one or more amino acids within the ELNN polypeptide’s sequence. In some embodiments, 2 or more instances are separated by three or more amino acids within the ELNN polypeptide’s sequence.In some embodiments, all of the instances are separated by three or more amino acids within the ELNN polypeptide’s sequence.

[0111] In some embodiments, an anti-ELNN antibody binds to ELNN polypeptides that comprise at least 3 instances of the sequence ESATPE (SEQ ID NO: 29) but does not bind to ELNN polypeptides that comprise at only 1 instance of the sequence ESATPE (SEQ ID NO: 29).

[0112] In some embodiments, antibodies and antigen-binding fragments thereof may comprise antibody sequences of different origins, comprising mouse, rat, rabbit, donkey, human, and / or camelid immunoglobulin sequences. In some embodiments, an antibody is fully human, humanized, or chimeric. In some embodiments, an antibody or antigen-binding fragment thereof is conjugated or linked to a tags or other functional moiety, e.g. a toxin, label, fluorophore, radiochemical, etc. In some embodiments an antibody or antigen-binding fragment thereof is conjugated or linked to biotin. In some embodiments, an antibody or antigen-binding fragment thereof is conjugated or linked to streptavidin. V. Pharmaceutical Compositions

[0113] In an aspect, the present disclosure is directed to pharmaceutical compositions of a chimeric protein, e.g., efanesoctocog alfa.

[0114] In some embodiments, the present disclosure is directed to a pharmaceutical composition comprising: efanesoctocog alfa, L-histidine, L-arginine hydrochloride, sucrose, calcium chloride, and polysorbate 80. In some embodiments, the pharmaceutical composition comprises 250, 500, 1000, 2000, 3000 or 4000 IU of efanesoctocog alfa. In some embodiments, the pharmaceutical composition comprises 1000 IU of efanesoctocog alfa. In some embodiments, the pharmaceutical composition comprises efanesoctocog alfa, about 10 mM L-histidine, about 250 mM Arginine-HCl, about 5mM CaCl2, about 5% (w / v) sucrose, and about 0.05% (w / v) polysorbate 80. In some embodiments, the pharmaceutical composition comprises efanesoctocog alfa, 10 mM L-histidine, 250 mM Arginine-HCl, 5mM CaCl2, 5% (w / v) sucrose, and 0.05% (w / v) polysorbate 80. In some embodiments, the pharmaceutical composition has a pH of about 7.0. In some embodiments, the pharmaceutical composition has a pH of 6.8 to 7.2. In some embodiments, the pharmaceutical composition has a pH of 6.8. In some embodiments, the pharmaceutical composition has a pH of 7.0.

[0115] In some embodiments, the chimeric protein is in a pharmaceutical composition that comprises about 250 IU, 500 IU, 1000 IU, 2000 IU, 3000 IU, or 4,000 IU of the chimeric protein. In some embodiments, the chimeric protein is efanesoctocog alfa. In some embodiments, the efanesoctocog alfa is in a pharmaceutical composition that comprises: (a) 45 mg / mL to 60 mg / mLsucrose; (b) 1.5 mg / mL to 2.0 mg / mL L-histidine; (c) 40 mg / mL to 60 mg / mL L-arginine; (d) 0.5 mg / mL to 0.9 mg / mL calcium chloride; and (e) 0.4 mg / mL to 0.7 mg / mL polysorbate 80. In some embodiments, the efanesoctocog alfa is in a pharmaceutical composition that comprises: (a) about 50 mg / ml sucrose; (b) about 1.55 mg / ml L-histidine; (c) about 52.67 mg / ml L-arginine-HCl; (d) about 0.73 mg / ml calcium chloride dihydrate; and (e) about 0.50 mg / ml polysorbate 80. In some embodiments, the efanesoctocog alfa is in a pharmaceutical composition that comprises: (a) about 56.12 mg / ml sucrose; (b) about 1.74 mg / ml L-histidine; (c) about 59.11 mg / ml L-arginine- HCl; (d) about 0.62 mg / ml calcium chloride; and (e) about 0.56 mg / ml polysorbate 80. In some embodiments, the efanesoctocog alfa is in a pharmaceutical composition that comprises: (a) 5% (w / v) to 7.5% (w / v) sucrose; (b) 7.5 mM to 12.5 mM histidine; (c) 225 mM to about 300 mM arginine; (d) 5 mM to 6 mM calcium chloride; and (e) 0.01% (w / v) to about 0.075% (w / v) polysorbate 80.

[0116] In some embodiments, the lyophilized pharmaceutical composition is in a lyophilized cake. In some embodiments, the lyophilized cake is white. In some embodiments, the lyophilized cake is less than Y4 in the European Pharmacopoeia color scale. See Degree of Coloration of Liquids (Method 2.2.2), European Pharmacopoeia, 10thEd. (2021).

[0117] In some embodiments, the pharmaceutical composition disclosed herein is a lyophilized pharmaceutical composition (e.g., powder). In some embodiments, the pharmaceutical composition is supplied in a sterile vial that requires reconstitution with sterile water.

[0118] In some embodiments, the lyophilized pharmaceutical composition and sterile water are combined to produce an injectable solution. In some embodiments, the lyophilized pharmaceutical composition is combined with about 2 mL to about 5 mL of sterile water. In some embodiments, the lyophilized pharmaceutical composition is combined with about 3 mL of sterile water. In some embodiments, the lyophilized pharmaceutical composition is combined with 3 mL of sterile water. In some embodiments, the sterile water is USP grade sterile water. In some embodiments, the sterile water is USP grade sterile water for injection. In some embodiments, the sterile water is pyrogen-free or nonpyrogenic. In some embodiments, the sterile water does not contain a bacteriostatic or antimicrobial agent. In some embodiments, the sterile water contains a bacteriostatic or antimicrobial agent. In some embodiments, the sterile water is sterilized using a filter. In some embodiments, the sterile water is sterilized using a 0.1μm filter. In some embodiments, the sterile water is distilled water. In some embodiments, the sterile water is sterile, nonpyrogenic, distilled water, hypotonic, with an osmolarity of zero mOsmol / L, and does not contain a bacteriostatic or antimicrobial agent.

[0119] In some embodiments, the chimeric protein is provided as part of a kit with a pharmaceutical label, wherein the pharmaceutical label comprises information that is sufficient to enable a human subject, a caregiver, or a medical practitioner to practice a method of administering a chimeric protein as described herein.

[0120] Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the disclosure. All patents, publications, and articles referred to herein are expressly and specifically incorporated herein by reference. EXAMPLES Example 1: A Phase 1, Open-Label Study to Assess the Pharmacokinetics, and Safety and Tolerability of a Single Intravenous Injection of efanesoctocog alfa in Adults with type 2N and 3 von Willebrand disease (VWD)

[0121] Extended half-life recombinant FVIII (rFVIII) products have reduced the frequency of FVIII administration for prophylaxis in hemophilia patients. Next-generation extended half-life rFVIII products that prevent and control bleeding episodes for longer periods of time could result in less frequent administration, which in turn could improve quality of life for VWD patients.

[0122] Efanesoctocog alfa, also known as “BIVV001”, “efanesoctocogum alfa”, “Efa”, and “rFVIIIFc-VWF-XTEN”, is a high-sustained blood clotting factor VIII (FVIII), engineered to have an extended half-life that is independent of VWF. Preclinical and clinical experience indicate that efanesoctocog alfa has an extended half-life, irrespective of VWF level, and maintains high sustained FVIII activity levels.

[0123] This study was a Phase 1, multicenter, open-label, single-arm, single-dose, non- randomized study in adult participants with type 2N (Normandy) or type 3 VWD. The rationale to conduct the Phase 1 study in patients with type 2N and type 3 VWD, in whom FVIII levels are substantially reduced, is to characterize the pharmacokinetics (PK) of efanesoctocog alfa, which should be independent from FVIII binding capacity and VWF levels in patients with type 2N and type 3 VWD. This study was also conducted to assess the safety and tolerability of efanesoctocog alfa in these populations. A. Study Design and Population

[0124] The object of the present study was to assess the safety, tolerability, and FVIII activity (e.g. PK) of a single dose of efanesoctocog alfa. After screening for up to 28 days, each participant was administered a single 25 IU / kg dose of efanesoctocog alfa. This was followed by a safety observation period, which included PK sampling up to 10 days post-dose, a visit at day 15 post-dose, and the end of study at 28 days post-dose. A graphical representation of the study design is presented as FIG.2.

[0125] The VWD patient population for this study was defined as having reduced levels of functioning VWF, thereby leading to reduced levels of FVIII activity. Participants were assessed as part of the screening criteria and those with endogenous FVIII activity equal to or less than 20% (<20 IU / dL) were included in this study.

[0126] Inclusion Criteria

[0127] 01. The patient population could include adult male and / or female patients between 18 and 65 years of age with type 2N or 3 VWD.02. Each participant was diagnosed with hereditary type 3 VWD (≤3 IU / dL VWF: Ag) or type 2N VWD (FVIII:C / VWF: Ag <0.6 and severely decreased VWF:FVIIIB) as documented in historical medical records; or a documented genotype known to produce VWD type 3 or 2N VWD.03. Type 3 VWD participants were included if they had a medical history of at least 25 exposure days to VWF-containing coagulation factor concentrates. 04. Contraceptive use by men or women is consistent with local regulations regarding the methods of contraception for those participating in clinical studies. a) Male participants - No contraceptive measures required for this study. b) Female participants - A female participant is eligible to participate if she is not pregnant or breastfeeding, and at least one of the following conditions applies: - Is not a WOCBP, defined as: underwent surgical sterilization OR no menses (amenorrhea) for 12 months without an alternative medical cause. In the absence of 12 months of amenorrhea, status of not WOCBP needs confirmation with more than one FSH measurement (>40 IU / L or mIU / mL), or - Is a WOCBP and using an acceptable contraceptive method namely, double contraception algorithm to be adapted to country specifications; For most European countries: The accepted double contraception methods include the use of intrauterine device or hormonal contraception in addition to one of the following contraceptive options: (1) condom; (2) diaphragm or cervical / vault cap; (3) spermicide], during the intervention period (at a minimum until the last day of the study). A WOCBP must have a negative highly sensitive pregnancy test before the dose administration of study intervention. A serum pregnancy test should be performed at screening with additional pregnancy tests just prior to efanesoctocog alfa administration and at end of study visit. For time points other than screening, serum or urine pregnancy testing may be performed at the discretion of the investigator. The investigator is responsible for review of medical history, menstrual history, and recent sexual activity to decrease the risk for inclusion of a woman with an early undetected pregnancy. 05. Type 2N VWD participants are included if the use of desmopressin acetate (1-deamino-8-D-arginine vasopressin) (DDAVP) is deemed insufficient or contraindicated, as assessed by the investigator, or if they have required prior use of VWF- and FVIII- containing coagulation factor concentrates.

[0128] Exclusion Criteria

[0129] Participants were excluded from the study if they met any one of the following criteria: 01. Hereditary or acquired coagulation disorder other than VWD (including qualitative and quantitative platelet disorders, and thrombocytopenia <100,000 cells / uL at Screening).02. The participant has a FVIII activity level >20 IU / dL (>20%) at Screening.03. History or presence of a VWF inhibitor or clinical suspicion of a VWF inhibitor.04. History of a positive FVIII inhibitor test, defined as ≥0.6 BU / mL (by Nijmegen-modified Bethesda assay) or a clinical suspicion of a FVIII inhibitor. 05. Positive FVIII inhibitor test, defined as ≥0.6 BU / mL, at Screening. 06. History of hypersensitivity or anaphylaxis associated with any FVIII- or VWF-containing product.07. Current serious bacterial, viral or fungal infection (other than chronic viral hepatitis or HIV).08. Abnormal renal function, defined as a serum creatinine level >2.0 mg / dL at Screening.09. Serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) >5 x upper limit of normal (ULN) at Screening.10. Serum total bilirubin >3 x ULN at Screening.11. History of an unprovoked venous thromboembolism, myocardial infarction within 12 months prior to Screening, or occlusive cerebral vascular accident within 12 months prior to Screening. 12. VWD-associated gastrointestinal angiodysplasia related bleeding within 6 months prior to Baseline. 13. HIV-positive with an absolute CD4 count <200 cells / mm3 or a viral load of >400 copies / mL within 6 months prior to baseline documented in medical records.14. Previous participation in this study; participants who fail Screening may re-screen.15. Presence of clinically relevant disease or any condition that, in the opinion of the investigator or sponsor, would make the participant unsuitable for enrollment. 16. The participant has received or anticipates receiving systemic immunosuppressive or immunomodulatory treatment within 12 weeks prior to baseline.17. The participant requires the use of acetylsalicylic acid, non-NSAID anti-platelets, and NSAIDs above the maximum dose.18. Any participant who, in the judgment of the investigator, is likely to be noncompliant during the study, or unable to cooperate because of a language problem or poor mental development.19. Any participant enrolled in or having participated in any other clinical study involving an investigational medicinal product or in any other type of medical research and is still in the exclusion period according to applicable regulations.20. Any participant who cannot be contacted in case of emergency.21. Any participant who is the investigator or any sub-investigator, research assistant, pharmacist, study coordinator, or other staff thereof, directly involved in conducting the study, or is an immediate family member of such individuals.22. The participant is accommodated in an institution because of regulatory or legal order; prisoners or participants who are legally institutionalized. 23. The participant has undergone or is anticipated to undergo major surgery within 30 days of baseline. For the purpose of this Example 1, major surgery is defined as any surgical procedure (elective or emergent) that usually, but not always, involves general anesthesiaand / or respiratory assistance, in which a major body cavity is penetrated and exposed, or a substantial impairment of physical or physiological functions is produced (e.g., laparotomy, thoracotomy, craniotomy, joint replacement, or limb amputation). 24. Patients currently on a prophylaxis regimen for the treatment of VWD that, in the investigator's opinion, would preclude participation in the study due to the possible increased risk of bleeding associated with the requirement to withhold prophylaxis during the study.

[0130] For statistical analysis, participants were analyzed according to the intervention received and classified as a member of one or more of the following sub-populations: (1) Safety population, which includes all enrolled participants who receive at least 1 dose of efanesoctocog alfa; (2) Pharmacokinetic population, which includes all enrolled and treated participants (safety population) with adequate blood samples enabling determination of at least one PK parameter; and / or (3) Anti-drug antibody (ADA) population, which includes all enrolled participants treated with efanesoctocog alfa with at least one post-baseline ADA result (positive, negative or inconclusive).

[0131] Primary Objectives and Endpoints

[0132] The primary objective of the study was to characterize the pharmacokinetics (PK) of efanesoctocog alfa after a single intravenous (IV) administration, as assessed by FVIII activity determined by the 1-stage activated partial thromboplastin time (aPPT) clotting assay, as well as the efanesoctocog alfa capture chromogenic Coatest FVIII activity assay. Details regarding these assays are provided elsewhere herein.

[0133] Primary endpoints were PK parameters, assessed as FVIII activity by two assay methods, which includes but not limited to the following: maximum activity (Cmax); terminal half- life (t1 / 2z); clearance (CL); volume of distribution at steady state (Vss); area under the activity time curve extrapolated to infinity (AUC∞); mean residence time (MRT); and incremental recovery (IR). A list of pharmacokinetic parameters and definitions is provided in Table 3.

[0134] The secondary objective of the study was to assess the safety and tolerability of a single IV dose of efanesoctocog alfa in adults with type 2N and 3 VWD.

[0135] Secondary endpoints were the occurrence of adverse events (AEs) and serious adverse events (SAEs); and the occurrence of clinically significant abnormalities in laboratory tests, including development of inhibitors (neutralizing antibodies directed against FVIII) as determined via the Nijmegen-modified Bethesda assay.

[0136] The exploratory objective was to perform additional assessments of immunogenicity. Exploratory endpoints were the occurrence of anti-drug antibodies (ADAs).B. Dosing

[0137] Participants received a single IV dose of efanesoctocog alfa, with an injection duration of 8 ±2 minutes. The dose of drug per administration was 25 IU / Kg. A single dose of efanesoctocog alfa at 25 IU / kg was selected to provide a robust PK profile while maintaining FVIII activity levels within normal physiological range. Administration occurred during the morning.

[0138] Formulation

[0139] Drug product was supplied as a lyophilized powder in a sterile vial requiring reconstitution with Sterile Water for Injection (diluent). For this study, each vial of drug product includes 1000 IU of efanesoctocog alfa along with excipients comprising L-histidine, L-arginine hydrochloride, sucrose, calcium chloride dihydrate, and polysorbate 80.

[0140] Safety Assessment

[0141] Drug product tolerability investigations at baseline and during the study are presented in Table 1.2. Non-limiting examples of these investigations include the following:

[0142] 1. Physical examination: assessment of the cardiovascular, respiratory, gastrointestinal, neurological, dermatological, and musculoskeletal systems.

[0143] 2. Vital signs: blood pressure, pulse rate, respiratory rate, and body temperature (°C).

[0144] 3. Laboratory tests: a list of laboratory assessments is provided in Table 1.

[0145] 4. Serology tests: Hepatitis B antigen, Hepatitis C antibodies, anti-HIV1 and anti-HIV2 antibodies.

[0146] 5. β-HCG or FSH measurement (if applicable, for female participants only)

[0147] 6. Inhibitor development: Blood samples were collected per the schedule in Table 1.2 for the detection of inhibitors. Samples were also collected at the time of any clinical event deemed relevant to inhibitor testing. Inhibitor development was assessed at a central laboratory by the Nijmegen-modified Bethesda assay and is defined as an initial test result of ≥0.6 BU / mL confirmed by a second test result from an independent blood sample collected within 2 to 4 weeks of the first positive sample. Initial samples (not for confirmation) were obtained at Screening, Day 1 (pre- dose), Day 15, and Day 29. 7. Anti-drug antibodies (ADA): Blood samples will be collected per the schedule in Section 1.2 for the detection and analysis of anti-efanesoctocog alfa antibodies. Samples may also be collected at the time of any clinical event deemed relevant to anti-efanesoctocog alfa antibody testing. Testing for potential antibody formation will be performed at a central laboratory using a validatedefanesoctocog alfa-specific ADA assay. Confirmed positive samples will be further characterized for antibodies specific to FVIII, Fc, ELNN (XTEN), or D’D3. Table 1: Laboratory assessments

[0148] All tests and the schedule of assessments over the course of the study are presented in Table 1.2.

[0149] Concomitant Medication: The use of concomitant medication should not be allowed during the study unless specified in the inclusion criteria or study procedures or medically required. However, if a specific treatment is required for any reason, an accurate record must be entered in the eCRF, including the name of the medication (INN), daily dosage, and duration of use. Permitted concomitant therapy: Participants taking medication routinely for a pre-existing condition should be on a treatment regimen that has been stable for at least 3 weeks prior to enrollment, and dosage changes should not be anticipated during the observation period for this study. Pre-study stable NSAID doses below the maximum dose specified in the local prescribing information at the time of enrollment are permitted. All concurrent prescription and nonprescription medications including over-the counter and alternative preparations (including herbal remedies,vitamins, and health food supplements) should be recorded at Screening and throughout the treatment and follow-up periods.

[0150] Prohibited concomitant therapy: No premedication for pain or pyrexia relief is to be given for administration of efanesoctocog alfa. Should pre-medications be contemplated, this will be discussed on a case-by-case basis with the Sanofi Study Medical Monitor or designee and before administration.

[0151] Medications prohibited during the study include: 1. Acetylsalicylic acid (ASA) or non- NSAID anti-platelet therapies 2. NSAIDs at doses above the maximum dose specified in local prescribing information 3. Systemic treatment with chemotherapy and / or other immunosuppressive drugs (except for the treatment of Hepatitis C virus [HCV] or HIV). The use of systemic steroids for the treatment of acute respiratory illness (eg, asthma), acute allergic episodes, or otherwise life-threatening episodes is allowed. Treatment in these circumstances should not exceed a 14-day duration. Local, topical, and / or inhaled steroid use is permitted. 4. Any other FVIII- or VWF-containing concentrate or DDAVP from at least 96 hours prior to BIVV001 dosing until blood samples for Day 15 laboratory assessments are collected. 5. Anticoagulant agents.

[0152] Adverse Events

[0153] Adverse events (AEs), reported by the participant or observed by the investigator, will be recorded. In this study, AEs were collected starting from signature of the informed consent form (ICF) through the end of the 28-day safety observation period. Laboratory assessments related to safety were collected as outlined in Table 1.2 and include evaluation for inhibitor development via a central laboratory using the Nijmegen-Modified Bethesda Assay at 14 and 28 days following administration of efanesoctocog alfa. The 14- and 28-day post-dose timepoints were selected based on published information on immunological responses (Shankar et al. AAPS J.2014;16(4):658-73).

[0154] In this study, an adverse event (AE) is defined as any untoward medical occurrence in a participant administered a pharmaceutical product and which does not necessarily have to have a causal relationship with this treatment. An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a pharmaceutical product, whether or not related to the pharmaceutical product. Determination of whether an abnormal laboratory value and / or vital sign result meets the definition of an AE will be made by the investigator. Abnormal results are not considered AEs unless one or more of the following criteria are met: (1) symptomatic, (2) requiring either corrective treatmentor consultation, (3) leading to IMP discontinuation or modification of dosing, (4) fulfilling a seriousness criterion, and / or (5) defined as an adverse event of special interest (AESI).

[0155] Bleeding episodes in this patient population were not considered AEs; however, the concomitant events associated with a bleeding episode were reported as AEs as appropriate (e.g., an elbow fracture). Bleeding episodes that meet the criteria for a serious adverse event (SAE) were be reported as a SAE as appropriate.

[0156] In this study, a serious adverse event (SAE) is defined as any untoward medical occurrence that at any dose results in death, or is life-threatening, requires inpatient hospitalization or prolongation of existing hospitalization, or results in persistent or significant disability / incapacity, or is a congenital anomaly / birth defect, or is a medically important event.

[0157] In this study, an adverse event of special interest (AESI) is defined as an adverse event (serious or nonserious) of scientific and medical concern, specific to the IMP or program, for which ongoing monitoring and rapid communication by the investigator to the sponsor may be appropriate. AESI may require further investigation in order to characterize and understand them. Examples of AESI include development of an inhibitor as defined herein and a Grade 3 or higher allergic reaction.Table 1.2: Study Period Flow Chart and Schedulet erapy spec ca y or entry nto t e study. b Visits at the site at screening (may have more than one visit to complete screening activities), D1 for predose activities and of use ior e R no lq Testing for potential antibody formation will be performed at a central laboratory using a validated rFVIIIFc-VWF-ELNN-specific ADA assay. Confirmed positive samples will be further characterized for antibodies specific to Fc, FVIII, D′D3, or ELNN; samples will be obtained at Screening, Day 1 (pre-dose), Day 15, and Day 29. r Only for type 2N participants: von Willebrand comprehensive panel (which includes assessments of von Willebrand Factor [VWF] ristocetin cofactor activity, and VWF antigen). Samples will be obtained at screening, Day 1 (pre-dose), Day 15, and Day 29. s Adverse events and SAEs occurring after signing of the ICF and until the End of Study visit will be recorded in the eCRF. t BIVV001 dose will be administered via slow, manual IV injections over a duration of 8 ±2 minutes. Samples will be collected for 2 bioanalytical assays: (1) 1-stage activated partial thromboplastin time (aPPT-based FVIII activity assay and (2) BIVV001 capture chromogenic Coatest FVIII activity assay; PK samples will be collected according to scheme as described here. The predose sample should be taken within 30 minutes prior to the BIVV001 injection. PK sampling time points are measured relative to the start of injection. Infusion start time and end time should be recorded in eCRF. Abbreviations: ADA: Antidrug antibodies; AE: Adverse event; D: Day; DDAVP: Desmopressin acetate (1-deamino-8-D-arginine vasopressin); eCRF: electronic Case Report Form; EOS: End of study; FSH: Follicle stimulating Hormone; FVIII: Blood clotting factor III; HCG: Human chorionic gonadotropin; HBV: Hepatitis B virus; HCV: Hepatitis C virus; HIV: Human immunodeficiency virus; IMP: Investigational medicinal product; IV: Intravenous; SAEs: Serious adverse events; WOCBP: Women of childbearing potential. C. Pharmacokinetic Assessment

[0158] Pharmacokinetic (PK) assessments were conducted on participant samples collected over the course of the study. The sampling times for blood collection were 10 minutes pre-dose, followed by 10 min, 30 min, 1H, 3H, 6H, 9H, 24H, 48H, 72H, 96H, 120H, 168H, 240H, 336H, and 672H post-dose (see Table 1.2). Blood samples were taken at a volume of 3 mL each, and each participant provided 14 total samples for each of the two bioanalytical methods used in the study.

[0159] Two PK bioanalytical methods were used in this study: (1) 1-stage aPTT-based FVIII activity assay and (2) efanesoctocog alfa capture chromogenic Coatest FVIII activity assay. The first assay to measure FVIII activity of efanesoctocog alfa uses a modification of the aPTT using Dade Actin FSL activated PTT reagent (1-stage aPTT) on the BCS XP analyzer (Siemens Healthcare Diagnostics). The assay was validated using plasma standard that has been calibrated against the WHO (World Health Organization) 6th International Standard for FVIII. The 1-stage aPTT assay is able to measure both native and efanesoctocog alfa FVIII activity. The LLOQ of the assay is at 1% of normal FVIII activity (0.010 IU / mL).

[0160] The second method for determining efanesoctocog alfa activity in plasma is a modified chromogenic Coatest FVIII activity assay. In this assay, the analyte (efanesoctocog alfa) is first captured using an anti-ELNN antibody. The activity of captured efanesoctocog alfa is then determined with Coatest FVIII chromogenic assay, which is provided in the form of a chromogenic substrate assay that quantifies the activated FVIII (FVIIIa)-mediated conversion of factor X (FX) to activated FX (FXa) in the presence of trace amounts of thrombin (for FVIII activation) and excess activated factor IX (FIXa), FX, Ca++, and phospholipids. Formation of FXa is measured by turnover of a chromogenic FXa peptide substrate. The LLOQ of the assay is at 0.4% of normal FVIII activity (0.004 IU / mL, or 4 mIU / mL). The assay was validated using efanesoctocog alfa self- standard.

[0161] Pharmacokinetic parameters

[0162] PK assessments were based on FVIII activity levels determined by 1-stage clotting assay and capture chromogenic assay performed at all scheduled visits (see Table 1.2). The PK of the single dose of efanesoctocog alfa was determined via the estimation of PK parameters, assessed as FVIII activity by 2 assay methods, which included the parameters shown in Table 3.substantially reduced FVIII levels. Hence the rationale for the study was to characterize the pharmacokinetics (PK) of efanesoctocog alfa, which should be independent of the FVIII binding capacity of VWF and the patient’s VWF levels within these subgroups. In addition, the safety andtolerability of efanesoctocog alfa was assessed. Additional details regarding the study and its design are provided above.

[0164] Participant Disposition

[0165] Six participants were enrolled and completed the study (Table 4). Five participants were female (83%), 2 participants had Type 2N VWD (33%), and 4 participants had Type 3 VWD (67%). In the 2 participants with Type 2N VWD, VWF:Ag levels at baseline were 38% and 159%, with VWF ristocetin cofactor (VWF:RCo) activity being 15% and 145%, respectively. Individuals with Type 2N VWD had FVIII activity levels of approximately 3% and 7% by OSA; and the FVIII activity was approximately 1%, 6%, 7% and 15% by OSA among those with Type 3 disease. Participants had a median (range) age of 47 (38–55) years and at diagnosis of VWD had a median (range) age of 7 (1–41) years. Fifty percent of participants (n=3) had a history of joint bleeding and no participant had a history of bleeding related to gastrointestinal angiodysplasia. Participants had a median (range) of 3 (0–4) bleeding episodes in the 12 months prior to screening. Four participants (67%) with Type 3 VWD had previously received prophylaxis against bleeding. The most common medical conditions reported among participants were hepatitis C (n=5; 83%), tooth avulsion (n=4; 67%), and epistaxis (n=3; 50%); all other medical histories were reported for ≤2 participants (33%) each.Table 4. Parficipant demographics and baseline characterisficsEfanesoctocog alfa 25 IU / kg (N=6) 3)Mean (SD) 12.8 (15.3) Median (range) 7 (1–41) Total number of bleeding episodes in the 12 months prior to screeningadministrafion on Day 1.

[0166] Five participants (83%) received ≥1 concomitant medication, other than bleeding medications, during the study (Table 5). Half of the participants (n=3) received concomitant analgesic medication; all other concomitant medication was reported for ≤2 participants (33%) each. Three participants received concomitant bleeding medication during the study (not during the proscribed PK period), including tranexamic acid (n=2), VWF (n=1), and vonicog alfa (n=1) (Table 5). Table 5. Concomitant medicafionsaEfanesoctocog alfa 25 IU / kgPsycholepfics 1 (16.7)Dermatologicals 1 (16.7)efanesoctocog alfa administrafion up to and including the end of study visit).

[0167] Pharmacokinetic Results

[0168] Plasma FVIII activity was measured by 2 bioanalytical methods. The first method (1) was a 1-stage aPTT-based FVIII activity assay using Actin-FSL reagent (referred to as ‘1-stage clotting assay’). The 1-stage aPTT assay was calibrated using World Health Organization (WHO) 6th international FVIII standard and its lower limit of quantification (LLOQ) value was 1% FVIII activity (1 IU / dL). The second method (2) was an efanesoctocog alfa capture chromogenic Coatest FVIII activity assay (a non-limiting example of a capture chromogenic assay). In the capture chromogenic assay, an anti-ELNN antibody was used to capture efanesoctocog alfa and theactivity of the captured efanesoctocog alfa was then tested using a chromogenic assay. The anti- ELNN antibody comprised two heavy chains comprising the amino acid sequence of SEQ ID NO:31 and two light chains comprising the amino acid sequence of SEQ ID NO:36. The 1-stage aPTT assay measures the activity of both endogenous FVIII and efanesoctocog alfa, while the capture chromogenic assay measures only efanesoctocog alfa activity.

[0169] As the capture chromogenic assay could not detect WHO plasma FVIII standard, it was calibrated using efanesoctocog alfa (i.e., self or product specific standard). The LLOQ of capture chromogenic assay was 0.4% (0.4 IU / dL) FVIII activity. Baseline-corrected FVIII activity levels and actual collection time were used to calculate the PK parameters by noncompartmental analysis.

[0170] Exemplary Capture Chromogenic Assay

[0171] A description and procedure for the capture chromogenic assay used in this Example 1 is as follows:

[0172] This is a chromogenic assay where the analyte (efanesoctocog alfa) is captured using an antibody. A biotinylated anti-ELNN antibody is coated onto a microplate. Samples containing efanesoctocog alfa are added to the precoated microwells. After washing away unbound substances, the activity of captured efanesoctocog alfa is determined with coatest factor VIII activity assay. Briefly, the factor VIII activity assay is a chromogenic assay where factor Xa generation is measured. Factor X is activated to factor Xa by factor IXa in the presence of calcium and phospholipids. This reaction is greatly stimulated by factor VIII. By using optimal amounts of Ca2+ and phospholipids and an excess of factors IXa and X, the rate of activation of factor X to factor Xa is solely dependent on the amount of factor VIII. Thus, Factor Xa generation is proportional to FVIII activity. Factor Xa generation is measured using chromogenic substrate S- 2765, which when hydrolyzed by Factor Xa liberates chromophoric group, pNA, that is measured photometrically at 405 nm.

[0173] Abbreviations: BSA: Bovine Serum Albumin CV: Coefficient of Variance HPC: High Positive Control LLOQ: Lower Limit Of Quantification LPC: Low Positive Control MPC: Medium Positive Control PBS: Phosphate Buffered SalineQC: Quality Control ULOQ: Upper Limit Of Quantification

[0174] Procedure: 1. Make Capture Antibody Working Solution (Concentration -1.2mg / ml, Final Dilution- 2μglmL). -Add 20 μl of biotinylated anti-ELNN antibody to 12ml of 1X PBS, in order to obtain a final concentration of approximately 2μg / ml. Mix by inversion. 2. Wash Neutravidin low bind clear plate with washing buffer. 3. COATING: Add 100μl of capture antibody working solution per well of a Neutravidin low bind clear plate. Seal plate and incubate at ambient room temperature for 45±10 min. 4. Prepare efanesoctocog alfa intermediate Stocks / Standards / QCs as indicated in Reagent Table 1, 2, 3, and 4. 5. Make 10-fold dilution of Standards / QCs / Samples: -Add 30μl of Standards / QCs / Samples to 270μl of low salt binding buffer. Mix well. 6. Wash plate with washing buffer. 7. Loading: transfer 100μl of the 10-fold diluted Standards / QCs / Samples in duplicate in the coated plate. Seal plate and incubate at ambient room temperature for 35±5 min. 8. During incubation and prior to washing (step 10), prepare 1X Buffer -Add 0.5 ml of Buffer Stock Solution (10x concentrated) to 4.5 ml of Nerl water. 9. Prepare FIXa / FX / Phospholipids (FFP) solution 5 minutes prior to use -Add 2 ml of Phospholipid to 10 ml of FIXa / FX mix. 10. Wash plate twice with washing buffer. 11. Add 25μl / well of 1x buffer stock solution. 12. Add 50μl / well of the FIXa / FX / Phospholipid solution (FFP) and tap the plate to mix. Incubate at 37°C in dry incubator for 5min. 13. Add 25μl / well of CaCl2, tap the plate to mix. Incubate at 37°C in dry incubator for 5 min. 14. Add 50μl / well of FXa substrate, tap the plate to mix. Incubate at 37°C in dry incubator for 10min. 15. Stopping: Add 25μl / well of 20% acetic acid solution to stop the colorimetric reaction in the well.

[0175] Reagents and Equipment: • Efanesoctocog alfa (Activity - 4000 IU Vial), received in lyophilized condition shipped at 2-8 °C and stored at -80°C until use. Efanesoctocog alfa was reconstituted in 1ml vehicle solution (10 mM Histidine, 250 mM Arginine HCl, 5% w / v sucrose, 5 mM Calcium Chloride, 0.05% PS80), aliquoted and stored at -80°C.• Biotinylated Anti-ELNN antibody (Concentration (1.2mg / ml) • BioTek plate Reader (SN 200701) • Single channel Rainin Pipettes (LTS20: 2-20μl, LTS200: 20-200μl, and LTS1000: 100- 1000μl) • Multichannel pipettes (12 channel 20-200μl) • Neutravidin Low Bind Clear Plate (Thermo Scientific, Cat#15129) • Microtiter tube 1.2ml (Fisher Scientific, Cat#02-681-376) • Acetic Acid 99.7%+ A.C.S. reagent (Sigma, Cat#242853) • 1X PBS pH7.4 without Calcium & magnesium • Sodium Chloride (Sigma, Cat#S9888) • Tween-20 (Bio-Rad Laboratory, M2595) • Bovine Serum Albumin (Sigma, Cat#A4503) • Chromogenix Coatest SP FVIII Kit (Diapharma, Cat# 82408663) • Pooled Human Normal Plasma: (Precision BioLogic, Cat# CCN-20)

[0176] Reagent Preparation:

[0177] Efanesoctocog alfa: was reconstituted in 1ml vehicle solution (10 mM Histidine, 250 mM Arginine HCl, 5% w / v sucrose, 5 mM Calcium Chloride, 0.05% PS80), aliquoted and stored at - 80°C. On the day of assay, one vial of efanesoctocog alfa was thawed quickly in a 37°C water bath.

[0178] Biotinylated Anti-ELNN antibody (Concentration - 1.2mg / ml): The test solution (1 ml per one Eppendorf tube, total 5 vials) was received in dry ice and stored at -80°C upon receipt. In order to prevent multiple freeze-thaw cycles it was quickly thawed in a 37°C water bath, aliquoted to and stored at -80°C until use. On the day of assay, one vial of biotinylated anti-ELNN antibody was thawed quickly in a 37°C water bath.20 μl of biotinylated anti ELNN antibody was added to 12 ml of 1X PBS in order to obtain a final concentration of approximately 2μg / ml.

[0179] Wash Buffer: 1X PBS, 0.05% Tween-20, pH 7.4

[0180] Low Salt Binding Buffer (1X PBS, 0.05 % Tween-20, pH 7.4, 0.25% BSA, 200mM NaCl)

[0181] 20% Acetic Acid

[0182] FXa substrate: Reconstitute a vial of S-2765 with 12.0 mL of Nerl water.

[0183] Factor IXa + factor X: Reconstitute the vial of Factor IXa + Factor X with 10 ml of cold Nerl water.

[0184] Prepare efanesoctocog alfa intermediate Stocks in normal human citrated plasma as indicated in Reagent Table 1. Reagent Table 1 StockTo make intermediate stock HumanTarget Identification Stock ID Stock Conc. Volume Plasma Vol Conc. L I Ld in Reagent Table 2. Reagent Table 2 StandardTo make Standards (STD) solution HumanTarget Identification Stock ID Stock Volume (µL) Plasma Vol Conc.curve.

[0186] Prepare efanesoctocog alfa Quality Controls (LPC, MPC, HPC) in human normal plasma as indicated in Reagent Table 3. Reagent Table 3 Quality To make Quality Controls (QC) solution Human Target Conc. ControlsStock ID Stock concV l m ( L) Plasma VolI / L

[0187] Prepare efanesoctocog alfa Quality Controls (QC) in human FVIII deficient plasma as indicated in Reagent Table 4. Reagent Table 4 QCQC Stock FVIII (-)Target identification Stock ID Conc.(mIU / mL) Vol Plasma Vol Conc. L L mIU / mL

[0188] Plasma baseline-corrected factor VIII activity levels

[0189] Mean plasma baseline-corrected FVIII activity level-time profiles, quantified by the 1- stage clotting assay and capture chromogenic assay are presented in Table 6.

[0190] Prior to receiving efanesoctocog alfa, mean (range) baseline endogenous FVIII activity level with the OSA was 6.7 IU / dL (1.3 IU / dL–14.6 IU / dL) and was under the LLOQ with the CCS. As quantified by both the OSA and CCS, a single dose of efanesoctocog alfa (25 IU / kg) maintained mean baseline-corrected FVIII activity level >1 IU / dL up to 10 days post-dose (Figure 3 and Table 6). Mean baseline-corrected FVIII activities were maintained >40 IU / dL for nearly 3 days post-dose (38.5 IU / dL at 72 hours) and >10 IU / dL up to 5 days post-dose when quantified with the OSA (Figure 3A and Table 6). When quantified by the CCS, mean baseline-corrected FVIII activity levels were maintained >40 IU / dL up to 1-day post-dose and >10 IU / dL up to 4 days post-dose (Figure 3B and Table 6). Table 6. Mean plasma baseline-corrected FVIII activity levels over time quantified by the one-stage clotting assay or capture chromogenic assay following a single dose of efanesoctocog alfa (25 IU / kg) One-stage assay Capture chromogenic Nominal time ost-dose0.1790.8a a 57.3 8 a a 0594558

[0191] As quantified by the 1-stage clotting assay, efanesoctocog alfa maintained a mean baseline-corrected FVIII activity level of >40 IU / dL (normal to near normal level) up to nearly 72 hours (3 days) postdose (38.5 IU / dL at 72 hours), >10 IU / dL up to 120 hours (5 days) postdose, and >1 IU / dL up to the last sampling time point of 240 hours (10 days) postdose.

[0192] As quantified by the capture chromogenic assay, efanesoctocog alfa maintained a mean baseline-corrected FVIII activity level of >40 IU / dL (normal to near normal level) up to 24 hours (1 day) postdose, >10 IU / dL up to 96 hours (4 days) postdose, and >1 IU / dL up to the last sampling time point of 240 hours (10 days) postdose.

[0193] Pharmacokinetic parameters for baseline-corrected factor VIII activity

[0194] A summary of plasma PK parameters for baseline-corrected FVIII activity is presented in Table 7.

[0195] Following a single dose of efanesoctocog alfa (25 IU / kg) the median (range) time to maximum (tmax) of the baseline-corrected FVIII activity level was 1.09 (0.17–2.95) hours and 2.96 (0.50–6.00) hours, for the OSA and CCS, respectively (Table 7). Mean (SD) t1 / 2z values were 39.1 (8.70) hours and 49.0 (10.2) hours, for the OSA and CCS, respectively. For the OSA, mean (SD) Cmax, AUC, and IR were 111 (30.9) IU / dL, 6910 (1550) IU∙h / dL, and 4.41 (1.24)(IU / dL) / (IU / kg), respectively. Corresponding values for the CCS were 63.8 (14.8) IU / dL, 3730 (419) IU∙h / dL, and 2.55 (0.596) (IU / dL) / (IU / kg). Between-participant variation in IR, Cmax, and AUC were low, with coefficient of variation values ranging from 11% to 28%. Table 7: Mean ±SD (geometric mean) [CV%] pharmacokinetic parameters following a 25 IU / kg dose of efanesoctocog alfa in participants with type 2N or 3 von Willebrand disease PK Parameters 1-Stage Clotting Assay Capture Chromogenic Assay N6 6CL,RT,mean residence fime; PK, pharmacokinefic; SD, standard deviafion; t1 / 2z, terminal half-life; tmax, fime toCmax; Vss, volume of distribufion at steady state; VWD, von Willebrand disease.

[0196] Summary – Pharmacokinetic Results

[0197] This Phase 1 study of participants with Type 2N and Type 3 VWD demonstrated that a single dose of efanesoctocog alfa (25 IU / kg) maintains FVIII activity levels >40 IU / dL (normal to near-normal levels) for nearly 3 days post-dose when assessed with the 1-stage clotting assay, and >40 IU / dL for up to 1-day post-dose when assessed by the capture chromogenic assay. Mean half-life values for baseline-corrected FVIII activity quantified by the 1-stage clotting assay and capture chromogenic assay were 39.1 and 49.0 hours, respectively.

[0198] The PK profile obtained using the capture chromogenic assay in this study of participants with Type 2N and Type 3 VWD is comparable to the profile observed in participants with severe haemophilia A in a Phase 1 / 2a study following a single 25 IU / kg dose.

[0199] In the haemophilia Phase 1 / 2a study, mean FVIII activity levels assessed by the OSA were 17 IU / dL at 4 days and >1 IU / dL for up to 10 days post-dose. There were no prominentdifferences in PK results between patients with type 2N and type 3 VWD, though it should be noted that patient numbers for this comparison were low.

[0200] The data obtained from the 1-stage clotting assay appeared to be higher than those obtained from the capture chromogenic assay. The reason for this discrepancy is not entirely clear, however, in this patient population the capture chromogenic assay results may be most accurate as interference from endogenous FVIII is avoided by use of a self-standard. Safety and Tolerability Results

[0201] A single dose of efanesoctocog alfa (25 IU / kg) was well-tolerated, inhibitor development to FVIII was not detected, and there were no reports of serious allergic reaction, anaphylaxis, or vascular thrombotic events. Three participants (50%) experienced a total of 8 treatment-emergent AEs (TEAEs) during the study (Tables 8 and 9). TEAEs reported included 5 events of headache in 2 participants, and 1 event each of gastrointestinal motility disorder, nausea, and arthropod bite (Table 9). All TEAEs were assessed by the investigator as non-serious, mild, and not related to efanesoctocog alfa treatment. Of the 8 TEAEs, 6 recovered / resolved, and 2 (gastrointestinal motility disorder and nausea) did not. No deaths, serious or severe TEAEs, AESIs, TEAEs leading to permanent discontinuation of study intervention or withdrawal from the study were reported (Table 8). There were no clinically meaningful patterns identified in clinical laboratory parameters, vital signs, or physical examinations throughout the study. No treatment-emergent ADAs, either treatment-induced or treatment-boosted, were identified. For the 2 participants with Type 2N VWD, 1 participant’s VWF levels were within the normal range for all assessments and 1 participant had VWF levels below the normal range at all assessments (including prior to efanesoctocog dosing). Following a single dose of efanesoctocog alfa (25 IU / kg), there was no change in either VWF antigen or activity levels. Table 8. Overview of treatment-emergent adverse eventsaEfanesoctocog alfa (25 IU / kg) eevent.aAn AE is considered treatment-emergent if it occurred any fime during the TEAE period (from efanesoctocog alfa administrafion up to and including the end of study visit).bAESIs included development of either an inhibitor, a Grade ≥3 allergic reacfion, or an embolic or thrombofic event (except for injecfion site thrombophlebifis), among others. Table 9. Number of parficipants with TEAE by primary system organ class and preferred termaEfanesoctocog alfa (25 IU / kg) N=6(from efanesoctocog alfa administrafion up to and including the end of study visit).

[0202] Factor VIII Inhibitor Development

[0203] Factor VIII inhibitor development was not detected during this study.

[0204] Antidrug Antibodies

[0205] No treatment-emergent ADA response against efanesoctocog alfa was observed as no participant had treatment-induced or treatment-boosted ADAs.

[0206] Summary – Safety and Tolerability

[0207] In this study, a single dose of efanesoctocog alfa (25 IU / kg) was well-tolerated in VWD patients. No participants developed inhibitors to FVIII or anti-drug antibodies (ADA) and there were no reports of SAEs, serious allergic reactions, anaphylaxis, or thrombotic events. These safety results are also consistent with Phase 1 / 2a single and repeat dose studies, and Phase 3 XTEND-1 and XTEND-Kids studies in haemophilia A. In these studies of patients with haemophilia A, efanesoctocog alfa was well-tolerated and FVIII inhibitor development was not detected. The results from this study demonstrate that a single dose of efanesoctocog alfa (25 IU / kg) is well- tolerated and maintains high FVIII activity levels for a prolonged period in patients with Type 2N or Type 3 VWD.Example 2: Characterization of an anti-ELNN antibody.

[0208] Anti-ELNN antibodies are needed. However, the properties of ELNNs minimize the potential for immunogenicity, as their lack of stable tertiary structure disfavors antibody binding and the absence of hydrophobic, aromatic and positively charged residues that serve as anchor residues for peptide MHC II binding reduces the potential for T cell epitopes.

[0209] This example relates to an anti-ELNN antibody (termed the “4D9G3 antibody”), which is a mouse IgG1 monoclonal antibody with a kappa light chain. The anti-ELNN antibody is produced from the mouse hybridoma clone 4D9G3.

[0210] The 4D9G3 antibody can be recombinantly expressed in host cells (e.g., CHO cells that are well-known in the art) and purified using well known procedures for antibody purification, including but not limited to processes comprising protein A chromatography.

[0211] To map the epitope of the 4D9G3 antibody, 183 unique peptides were designed based on ELNN_AE864 (SEQ ID NO: 26) (1 amino acid offset).

[0212] The 4D9G3 antibody recognizes the amino acid sequence (E)SATPE, which is within one of the four building blocks of ELNN_AE864. ELNN_AE864 contains 23 copies and ELNN_144 contains 3 copies (see FIG.4). Of all 183 unique peptides, 43 peptides containing the ESATPE epitope showed significant binding while 5 peptides containing only SATPE showed reduced binding. The remaining 135 peptides, which did not contain the epitope, showed very low binding. (FIG.5).

[0213] Within the 43 peptides containing the epitope ESATPE, there is also variability in binding. By examining the corresponding peptide sequence, it appears that all peptides with less than or equal to 2 amino acids on the C-terminus of the epitope showed inferior signal. On the other hand, if the epitope is on the very N-terminus, binding to the 4D9G3 antibody was not affected (FIG.6).

[0214] The epitope of the antibody is (E)SATPE.

[0215] Non-limiting uses of the 4D9G3 antibody and antibodies derived therefrom include: a. Use as a capture antibody, e.g., in ELISA or in capture chromatography assays b. Biotin-4D9G3 can be used as detection antibody in ELISA c. ELNNs that contain multiple copies of the epitope can be quantified by ELISA using 4D9G3 for capture and biotin-4D9G3 for detectionExample 3. Transfer of 4D9G3 CDRs into alternative IgG backbones

[0216] CDRs, variable regions, and / or other parts of the 4D9G3 antibody can be moved into different antibody backbones. In a non-limiting example, certain sequences from the 4D9G3 antibody (which is in a mouse IgG1 background) can be moved into a mouse IgG2a backbone. The IgG2a backbone has advantages with respect to protein A purification, as well as potential advantages with respect to stability. In another non-limiting example, sequences of the 4D9G3 antibody were moved into a human IgG1 antibody backbone, which was the antibody used in Example 1. The sequences of this antibody are disclosed elsewhere in the application (see Table C). VI. Embodiments of the Disclosure

[0217] The present disclosure includes (and is not limited to) the following exemplary embodiments (E):

[0218] E1. A method for determining the level of exogenous Clotting Factor VIII (FVIII) activity in the blood or plasma of a human subject who has von Willebrand disease (VWD) and to whom a chimeric protein has been administered, wherein the chimeric protein comprises a FVIII protein and an ELNN polypeptide, the method comprising (i) separating the chimeric protein from a biological sample from the subject with an anti-ELNN antibody or antigen-binding fragment thereof to obtain a captured chimeric protein sample, wherein the biological sample is a blood sample or a plasma sample; (ii) assaying the level of FVIII activity in the captured chimeric protein with a chromogenic assay to obtain an assayed FVIII activity value; and (iii) reporting the assayed FVIII activity value or an approximated value thereof without correcting the assayed FVIII activity value or approximated value thereof.

[0219] E2. The method of E1, wherein the chimeric protein was administered to the subject less than one week before the sample was taken from the subject.

[0220] E3. The method of E1 or E2, wherein the biological sample is a blood sample.

[0221] E4. The method of E1 or E2, wherein the biological sample is a plasma sample.

[0222] E5. The method of any one of E1 to E4, wherein correcting the assayed FVIII activity value or approximated value thereof comprises reducing the value.

[0223] E6. The method of any one of E1 to E5, wherein correcting the assayed FVIII activity value or approximated value thereof comprises reducing the value, as if an overestimation of the FVIII activity needs to be accounted for.

[0224] E7. The method of any one of E1 to E6, wherein an activated partial thromboplastin time (aPTT)-based one stage clotting assay is not used to assess the level of FVIII activity.

[0225] E8. The method of any one of E1 to E7, wherein the anti-ELNN antibody or antigen- binding fragment thereof comprises three light chain complementarity determining region (LCDR) sequences, wherein LCDR1, LCDR2, and LCDR3 comprises SEQ ID NOs: 38, 39 and 40, respectively, and wherein the anti-ELNN antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining region (HCDR) sequences, wherein HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 32, 33 and 34, respectively.

[0226] E9. The method of any one of E1 to E8, wherein the anti-ELNN antibody or an antigen binding fragment thereof comprises a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:32 and a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:37.

[0227] E10. The method of any one of E1 to E9, wherein the anti-ELNN antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising three heavy-chain CDRs, and a light chain variable region comprising three light-chain CDRs, wherein said three heavy-chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:32 (HCVR), and said three light-chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:37 (LCVR).

[0228] E11. The method of any one of E1 to E10, wherein the anti-ELNN antibody or antigen- binding fragment thereof comprises a heavy chain variable region (HCVR) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:32 and a light chain variable region (LCVR) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:37.

[0229] E12. The method of any one of E1 to E11, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:37.

[0230] E13. The method of any one of E1 to E12, wherein the antibody comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:31 and a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:36.

[0231] E14. The method of any one of E1 to E13, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:31 and a light chain comprising the amino acid sequence of SEQ ID NO:36.

[0232] E15. The method of any one of E1 to E14, wherein the antibody comprises two heavy chains comprising the amino acid sequence of SEQ ID NO:31, and two light chains comprising the amino acid sequence of SEQ ID NO:36.

[0233] E16. The method of any one of E1 to E15, wherein the antibody comprises two heavy chains consisting of the amino acid sequence of SEQ ID NO:31, and two light chains consisting of the amino acid sequence of SEQ ID NO:36.

[0234] E17. The method of any one of E1 to E16, wherein the antibody or antigen binding fragment thereof binds to an ELNN polypeptide and comprises (i) a heavy chain variable region which is at least 98% identical to SEQ ID NO: 32 and (ii) a light chain variable region which is at least 98% identical to SEQ ID NO: 37.

[0235] E18. The method of any one of E1 to E8, wherein the anti-ELNN antibody or antigen binding portion thereof comprises (i) a light chain variable domain comprising an amino acid sequence at least 90% identical to a light chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs 38, 39 and 40, and (ii) a heavy chain variable domain comprising an amino acid sequence at least 90% identical to a heavy chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs: 32, 33, and 34.

[0236] E19. The method of any one of E1 to E18, wherein the ELNN polypeptide comprises at least three instances of the sequence: SATPE (SEQ ID NO: 28).

[0237] E20. The method of any one of E1 to E19, wherein the ELNN polypeptide comprises at least 3 instances of the sequence: ESATPE (SEQ ID NO: 29).

[0238] E21. The method of any one of E1 to E20, wherein the ELNN polypeptide comprises at least 4 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0239] E22. The method of any one of E1 to E21, wherein the ELNN polypeptide comprises at least 5 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0240] E23. The method of any one of E1 to E22, and wherein the ELNN polypeptide comprises at least 6 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0241] E24. The method of E21 to E23, wherein each of the at least 3, at least 4, at least 5, or at least 6 instances of SATPE (SEQ ID NO: 28) are as part of the following sequence: ESATPE (SEQ ID NO: 29).

[0242] E25. The method of E21 to E24, wherein each instance of SATPE (SEQ ID NO: 28) and / or ESATPE (SEQ ID NO: 29) in the sequence of the ELNN polypeptide is followed by at least 3 amino acids at the C-terminal end thereof.

[0243] E26. The method of any one of E1 to E25, wherein the ELNN polypeptide comprises the sequence of SEQ ID NO: 14, 24, 25, 26, and / or 27.

[0244] E27. The method of any one of E18 to E26, wherein the anti-ELNN antibody or antigen- binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:37.

[0245] E28. The method of any one of E18 to E27, wherein the anti-ELNN antibody or binding portion thereof comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:31, wherein the anti-ELNN antibody or binding portion thereof comprises a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:36.

[0246] E29. The method of any one of E18 to E27, wherein the anti-ELNN antibody or binding portion thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:31, and wherein the anti-ELNN antibody or binding portion thereof a light chain comprising the amino acid sequence of SEQ ID NO:36.

[0247] E30. The method of E29, wherein the anti-ELNN antibody or binding portion thereof comprises two heavy chains comprising the amino acid sequence of SEQ ID NO:31, and wherein the anti-ELNN antibody or binding portion thereof comprises two light chains comprising the amino acid sequence of SEQ ID NO:36.

[0248] E31. The method of any one of E1 to E30, wherein the chimeric protein further comprises a von Willebrand Factor (VWF) fragment.

[0249] E32. A method for determining the level of Clotting Factor VIII (FVIII) activity in the blood or plasma of a human subject who has von Willebrand disease (VWD) and to whom a chimeric protein has been administered, wherein the chimeric protein comprises (a) a FVIII protein and (b) an ELNN polypeptide and / or a von Willebrand Factor (VWF) fragment, the method comprising (i) assaying the level of FVIII activity in the chimeric protein with an activated partial thromboplastin time (aPTT)-based one stage clotting assay to obtain an assayed FVIII activity value, wherein the aPTT-based one stage clotting assay does;(ii) reducing the assayed FVIII activity value to obtain an adjusted FVIII activity value; and (iii) reporting the adjusted FVIII activity value or an approximated value thereof.

[0250] E33. The method of E32, wherein the chimeric protein comprises a FVIII protein and an ELNN polypeptide.

[0251] E34. The method of any one of E32 to E33, wherein the ELNN polypeptide is inserted into the FVIII protein of the chimeric protein.

[0252] E35. The method of any one of E32 to E34, wherein the FVIII activity value or the approximated value thereof is reported to the subject, a caregiver of the subject, or a medical professional.

[0253] E36. The method of any one of E32 to E35, wherein the FVIII activity value or the approximated value thereof is reported to the subject.

[0254] E37. The method any one of E32 to E36, wherein the subject has a body mass index of ≥30 kg / m2.

[0255] E38. The method any one of E32 to E37, wherein the subject is a female.

[0256] E39. The method any one of E32 to E37, wherein the subject is a male.

[0257] E40. The method any one of E32 to E39, wherein the subject is human.

[0258] E41. The method any one of E1 to E40, wherein the VWD is Type 2N VWD.

[0259] E42. The method any one of E1 to E40, wherein the VWD is Type 3 VWD.

[0260] E43. A method for determining the level of Clotting Factor VIII (FVIII) activity from a chimeric protein in a biological sample, wherein the chimeric protein comprises a FVIII protein and an ELNN polypeptide, the method comprising (i) separating the chimeric protein from the biological sample from the subject with an anti-ELNN antibody to obtain a captured chimeric protein sample, wherein the biological sample is a blood sample or a plasma sample; and (ii) assaying the level of FVIII activity in the captured chimeric protein with a chromogenic assay to obtain an assayed FVIII activity value.

[0261] E44. The method of any one of E1 to E43, wherein the chimeric protein is in a pharmaceutical composition comprising sucrose, histidine, arginine, calcium chloride, and polysorbate 80.

[0262] E45. The method of any one of E1 to 44, wherein the chimeric protein is in a pharmaceutical composition comprising: (a) about 5% (w / v) sucrose; (b) about 10 mM histidine; (c) about 250 mM arginine; (d) about 5 mM calcium chloride; and (e) about 0.05% polysorbate 80, and a pH of about 7.0.

[0263] E46. The method of E50 or E51, wherein the pharmaceutical composition is lyophilized.

[0264] E47. The method of E52, wherein the lyophilized pharmaceutical composition is reconstituted with sterile water prior to administering to the subject.

[0265] E48. The method of any one of E1 to E53, wherein the chimeric protein comprises a VWF fragment.

[0266] E49. The method of E54, wherein the VWF fragment comprises a D’D3 fragment of VWF.

[0267] E50. The method of E55, wherein the VWF fragment consists of a D’D3 fragment of VWF.

[0268] E51. The method of any one of E55 to E56, wherein the D’D3 fragment of VWF is mutated such that it cannot form disulfide binds with another D’D3 fragment of VWF.

[0269] E52. The method of any one of E55 to E57, wherein the D' domain of the D’D3 fragment of VWF comprises the sequence of SEQ ID NO: 21.

[0270] E53. The method of any one of E55 to E58, wherein the D3 domain of the D’D3 fragment of VWF comprises the sequence of SEQ ID NO: 23.

[0271] E54. The method of any one of E1 to E59, wherein the FVIII protein comprises at least a partial deletion of the wild-type FVIII B domain.

[0272] E55. The method of any one of E1 to E60, wherein the chimeric protein comprises an ELNN polypeptide comprising the sequence of SEQ ID NO: 14.

[0273] E56. The method of any one of E1 to E60, wherein the chimeric protein comprises an ELNN polypeptide comprising the sequence of SEQ ID NO: 24.

[0274] E57. The method of any one of E1 to E62, wherein the chimeric protein comprises a first polypeptide which comprises a FVIII protein and a first immunoglobulin (“Ig”) constant region or a portion thereof, and a second polypeptide which comprises a VWF fragment and a second Ig constant region or a portion thereof.

[0275] E58. The method of E63, wherein the chimeric protein comprises (i) a first polypeptide comprising a FVIII protein, an ELNN polypeptide inserted within and replacing a portion of the B domain, and a first Fc region; and (ii) a second polypeptide comprising a VWF fragment, an additional ELNN polypeptide, an a2 linker, and a second Fc region.

[0276] E59. The method of E64, wherein the chimeric protein is a FVIII-ELNN-Fc / D’D3-ELNN- Fc heterodimer.

[0277] E60. The method of any one of E63 to E65, wherein the first polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 1 and the second polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 2, and wherein the first polypeptide chain and the second polypeptide chain are covalently linked by two disulfide bonds between the Ig constant region domains of the first and second polypeptide chains.

[0278] E61. The method of any one of E1 to E66, wherein the chimeric protein is efanesoctocog alfa.

[0279] E62. A method of monitoring the level of FVIII in the blood or plasma of a human subject who is receiving treatment for Willebrand disease (VWD), the method comprising performing the method of any one of E1 to E42 or E49 to E67 at least once per year.

[0280] E63. The method of E43, comprising performing the method of any one of E1 to E42 at least once every six months.

[0281] E64. The method of E44, comprising performing the method of any one of E1 to E42 at least once per month.

[0282] E65. The method of any one of E1 to E45, wherein multiple doses of the chimeric protein are administered to the subject at a dosing interval.

[0283] E66. The method of E46, wherein at least one of the multiple doses is from about 10 IU / kg to about 30 IU / kg and the dosing interval is at least about 7 days.

[0284] E67. The method of E46, wherein at least one of the multiple doses is from about 25 IU / kg and the dosing interval is at least about 7 days.

[0285] E68. An antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, wherein the antibody or antigen-binding fragment thereof comprises three light chaincomplementarity determining region (CDR) sequences of SEQ ID NOs: 38, 39 and 40, and three heavy chain complementarity determining region (CDR) sequences of SEQ ID NOs: 32, 33 and 34, and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0286] E69. An antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, comprising three heavy-chain CDRs of a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:32 and three light-chain CDRs of a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:37 and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0287] E70. An antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, comprising a heavy chain variable region comprising three heavy-chain CDRs, and a light chain variable region comprising three light-chain CDRs, wherein said three heavy-chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:32 (HCVR), and said three light- chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:37 (LCVR), and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: ESATPE (SEQ ID NO: 29).

[0288] E71. The antibody or antigen-binding fragment of any one of claims 68 to 70, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:32 and a light chain variable region (LCVR) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:37.

[0289] E72. The antibody or antigen-binding fragment of any one of claims 68 to 71, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:37.

[0290] E73. The antibody or antigen-binding fragment of any one of E68 to E72, wherein the antibody comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:31 and a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:36.

[0291] E74. The antibody or antigen-binding fragment of any one of E68 to E73, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:31 and a light chain comprising the amino acid sequence of SEQ ID NO:36.

[0292] E75. The antibody of any one of E68 to E74, wherein the antibody comprises two heavy chains comprising the amino acid sequence of SEQ ID NO:31, and two light chains comprising the amino acid sequence of SEQ ID NO:36.

[0293] E76. The antibody of any one of E68 to E75, wherein the antibody comprises two heavy chains consisting of the amino acid sequence of SEQ ID NO:31, and two light chains consisting of the amino acid sequence of SEQ ID NO:36.

[0294] E77. A monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen binding fragment thereof binds to an ELNN polypeptide and comprises (i) a heavy chain variable region which is at least 98% identical to SEQ ID NO: 32 and (ii) a light chain variable region which is at least 98% identical to SEQ ID NO: 37, and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0295] E78. An antigen binding protein comprising (i) a light chain variable domain comprising an amino acid sequence at least 90% identical to a light chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs 38, 39 and 40, and (ii) a heavy chain variable domain comprising an amino acid sequence at least 90% identical to a heavy chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs: 32, 33, and 34, and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0296] E79. The antibody, antigen-binding fragment, or antigen binding protein of any one of E68 to E78, wherein the ELNN polypeptide comprises the sequence: ESATPE (SEQ ID NO: 29).

[0297] E80. The antibody, antigen-binding fragment, or antigen binding protein of any one of E68 to E79, wherein each of the instances of the sequence SATPE (SEQ ID NO: 28) is ESATPE (SEQ ID NO: 29).

[0298] E81. The antibody, antigen-binding fragment, or antigen binding protein of any one of E68 to E79, wherein the ELNN polypeptide comprises at least 4 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0299] E82. The antibody, antigen-binding fragment, or antigen binding protein of any one of E68 to E79, and wherein the ELNN polypeptide comprises at least 5 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0300] E83. The antibody, antigen-binding fragment, or antigen binding protein of any one of E68 to E79, and wherein the ELNN polypeptide comprises at least 6 instances of the following sequence: SATPE (SEQ ID NO: 28).

[0301] E84. The antibody, antigen-binding fragment, or antigen binding protein of any one of E68 to E83, wherein each of the at least 3, at least 4, at least 5, or at least 6 instances of SATPE (SEQ ID NO: 28) are part of the following sequence: ESATPE (SEQ ID NO: 29).

[0302] E85. The antibody, antigen-binding fragment, or antigen binding protein of any one of E68 to E84, wherein each instance of SATPE (SEQ ID NO: 28) and / or ESATPE (SEQ ID NO: 29) in the sequence of the ELNN polypeptide is followed by at least 3 amino acids at the C-terminal end thereof.

[0303] E86. The antibody, antigen-binding fragment, or antigen binding protein of any one of E68 to E85, wherein the ELNN polypeptide comprises the sequence of SEQ ID NO: 14, 24, 25, 26, and / or 27.

[0304] E87. The antibody or antigen-binding fragment of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:37.

[0305] E88. An antibody or antigen-binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:31 and a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:36.

[0306] E89. An antibody or antigen-binding fragment comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:31 and a light chain comprising the amino acid sequence of SEQ ID NO:36.

[0307] E90. An antibody comprising two heavy chains comprising the amino acid sequence of SEQ ID NO:31, and two light chains comprising the amino acid sequence of SEQ ID NO:36.

[0308] E91. The antibody, antigen-binding fragment, or antigen binding protein of any one of E66 to E88, which is an isolated antibody, antigen-binding fragment, or antigen binding protein.

[0309] E92. A polynucleotide or set of polynucleotides encoding the antibody, antigen-binding fragment, or antigen binding protein of any one of E68 to E91.

[0310] E93. A vector comprising the polynucleotide or set of polynucleotides of E92.

[0311] E94. A host cell comprising the vector of E93.

[0312] E95. The host cell comprising of E94, which is a CHO or HEK293 cell.VII. References

[0313] 1 Leebeek FW, Eikenboom JC. Von Willebrand's Disease. N Engl J Med.2016; 375: 2067-80.10.1056 / NEJMra1601561.

[0314] 2 Weyand AC, Flood VH. Von Willebrand Disease: Current Status of Diagnosis and Management. Hematol Oncol Clin North Am.2021; 35: 1085-101.10.1016 / j.hoc.2021.07.004.

[0315] 3 Dong X, Leksa NC, Chhabra ES, Arndt JW, Lu Q, Knockenhauer KE, Peters RT, Springer TA. The von Willebrand factor D'D3 assembly and structural principles for factor VIII binding and concatemer biogenesis. Blood.2019; 133: 1523-33.10.1182 / blood-2018-10-876300.

[0316] 4 Lenting PJ, CJ VANS, Denis CV. Clearance mechanisms of von Willebrand factor and factor VIII. J Thromb Haemost.2007; 5: 1353-60.10.1111 / j.1538-7836.2007.02572.x.

[0317] 5 Pipe SW, Montgomery RR, Pratt KP, Lenting PJ, Lillicrap D. Life in the shadow of a dominant partner: the FVIII-VWF association and its clinical implications for hemophilia A. Blood. 2016; 128: 2007-16.10.1182 / blood-2016-04-713289.

[0318] 6 Goodeve A, James P. von Willebrand Disease. In: Adam MP, Feldman J, Mirzaa GM, Pagon RA, Wallace SE, Bean LJH, Gripp KW, Amemiya A, eds. GeneReviews(®). Seattle (WA): University of Washington, Seattle. Copyright © 1993-2024, University of Washington, Seattle. GeneReviews is a registered trademark of the University of Washington, Seattle. All rights reserved., 2017.

[0319] 7 Lillicrap D. von Willebrand disease: advances in pathogenetic understanding, diagnosis, and therapy. Hematology Am Soc Hematol Educ Program. 2013; 2013: 254-60. 10.1182 / asheducation-2013.1.254.

[0320] 8 Connell NT, Flood VH, Brignardello-Petersen R, Abdul-Kadir R, Arapshian A, Couper S, Grow JM, Kouides P, Laffan M, Lavin M, Leebeek FWG, O'Brien SH, Ozelo MC, Tosetto A, Weyand AC, James PD, Kalot MA, Husainat N, Mustafa RA. ASH ISTH NHF WFH 2021 guidelines on the management of von Willebrand disease. Blood Adv. 2021; 5: 301-25. 10.1182 / bloodadvances.2020003264.

[0321] 9 Seidizadeh O, Peyvandi F, Mannucci PM. Von Willebrand disease type 2N: An update. J Thromb Haemost.2021; 19: 909-16.10.1111 / jth.15247.

[0322] 10 Peyvandi F, Castaman G, Gresele P, De Cristofaro R, Schinco P, Bertomoro A, Morfini M, Gamba G, Barillari G, Jiménez-Yuste V, Königs C, Iorio A, Federici AB. A phase III study comparing secondary long-term prophylaxis versus on-demand treatment with vWF / FVIIIconcentrates in severe inherited von Willebrand disease. Blood Transfus. 2019; 17: 391-8. 10.2450 / 2019.0183-18.

[0323] 11 Therapeutics B. ALTUVIIIO Prescribing Information. https: / / www.altuviiio.com / assets / pdf / ALTUVIIIO-Prescribing-Information.pdf, 2023.

[0324] 12 von Drygalski A, Chowdary P, Kulkarni R, Susen S, Konkle BA, Oldenburg J, Matino D, Klamroth R, Weyand AC, Jimenez-Yuste V, Nogami K, Poloskey S, Winding B, Willemze A, Knobe K. Efanesoctocog Alfa Prophylaxis for Patients with Severe Hemophilia A. N Engl J Med.2023; 388: 310-8.10.1056 / NEJMoa2209226.

[0325] 13 Seth Chhabra E, Liu T, Kulman J, Patarroyo-White S, Yang B, Lu Q, Drager D, Moore N, Liu J, Holthaus AM, Sommer JM, Ismail A, Rabinovich D, Liu Z, van der Flier A, Goodman A, Furcht C, Tie M, Carlage T, Mauldin R, Dobrowsky TM, Liu Z, Mercury O, Zhu L, Mei B, Schellenberger V, Jiang H, Pierce GF, Salas J, Peters R. BIVV001, a new class of factor VIII replacement for hemophilia A that is independent of von Willebrand factor in primates and mice. Blood.2020; 135: 1484-96.10.1182 / blood.2019001292.

[0326] 14 Konkle BA, Shapiro AD, Quon DV, Staber JM, Kulkarni R, Ragni MV, Chhabra ES, Poloskey S, Rice K, Katragadda S, Fruebis J, Benson CC. BIVV001 Fusion Protein as Factor VIII Replacement Therapy for Hemophilia A. N Engl J Med. 2020; 383: 1018-27. 10.1056 / NEJMoa2002699.

[0327] 15 Staber JM, Lissitchkov T, Konkle BA, Shapiro AD, Quon DV, Kulkarni R, Hamilton M, Chhabra ES, Katragadda S, Altincatal A, Willemze A, Dumont J, Ragni MV. Efanesoctocog Alfa Half-Life and Clearance Are Independent of von Willebrand Factor in Severe Hemophilia A: A Post Hoc Analysis from Phase 1 / 2a Studies. Blood. 2021; 138: 1035-. 10.1182 / blood-2021- 148534.

[0328] 16 Pipe S, Sadeghi-Khomami A, Konkle BA, Kitchen S, Negrier C, Liu M, Santagostino E, Willemze A, Abad-Franch L, Knobe K, Seth Chhabra E. A global comparative field study to evaluate the factor VIII activity of efanesoctocog alfa by one-stage clotting and chromogenic substrate assays at clinical haemostasis laboratories. Haemophilia.2024; 30: 214- 23.10.1111 / hae.14831.

[0329] 17 Lissitchkov T, Willemze A, Katragadda S, Rice K, Poloskey S, Benson C. Efanesoctocog alfa for hemophilia A: results from a phase 1 repeat-dose study. Blood Adv.2022; 6: 1089-94.10.1182 / bloodadvances.2021006119.

[0330] 18 Malec L, Peyvandi F, Chan AKC, Königs C, Zulfikar B, Yuan H, Simpson M, Román MTÁ, Carcao M, Staber JM, Dunn AL, Chou S-C, d’Oiron R, Albisetti M, Demissie M, SantagostinoE, Yarramaneni A, Wong N, Abad-Franch L, Gunawardena S, Fijnvandraat K. Efanesoctocog Alfa Prophylaxis for Children with Severe Hemophilia A. N Engl J Med. 2024; 391: 235-46. doi:10.1056 / NEJMoa2312611.

[0331] 19 Coppola A, Franchini M, Makris M, Santagostino E, Di Minno G, Mannucci PM. Thrombotic adverse events to coagulation factor concentrates for treatment of patients with haemophilia and von Willebrand disease: a systematic review of prospective studies. Haemophilia.2012; 18: e173-87.10.1111 / j.1365-2516.2012.02758.x.

[0332] 20 Ryu JH, Bauer KA, Schulman S. Periprocedural management of type 2N von Willebrand disease with efanesoctocog alfa. J Thromb Haemost. 2023; 21: 3508-10. 10.1016 / j.jtha.2023.09.009.SEQUENCES Table A: Exemplary chimeric protein sequences A-FVIII(ELNN)-Fc: SEQ ID NO: 1 3301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY1601 TGACTGTAGA AGATGGGCCA ACTAAATCAG ATCCTCGGTG CCTGACCCGC1651 TATTACTCTA GTTTCGTTAA TATGGAGAGA GATCTAGCTT CAGGACTCAT 1701 TGGCCCTCTC CTCATCTGCT ACAAAGAATC TGTAGATCAA AGAGGAAACC5151 CCAGAGTTGG GTGCACCAGA TTGCCCTGAG GATGGAGGTT CTGGGCTGCG5201 AGGCACAGGA CCTCTACGAC AAAACTCACA CATGCCCACC GTGCCCAGCT 5251 CCAGAACTCC TGGGCGGACC GTCAGTCTTC CTCTTCCCCC CAAAACCCAA :651 GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT701 TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT 751 GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC 801 CCTCCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACG4251 AAGCTTCTCT GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCAGAAC4301 TCCTGGGCGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC 4351 CTCATGATCT CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG 4401 CCACGAAGAC CCTGAGGTCA AGTTCAACTG GTATGTGGAC GGCGTGGAAGTable B. Additional chimeric protein sequences Description / SequenceKMVYEDTLTL FPFSGETVFM SMENPGLWIL GCHNSDFRNR GMTALLKVSS CDKNTGDYYE 720 DSYEDISAYL LSKNNAIEPR SFSQNSRHPS TRQKQFNATT IPENDIEKTD PWFAHRTPMP 780 KIQNVSSSDL LMLLRQSPTP HGLSLSDLQE AKYETFSDDP SPGAIDSNNS LSEMTHFRPQ 840SCSMPLGMES KAISDAQITA SSYFTNMFAT WSPSKARLHL QGRSNAWRPQ VNNPKEWLQV 1620 DFQKTMKVTG VTTQGVKSLL TSMYVKEFLI SSSQDGHQWT LFFQNGKVKV FQGNQDSFTP 1680 VVNSLDPPLL TRYLRIHPQS WVHQIALRME VLGCEAQDLY DKTHTCPPCP APELLGGPSV 1740SVTVRLPGLH NSLVKLKHGA GVAMDGQDIQ LPLLKGDLRI QHTVTASVRL SYGEDLQMDW 480 DGRGRLLVKL SPVYAGKTCG LCGNYNGNQG DDFLTPSGLA EPRVEDFGNA WKLHGDCQDL 540 QKQHSDPCAL NPRMTRFSEE ACAVLTSPTF EACHRAVSPL PYLRNCRYDV CSCSDGRECL 600Table C. Additional Sequences Description / SEQ ID NO Sequence 60120 180 240 300 360 420 480 540 576 240 300 360 420 480 540 600 660 720 780 840 864 5ELNN polypeptide ESATPE Binding Sequence 6 SEQ ID NO: 28 60120 180 240 300 360 420 480 540 600 660 720 60 120 180 240 300 360 420 445 60116FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL 180 SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGECAmino Acid sequence DVVMTQTPLS LPVSLGDQAS ISCRSSQSLA HSNGNTYLHW YLQKTGQSPK LLIYKVSNRF of aELNN-mIgG1 60 Light Chain SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTYDP FTFGSGTKLE IKLight Chain SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTYDP FTFGSGTKLE IK Variable Region 112 SEQ ID NO: 57

[0333] The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0334] Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims.

[0335] All patents and publications cited herein are incorporated by reference herein in their entirety.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for determining the level of exogenous Clotting Factor VIII (FVIII) activity in the blood or plasma of a human subject who has von Willebrand disease (VWD) and to whom a chimeric protein has been administered, wherein the chimeric protein comprises a FVIII protein and an ELNN polypeptide, the method comprising (i) separating the chimeric protein from a biological sample from the subject with an anti-ELNN antibody or antigen-binding fragment thereof to obtain a captured chimeric protein sample, wherein the biological sample is a blood sample or a plasma sample; (ii) assaying the level of FVIII activity in the captured chimeric protein with a chromogenic assay to obtain an assayed FVIII activity value; and (iii) reporting the assayed FVIII activity value or an approximated value thereof without correcting the assayed FVIII activity value or approximated value thereof.

2. The method of claim 1, wherein the chimeric protein was administered to the subject less than one week before the sample was taken from the subject.

3. The method of claim 1 or 2, wherein the biological sample is a plasma sample.

4. The method of any one of claims 1 to 3, wherein correcting the assayed FVIII activity value or approximated value thereof comprises reducing the value as if an overestimation of the FVIII activity needs to be accounted for.

5. The method of any one of claims 1 to 4, wherein the anti-ELNN antibody or an antigen binding fragment thereof comprises a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:32 and a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:

37.

6. The method of any one of claims 1 to 5, wherein the ELNN polypeptide comprises at least 3 instances of the sequence: ESATPE (SEQ ID NO: 29).

7. The method of any one of claims 1 to 6, wherein the ELNN polypeptide comprises the sequence of SEQ ID NO: 14, 24, 25, 26, and / or 27.

8. A method for determining the level of Clotting Factor VIII (FVIII) activity in the blood or plasma of a human subject who has von Willebrand disease (VWD) and to whom a chimeric protein has been administered, wherein the chimeric protein comprises (a) a FVIII protein and (b) an ELNN polypeptide and / or a von Willebrand Factor (VWF) fragment, the method comprising (i) assaying the level of FVIII activity in the chimeric protein with an activated partial thromboplastin time (aPTT)-based one stage clotting assay to obtain an assayed FVIII activity value, wherein the aPTT-based one stage clotting assay does; (ii) reducing the assayed FVIII activity value to obtain an adjusted FVIII activity value; and (iii) reporting the adjusted FVIII activity value or an approximated value thereof.

9. The method of claim 8, wherein the chimeric protein comprises a FVIII protein and an ELNN polypeptide.

10. The method of any one of claims 8-9, wherein the ELNN polypeptide is inserted into the FVIII protein of the chimeric protein.

11. The method any one of claims 8-10, wherein the subject is human.

12. The method any one of claims 1 to 11, wherein the VWD is Type 2N VWD.

13. The method any one of claims 1 to 11, wherein the VWD is Type 3 VWD.

14. A method for determining the level of Clotting Factor VIII (FVIII) activity from a chimeric protein in a biological sample, wherein the chimeric protein comprises a FVIII protein and an ELNN polypeptide, the method comprising (i) separating the chimeric protein from the biological sample from the subject with an anti-ELNN antibody to obtain a captured chimeric protein sample, wherein the biological sample is a blood sample or a plasma sample; and (ii) assaying the level of FVIII activity in the captured chimeric protein with a chromogenic assay to obtain an assayed FVIII activity value.

15. The method of any one of claims 1 to 14, wherein the chimeric protein comprises a VWF fragment.

16. The method of claim 15, wherein the VWF fragment comprises a D’D3 fragment of VWF.

17. The method of claim 16, wherein the VWF fragment consists of a D’D3 fragment of VWF.

18. The method of any one of claims 16-17, wherein the D’D3 fragment of VWF is mutated such that it cannot form disulfide binds with another D’D3 fragment of VWF.

19. The method of any one of claims 1 to 18, wherein the FVIII protein comprises at least a partial deletion of the wild-type FVIII B domain.

20. The method of any one of claims 1 to 19wherein the chimeric protein is a FVIII-ELNN- Fc / D’D3-ELNN-Fc heterodimer.

21. The method of any one of claims 1 to 20, wherein the chimeric protein is efanesoctocog alfa.

22. A method of monitoring the level of FVIII in the blood or plasma of a human subject who is receiving treatment for Willebrand disease (VWD), the method comprising performing the method of any one of claims 1-21 at least once per year.

23. The method of any one of claims 1 to 22, wherein multiple doses of the chimeric protein are administered to the subject at a dosing interval.

24. The method of claim 23, wherein at least one of the multiple doses is from about 10 IU / kg to about 30 IU / kg and the dosing interval is at least about 7 days.

25. The method of claim 24, wherein at least one of the multiple doses is from about 25 IU / kg and the dosing interval is at least about 7 days.

26. An antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, wherein the antibody or antigen-binding fragment thereof comprises three light chain complementarity determining region (CDR) sequences of SEQ ID NOs: 38, 39 and 40, and three heavy chain complementarity determining region (CDR) sequences of SEQ ID NOs: 32, 33 and 34, and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28).

27. An antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, comprising three heavy-chain CDRs of a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:32 and three light-chain CDRs of a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:37 and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28).

28. An antibody or antigen-binding fragment thereof, which binds an ELNN polypeptide, comprising a heavy chain variable region comprising three heavy-chain CDRs, and a light chain variable region comprising three light-chain CDRs, wherein said three heavy-chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:32 (HCVR), and said three light-chain CDRs comprise the CDR1, CDR2 and CDR3 from SEQ ID NO:37 (LCVR), and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: ESATPE (SEQ ID NO: 29).

29. The antibody or antigen-binding fragment of any one of claims 66 to 69, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

37.

30. A monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen binding fragment thereof binds to an ELNN polypeptide and comprises (i) a heavy chain variable region which is at least 98% identical to SEQ ID NO: 32 and (ii) a light chain variable region which is at least 98% identical to SEQ ID NO: 37, and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28).

31. An antigen binding protein comprising (i) a light chain variable domain comprising an amino acid sequence at least 90% identical to a light chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs 38, 39 and 40, and (ii) a heavy chain variable domain comprising an amino acid sequence at least 90% identical to a heavy chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs: 32, 33, and 34, and wherein the ELNN polypeptide comprises at least 3 instances of the following sequence: SATPE (SEQ ID NO: 28).

32. An antibody or antigen-binding fragment comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:31 and a light chain comprising the amino acid sequence of SEQ ID NO:36.

33. A polynucleotide or set of polynucleotides encoding the antibody, antigen-binding fragment, or antigen binding protein of any one of claims 26-32.

34. A vector comprising the polynucleotide or set of polynucleotides of claim 33.

35. A host cell comprising the vector of claim 34.

36. The host cell of claim 35, wherein the host cell is a CHO or HEK293 cell.

Images