RELATED APPLICATIONS

5 [0001] This application claims priority to U.S. Provisional Patent Application No. 63/041,399, filed June 19, 2020, U.S. Provisional Application No. 63/044,546, filed June 26, 2020, and U.S. Provisional Application No. 63/046,344, filed June 30, 2020, the disclosures of which are hereby incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing in ASCII text file (Name:

10 719087_SA9_476PC_ST25.txt; Size: 71,408 bytes; and Date of Creation: June 17, 2021) is incorporated herein by reference in its entirety.

BACKGROUND

[0003] While plasma-derived and recombinant clotting factor products allow hemophilia patients to live longer and healthier lives, hemophilia still remains one of the most costly and complex conditions to manage.

15 Due to its complexity, treatment of hemophilia A using FVIII replacement therapy requires a special therapeutic management process for doctors, pharmacies, and patients. Clinicians often assess lifestyle, psychosocial requirements, and the home environment when evaluating a patient's or guardian's ability to provide adequate care.

[0004] The current recommended standard of care involves the regular administration (routine prophylaxis)

20 of FVIII to minimize the number of bleeding episodes. Routine prophylaxis has been associated with improvements in long-term outcomes, but is a demanding regimen limited by the need for frequent intravenous (IV) administration. See Manco-Johnson et al., N Engl J Med. 357(6)·.535-44 (2007). Extended half- life FVIII products have reduced the frequency of FVIII administration for prophylaxis; however, currently- available FVIII products interact with endogenous von Willebrand factor (VWF) and have comparable circulating

25 half-lives, consistent with an upper limit on the half-life of rFVIII variants due to the half-life of endogenous

VWF. See, e.g., Pipe et al., Blood. 128(16):2007-16 (2016). BRIEF SUMMARY

[0005] Provided herein are, inter alia, methods of treating hemophilia A, as well as software-based pharmacokinetics systems and their use to provide dosing information (such as a dose and a dosing interval) for a subject in need of treatment for hemophilia A.

[0006] The present disclosure includes a method of treating haemophilia A in a subject in need thereof, comprising selecting a dose and dosing interval for the administration of rFVIIIFc-VWF-XTEN to the subject, followed by administration of the rFVIIIFc-VWF-XTEN according to the dose and dosing interval, wherein the body weight and hematocrit of the subject are used (e.g., assessed, assayed, or considered) when selectingthe dose and dosing interval, and wherein the level of Von Willebrand factor (VWF) in the blood or plasma of the subject is not considered when selecting the dose and dosing interval. For example, a subject with haemophilia A who has a VWF level of from 50 to 75 IU/dL may receive the same dose and dosing interval as a corresponding subject with haemophilia A (e.g., a subject of the same age and body weight, and who has the same hematocrit) with a VWF level of higher than 75 IU/dL (such as from 100 IU/dLto 200 IU/dL). In some embodiments, selecting the dose and dosing interval comprises calculating an estimated rFVIIIFc-VWF-XTEN clearance and central volume (also referred to herein as the "volume of the central compartment" and "central volume of distribution") for the subject, wherein body weight is used when estimating rFVIIIFc-VWF-XTEN clearance and hematocrit is used when estimating central volume. In some embodiments, selecting the dose and dosing interval comprises calculating an estimated rFVIIIFc-VWF-XTEN clearance and central volume for the subject, wherein body weight is used when estimating rFVIIIFc-VWF-XTEN clearance and hematocrit and body weight are used when estimating central volume. Included herein is rFVIIIFc-VWF-XTEN for use in a method for treating hemophilia A, comprising administering rFVIIIFc-VWF-XTEN to a subject according to a dose and a dosing interval, wherein the dose and dosing interval are selected by using the body weight and hematocrit of the subject, and wherein the level of Von Willebrand factor (VWF) in the plasma of the subject is not considered when selecting the dose and dosing interval.

[0007] In some aspects, the present disclosure comprises a method (e.g., a computer-implemented method) of estimating dosing information of rFVIIIFc-VWF-XTEN, individualized for a subject. In some embodiments, the method comprises (a) receiving subject information; and (b) calculating the dosing information of the rFVIIIFc- VWF-XTEN using a software-based system, wherein the system is programmed to implement a one- compartment rFVIIIFc-VWF-XTEN popPK model comprising body weight and/or hematocrit as covariates. In some embodiments, the subject information includes the subject's body weight and hematrocrit. In some embodiments, the method further comprises outputting, by the software-based system, the dosing information. In some embodiments, the system is programmed to implement a one-compartment rFVIIIFc- VWF-XTEN popPK model comprising body weight and/or hematocrit as covariates and a Bayesian estimation program to calculate the dosing information. In some embodiments, the one-compartment rFVIIIFc-VWF-XTEN popPK model comprises body weight as a paremeter. In some embodiments, the one-compartment rFVIIIFc- VWF-XTEN popPK model comprises hematocrit as a covariate. In some embodiments, the one-compartment rFVIIIFc-VWF-XTEN popPK model comprises body weight and hematocrit as covariates. In some embodiments, the one-compartment rFVIIIFc-VWF-XTEN popPK model does not include a level of VWF (e.g., in the blood or plasma) as a covariate. In some embodiments, the dosing information comprises estimated or predicted rFVIIIFc-VWF-XTEN PK information over time after administration of the rFVIIIFc-VWF-XTEN. In some embodiments, the dosing information comprises estimated or predicted FVIII activity levels over time after administration of the rFVIIIFc-VWF-XTEN. In some embodiments, the dosing information comprises a dosing regimen. In some embodiments, the dosing regimen is a prophylaxis regimen. In some embodiments, the dosing regimen is an on-demand regimen. In some embodiments, (a) further comprises receving desired treatment outcome information. In some embodiments, the desired treatment outcome information comprises a desired rFVIIIFc-VWF-XTEN level or FVIII activity level at a desired timepoint.

[0008] In some aspects, the present disclosure provides a method of estimating (e.g., a computer- implemented method) dosing information of rFVIIIFc-VWF-XTEN, individualized for a subject, the method comprising: (a) receiving subject information and/or desired treatment outcome information by a software- based system containing a rFVIIIFc-VWF-XTEN popPK model, (b) calculating, by the software-based system, individualized dosing information using the rFVIIIFc-VWF-XTEN popPK model and the received information, and (c) outputting, by the software-based system, the individualized dosing information. Further disclosed is the method as described herein, further comprising selecting a dosing regimen based on the output individualized dosing information of (c) and administering the rFVIIIFc-VWF-XTEN to the subject according to the selected dosing regimen. In some embodiments, the software-based system of the methods described herein also contains a Bayesian estimation program.

[0009] Some embodiments include a method of estimating (e.g., a computer-implemented method) a rFVIIIFc-VWF-XTEN dosing regimen based on median popPK, the method comprising: (a) receiving subject information and/or desired treatment outcome information by a software-based system containing a rFVIIIFc- VWF-XTEN popPK model, (b) calculating, by the software-based system, median PK information using the rFVIIIFc-VWF-XTEN popPK model and the received information, and (c) outputting, by the software-based system, the median PK information. Also disclosed is the method as described herein, further comprising selecting a dosing regimen based on the output median PK information of (c), and administering rFVIIIFc-VWF- XTEN to a subject according to the selected dosing regimen. In some embodiments, the software-based system of the methods described herein also contains a Bayesian estimation program.

[0010] Some embodiments include a method (e.g., a computer-implemented method) of estimating individual subject PK of rFVIIIFc-VWF-XTEN, the method comprising: (a) receiving individual rFVIIIFc-VWF-XTEN PK information by a software-based system containing a rFVIIIFc-VWF-XTEN popPK model, b) estimating, by the software-based system, individualized subject PK information using the rFVIIIFc-VWF-XTEN popPK model and the received information, and c) outputting, by the software-based system, the individualized subject PK information. Also disclosed is the method as described herein, further comprising selecting a dosing regimen based on the output individualized subject PK information of (c), and administering rFVIIIFc-VWF-XTEN to the subject according to the selected regimen. In some embodiments, the software-based system of the methods described herein also contains a Bayesian estimation program.

[0011] Also disclosed is a method (e.g., a computer-implemented method) of estimating rFVIIIFc-VWF-XTEN dosing information individualized for a subject, the method comprising: (a) receiving subject information and/or desired treatment outcome information by one or more electronic devices, (b) transmitting, by a processing device, the subject information and/or desired treatment outcome information to an application program, wherein the application is programmed to implement a rFVIIIFc-VWF-XTEN popPK model, (c) receiving from the application program, individualized dosing information calculated using the rFVIIIFc-VWF-XTEN popPK model and the transmitted information of (b), and (d) outputting, by the one or more electronic devices, the individualized dosing information.

[0012] Also disclosed herein is a method (e.g., a computer-implemented method) of estimating rFVIIIFc-VWF- XTEN dosing information individualized for a subject, the method comprising: (a) receiving, by a processing device, subject information and/or desired treatment outcome information by an application program programmed to implement a rFVIIIFc-VWF-XTEN popPK model, wherein the received information is transmitted by one or more electronic devices, (b) calculating, by the application program, individualized rFVIIIFc-VWF-XTEN dosing information using the rFVIIIFc-VWF-XTEN popPK model and the received information, and (c) transmitting, by a processing device, the individualized calculated dosing information of (b) to one or more electronic devices for output of the information. In some embodiments, the method as described herein further comprises selecting a dosing regimen based on the output individualized dosing information of (c) and administering the rFVIIIFc-VWF-XTEN to the subject according to the selected dosing regimen.

[0013] Also disclosed is a method (e.g., a computer-implemented method) of estimating a rFVIIIFc-VWF-XTEN dosing regimen based on median rFVIIIFc-VWF-XTEN popPK, the method comprising: (a) receiving subject information and/or desired treatment outcome information by one or more electronic devices, (b) transmitting, by a processing device, the subject information and/or desired treatment outcome information to an application program, wherein the application is programmed to implement a rFVIIIFc-VWF-XTEN popPK model, (c) receiving from the application program, median rFVIIIFc-VWF-XTEN PK dosing information calculated using the rFVIIIFc-VWF-XTEN popPK model and the received information, and (d) outputting, by the one or more electronic devices, the median PK information.

[0014] Also disclosed is a method of estimating a rFVIIIFc-VWF-XTEN dosing regimen based on median rFVIIIFc-VWF-XTEN popPK, the method comprising: (a) receiving, by a processing device, subject information and/or desired treatment outcome information by an application program programmed to implement a rFVIIIFc-VWF-XTEN population pharmacokinetic (popPK) model, wherein the received information is transmitted by one or more electronic devices, (b) calculating, by the application program, individualized rFVIIIFc-VWF-XTEN dosing information using the rFVIIIFc-VWF-XTEN popPK model and the received information, and (c) transmitting, by a processing device, the individualized calculated dosing information of (b) to one or more electronic devices for output of the information. Also disclosed is the method as described herein, further comprising selecting a dosing regimen based on the output median PK information of (c), and (d) administering rFVIIIFc-VWF-XTEN to a subject according to the selected dosing regimen. Some embodiments include the method as described herein, further comprising selecting a dosing regimen based on the output median PK information of (c), and (d) administering rFVIIIFc-VWF-XTEN to a subject according to the selected dosing regimen.

[0015] Also disclosed herein is a method (e.g., a computer-implemented method) of estimating individual subject PK of rFVIIIFc-VWF-XTEN, the method comprising: (a) receiving, by one or more electronic devices, individual rFVIIIFc-VWF-XTEN PK information, (b) transmitting, by a processing device, the individual rFVIIIFc- VWF-XTEN PK information to an application program, wherein the application is programmed to implement a rFVIIIFc-VWF-XTEN popPK model, (c) receiving from the application program, individualized subject rFVIIIFc- VWF-XTEN PK information using the rFVIIIFc-VWF-XTEN popPK model and the transmitted information of (b), and (d) outputting, by the one or more electronic devices, the individualized subject PK information.

[0016] Some embodiments include a method (e.g., a computer-implemented method) of estimating individual subject PK of rFVIIIFc-VWF-XTEN, the method comprising: (a) receiving individual rFVIIIFc-VWF-XTEN PK information by an application program which is programmed to implement a rFVIIIFc-VWF-XTEN popPK model, wherein the received information is transmitted by one or more electronic devices, (b) calculating, by the application program, individualized subject PK information of rFVIIIFc-VWF-XTEN using the rFVIIIFc-VWF- XTEN popPK model and the received information, and (c) transmitting, by a processing device, the estimated individualized subject rFVIIIFc-VWF-XTEN PK information of (b) to one or more one or more electronic devices, for output of the information. In certain embodiments, the method as described herein further comprises selecting a dosing regimen based on the output estimated subject PK information of (c), and administering rFVIIIFc-VWF-XTEN to the subject according to the selected regimen.

[0017] Also included is a method (e.g., a computer-implemented method) of estimating individual subject PK of rFVIIIFc-VWF-XTEN, the method comprising: (a) receiving, by one or more electronic devices, information regarding individual body weight and/or subject hematocrit and (i) desired raise of plasma factor activity level following the dose or (ii) desired dose or desired dose interval, (b) transmitting, by a processing device, the information of (a) to an application program, wherein the application is programmed to implement a rFVIIIFc- VWF-XTEN popPK model, (c) receiving from the web based server and program, individualized subject PK information of rFVIIIFc-VWF-XTEN calculated using the rFVIIIFc-VWF-XTEN popPK model and the transmitted information of (b), and (d) outputting, by the one or more electronic devices, the estimated subject PK information. In some embodiments, the subject information received in (a) includes subject hematocrit. In some embodiments, the rFVIIIFc-VWF-XTEN popPK model calculates or estimates the subject's central volume based on the subject's hematocrit. In some embodiments, the subject information received in (a) includes subject body weight. In some embodiments, the rFVIIIFc-VWF-XTEN popPK model calculates or estimates the subject's central volume based on the subject's hematocrit and body weight. In some embodiments, the rFVIIIFc-VWF-XTEN popPK model calculates or estimates the subject's clearance based on the subject's body weight.

[0018] In some embodiments, the rFVIIIFc-VWF-XTEN popPK model uses the subject's body weight and hematocrit of the subject to provide the estimated subject PK information. In some embodiments, the rFVIIIFc- VWF-XTEN popPK model uses the subject's body weight and hematocrit to provide the estimated dosing information. In some embodiments, the concentration of VWF in the blood or plasma of the subject is not used by the rFVIIIFc-VWF-XTEN popPK model. In some embodiments, the rFVIIIFc-VWF-XTEN popPK model calculates or estimates rFVIIIFc-VWF-XTEN clearance and central volume for the subject, wherein body weight is used to estimate rFVIIIFc-VWF-XTEN clearance and hematocrit is used to estimate central volume. In some embodiments, the rFVIIIFc-VWF-XTEN popPK model calculates or estimates rFVIIIFc-VWF-XTEN clearance and central volume for the subject, wherein body weight is used to estimate rFVIIIFc-VWF-XTEN clearance and hematocrit and body weight are used to estimate central volume.

[0019] In some embodiments, the rFVIIIFc-VWF-XTEN popPK model is a one-compartment rFVIIIFc-VWF-XTEN popPK model. In some embodiments, the rFVIIIFc-VWF-XTEN popPK model comprises body weight and hematocrit as covariates. In some embodiments the rFVIIIFc-VWF-XTEN popPK model does not comprise VWF level (e.g., in the blood or plasma of a subject) as a covariate.

[0020] In some embodiments, the rFVIIIFc-VWF-XTEN popPK model incorporates or calculates clearance (e.g., in dL/hour), central compartment volume (e.g., in d L), allometric exponent of body weight on clearance, allometric exponent of body weight on central volume, and/or exponent of hematocrit on central volume.

[0021] In some embodiments, the rFVIIIFc-VWF-XTEN popPK model is the rFVIIIFc-VWF-XTEN popPK model [A]

[0022] In some aspects, the present disclosure provides a method (e.g., a computer-implemented method) of estimating dosing information of rFVIIIFc-VWF-XTEN, individualized for a subject, the method comprising: (a) receiving subject information and/or desired treatment outcome information by a software-based system containing a rFVIIIFc-VWF-XTEN popPK model [A], (b) calculating, by the software-based system, individualized dosing information using the rFVIIIFc-VWF-XTEN popPK model [A] and the received information, and c) outputting, by the software-based system, the individualized dosing information. Further disclosed is the method as described herein, further comprising selecting a dosing regimen based on the output individualized dosing information of (c) and administering the rFVIIIFc-VWF-XTEN to the subject according to the selected dosing regimen. In some embodiments, the software-based system of the methods described herein also contains a Bayesian estimation program.

[0023] Some embodiments include a method (e.g., a computer-implemented method) of estimating a rFVIIIFc-VWF-XTEN dosing regimen based on median popPK, the method comprising: (a) receiving subject information and/or desired treatment outcome information by a software-based system containing the rFVIIIFc-VWF-XTEN popPK model [A], (b) calculating, by the software-based system, median PK information using the rFVIIIFc-VWF-XTEN popPK model [A] and the received information, and (c) outputting, by the software-based system, the median PK information. Also disclosed is the method as described herein, further comprising selecting a dosing regimen based on the output median PK information of (c), and administering rFVIIIFc-VWF-XTEN to a subject according to the selected dosing regimen. In some embodiments, the software- based system of the methods described herein also contains a Bayesian estimation program.

[0024] Some embodiments include a method (e.g., a computer-implemented method) of estimating individual subject PK of rFVIIIFc-VWF-XTEN, the method comprising: (a) receiving individual rFVIIIFc-VWF-XTEN PK information by a software-based system containing the rFVIIIFc-VWF-XTEN popPK model [A], b) estimating, bythe software-based system, individualized subject PK information using the rFVIIIFc-VWF-XTEN popPK model [A] and the received information, and c) outputting, by the software-based system, the individualized subject PK information. Also disclosed is the method as described herein, further comprising selecting a dosing regimen based on the output individualized subject PK information of (c), and administering rFVIIIFc-VWF-XTEN to the subject according to the selected regimen. In some embodiments, the software-based system of the methods described herein also contains a Bayesian estimation program.

[0025] Also disclosed is a method (e.g., a computer-implemented method) of estimating rFVIIIFc-VWF-XTEN dosing information individualized for a subject, the method comprising: (a) receiving subject information and/or desired treatment outcome information by one or more electronic devices, (b) transmitting, by a processing device, the subject information and/or desired treatment outcome information to an application program, wherein the application is programmed to implement the rFVIIIFc-VWF-XTEN popPK model [A], (c) receiving from the application program, individualized dosing information calculated using the rFVIIIFc-VWF- XTEN popPK model [A] and the transmitted information of (b), and (d) outputting, bythe one or more electronic devices, the individualized dosing information.

[0026] Also disclosed herein is a method (e.g., a computer-implemented method) of estimating rFVIIIFc-VWF- XTEN dosing information individualized for a subject, the method comprising: (a) receiving, by a processing device, subject information and/or desired treatment outcome information by an application program programmed to implement the rFVIIIFc-VWF-XTEN popPK model [A], wherein the received information is transmitted by one or more electronic devices, (b) calculating, by the application program, individualized rFVIIIFc-VWF-XTEN dosing information using the rFVIIIFc-VWF-XTEN popPK model [A] and the received information, and (c) transmitting, by a processing device, the individualized calculated dosing information of (b) to one or more electronic devices for output of the information. In some embodiments, the method as described herein further comprises selecting a dosing regimen based on the output individualized dosing information of (c). In other embodiments, the method as described herein further comprises selecting a dosing regimen based on the output individualized dosing information of (c) and administering the rFVIIIFc-VWF-XTEN to the subject according to the selected dosing regimen.

[0027] Also disclosed is a method (e.g., a computer-implemented method) of estimating a rFVIIIFc-VWF-XTEN dosing regimen based on median rFVIIIFc-VWF-XTEN popPK, the method comprising: (a) receiving subject information and/or desired treatment outcome information by one or more electronic devices, (b) transmitting, by a processing device, the subject information and/or desired treatment outcome information to an application program, wherein the application is programmed to implement the rFVIIIFc-VWF-XTEN popPK model [A], (c) receiving from the application program, median rFVIIIFc-VWF-XTEN PK dosing information calculated using the rFVIIIFc-VWF-XTEN popPK model [A] and the received information, and (d) outputting, by the one or more electronic devices, the median PK information.

[0028] Also disclosed is a method (e.g., a computer-implemented method) of estimating a rFVIIIFc-VWF-XTEN dosing regimen based on median rFVIIIFc-VWF-XTEN popPK, the method comprising: (a) receiving, by a processing device, subject information and/or desired treatment outcome information by an application program programmed to implement a rFVIIIFc-VWF-XTEN population pharmacokinetic (popPK) model, wherein the received information is transmitted by one or more electronic devices, (b) calculating, by the application program, individualized rFVIIIFc-VWF-XTEN dosing information using the rFVIIIFc-VWF-XTEN popPK model [A] and the received information, and (c) transmitting, by a processing device, the individualized calculated dosing information of (b) to one or more electronic devices for output of the information. Also disclosed is the method as described herein, further comprising selecting a dosing regimen based on the output median PK information of (c). Some embodiments include the method as described herein, further comprising selecting a dosing regimen based on the output median PK information of (c), and (d) administering rFVIIIFc-VWF-XTEN to a subject according to the selected dosing regimen.

[0029] Also disclosed herein is a method (e.g., a computer-implemented method) of estimating individual subject PK of rFVIIIFc-VWF-XTEN, the method comprising: (a) receiving, by one or more electronic devices, individual rFVIIIFc-VWF-XTEN PK information, (b) transmitting, by a processing device, the individual rFVIIIFc- VWF-XTEN PK information to an application program, wherein the application is programmed to implement the rFVIIIFc-VWF-XTEN popPK model [A], (c) receiving from the application program, individualized subject rFVIIIFc-VWF-XTEN PK information using the rFVIIIFc-VWF-XTEN popPK model [A] and the transmitted information of (b), and (d) outputting, by the one or more electronic devices, the individualized subject PK information. [0030] Some embodiments include a method of estimating individual subject PK of rFVIIIFc-VWF-XTEN, the method comprising: (a) receiving individual rFVIIIFc-VWF-XTEN PK information by an application program which is programmed to implement the rFVIIIFc-VWF-XTEN popPK model [A], wherein the received information is transmitted by one or more electronic devices, (b) calculating, by the application program, individualized subject PK information of rFVIIIFc-VWF-XTEN using the rFVIIIFc-VWF-XTEN popPK model [A] and the received information, and (c) transmitting, by a processing device, the estimated individualized subject rFVIIIFc-VWF- XTEN PK information of (b) to one or more one or more electronic devices, for output of the information. In certain embodiments, the method as described herein further comprises selecting a dosing regimen based on the output estimated subject PK information of (c), and administering rFVIIIFc-VWF-XTEN to the subject according to the selected regimen.

[0031] Also included is a method (e.g., a computer-implemented method) of estimating individual subject PK of rFVIIIFc-VWF-XTEN, the method comprising: (a) receiving, by one or more electronic devices, information regarding individual body weight and/or subject hematocrit and (i) desired raise of plasma factor activity level following the dose or (ii) desired dose or desired dose interval, (b) transmitting, by a processing device, the information of (a) to an application program, wherein the application is programmed to implement the rFVIIIFc- VWF-XTEN popPK model [A], (c) receiving from the web based server and program, individualized subject PK information of rFVIIIFc-VWF-XTEN calculated using the rFVIIIFc-VWF-XTEN popPK model [A] and the transmitted information of (b), and (d) outputting, by the one or more electronic devices, the estimated subject PK information. In some embodiments, the subject information received in (a) includes subject hematocrit and/or body weight. In some embodiments, the rFVIIIFc-VWF-XTEN popPK model calculates or estimates the subject's central volume based on the subject's hematocrit. In some embodiments, the rFVIIIFc-VWF-XTEN popPK model calculates or estimates the subject's central volume based on the subject's haematocrit and body weight. In some embodiments, the rFVIIIFc-VWF-XTEN popPK model calculates or estimates the subject's clearance based on body weight.

[0032] Also disclosed is the method as described herein, wherein the individualized subject PK of rFVIIIFc- VWF-XTEN includes a PK curve or a PK parameter selected from incremental recovery (Cmax/Dose), mean residence time, terminal ti/ , clearance, Vss and AUC/Dose.

[0033] In some embodiments, the application program of the methods described herein is also programmed to implement a Bayesian estimation program. In some embodiments, the Bayesian estimation program is used by the application program along with the rFVIIIFc-VWF-XTEN popPK model (e.g., rFVIIIFc-VWF-XTEN popPK model [A]) and the received information in the calculating step. In some embodiments, the application program is accessible through a web server or network server.

[0034] Some embodiments include a computer readable storage medium having instructions stored thereon that, when executed by a processor, causes the processor to perform the methods as described herein. [0035] Also disclosed is a system comprising a processor and memory, the memory having instructions stored thereon that, when executed by the processor, cause the processor to perform the methods as described herein.

[0036] In some embodiments of the methods described herein, the desired treatment outcome information is desired rise in plasma FVIII level following dosing, and the output information is dose for acute treatment.

[0037] In some embodiments of the methods described herein, the desired treatment outcome information is desired dosing interval, and the output information is dose for prophylaxis.

[0038] In some embodiments of the methods described herein, the desired treatment outcome information is desired dose, and the output information is dosing interval for prophylaxis.

[0039] In some embodiments, the methods further comprise receiving, by the software-based system, additional subject information. In some embodiments, the subject information is body weight and/or subject hematocrit.

[0040] In some embodiments of the methods, step (a) further comprises receiving information by the electronic device information relating to diagnostic (baseline) factor level, dosing history, actual dose, actual time of PK sampling or factor activity level, and (b) further comprises transmitting, by a processing device, the information to the application program.

[0041] In some embodiments of the methods disclosed herein, the electronic device is a digital pen, a smart phone, a tablet computer, a personal digital assistant, a handheld computer, a laptop computer, a scanner, a camera, and/or a fax machine.

[0042] In some embodiments, the software-based system is a network-based system. In some embodiments, the software-based system is a web-based system.

[0043] In some embodiments, dosing information includes a recommended dose (e.g., in lU/kg) and dosing interval. In some embodiments, the software-based system provides a graph, table, or other depiction of simulated or estimated FVIII activity levels in the subject over time based on the dosing information.

[0044] In some embodiments, the dosing information includes an estimated minimum FVIII activity level that will be maintained over the entire dosing interval. In some embodiments, the rFVIIIFc-VWF-XTEN is administered by self-administration.

[0045] In some embodiments, the rFVIIIFc-VWF-XTEN is administered by a healthcare provider such as a nurse or a physician. In some embodiments, the rFVIIIFc-VWF-XTEN is administered by a caregiver, such as a family member (e.g., a parent).

[0046] Also provided herein is a combination comprising a kit or tool for estimating hematocrit and software that is or is part of a software-based system that uses a rFVIIIFc-VWF-XTEN model (e.g., rFVIIIFc-VWF-XTEN model [A]) to provide dosing or PK information in accordance with one or more of the methods disclosed herein.

[0047] Also disclosed herein are computer-implemented embodiments of each of the methods disclosed herein.

[0048] Also disclosed herein are methods for treating hemophilia A in a subject in need thereof, comprising administering to the subject a dose of rFVIIIFc-VWF-XTEN that is selected according to any of the disclosed methods. Also disclosed herein are methods for treating hemophilia A in a subject in need thereof, comprising administering to the subject a dose regimen of rFVIIIFc-VWF-XTEN that is selected according to any of the disclosed methods. Also disclosed are uses of rFVIIIFc-VWF-XTEN for the treatment of hemophilia A comprising administering to the subject a dose or dose regimen of rFVIIIFc-VWF-XTEN that is selected according to any of the disclosed methods.

[0049] Also disclosed herein are uses of rFVIIIFc-VWF-XTEN accordingto any of the methods disclosed herein.

[0050] Also disclosed is a combination comprising a kit or tool for estimating hematrocrit levels and software that is or is part of a software-based system that uses a rFVIIIFc-VWF-XTEN popPK model to provide dosing or PK information according to any of the methods disclosed herein. In some embodiments, the rFVIIIFc-VWF- XTEN popPK model is the rFVIIIFc-VWF-XTEN popPK model [A]

[0051] Also disclosed is a data processing apparatus, device, or system comprising a processor configured to provide dosing or PK information according to any of the methods disclosed herein. In some embodiments the data processing apparatus, device, or system comprises a means for carrying out the methods disclosed herein.

[0052] Also disclosed herein is a data processing apparatus, device, or system comprising a processor configured to implement a one-compartment rFVIIIFc-VWF-XTEN popPK model comprising a hemophilia A subject's body weight and hematocrit, but not the level of VWF in the plasma of the subject, as covariates. In some embodiments, the rFVIIIFc-VWF-XTEN popPK model is the rFVIIIFc-VWF-XTEN popPK model [A].

[0053] Also disclosed herein is a computer program comprising instructions which, when the program is executed by a computer, cause the computer to carry out any of the methods disclosed herein. In some embodiments, the computer program implements a one-compartment rFVIIIFc-VWF-XTEN popPK model comprising a hemophilia A subject's body weight and hematocrit, but not the level of VWF in the plasma of the subject, as covariates. In some embodiments, the rFVIIIFc-VWF-XTEN popPK model is the rFVIIIFc-VWF-XTEN popPK model [A].

[0054] Also disclosed herein is a computer-readable medium comprising instructions which, when executed by a computer, cause the computer to provide carry out any of the methods disclosed herein. In some embodiments, the computer-readable medium comprises instructions which, when executed by a computer, cause the computer to implement a one-compartment rFVIIIFc-VWF-XTEN popPK model comprising a hemophilia A subject's body weight and hematocrit, but not the level of VWF in the plasma of the subject, as covariates. In some embodiments, the rFVIIIFc-VWF-XTEN popPK model is the rFVIIIFc-VWF-XTEN popPK model [A]

[0055] Also disclosed herein are the methods, combinations, data processing apparatus, devices, systems, computer programs, or computer-readable mediums disclosed herein, wherein the rFVIIIFc-VWF-XTEN comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 3 covalently bound to a second polypeptide comprising the amino acid sequence of SEQ ID NO: 6, wherein the first polypeptide and the second polypeptide are covalently bound to each other via disulfide bonds. In some embodiments, the rFVIIIFc-VWF- XTEN is BIVV001 or a biosimilar thereof.

[0056] Also disclosed herein are the methods, combinations, data processing apparatus, devices, systems, computer programs, or computer-readable mediums disclosed herein, wherein the rFVIIIFc-VWF-XTEN comprises a first polypeptide whose amino acid sequence is identical to SEQ ID NO: 3 covalently bound to a second polypeptide whose amino acid sequence is identical to SEQ ID NO: 6, wherein the first polypeptide and the second polypeptide are covalently bound to each other via disulfide bonds. In some embodiments, the rFVIIIFc-VWF-XTEN is BIVV001 or a biosimilar thereof.

BRIEF DESCRIPTION OF DRAWINGS/FIGURES

[0057] Figures 1A and IB are schematic representations of study designs of the single-dose clinical trials for rFVIIIFc-VWF-XTEN. Fig. 1A is a schematic representation of the clinical trial study design for a single, 25 lU/kg dose of rFVIIIFc-VWF-XTEN. Fig. IB is a schematic representation of the clinical trial study design for a single, 65 lU/kg dose of rFVIIIFc-VWF-XTEN.

[0058] Figure 2 is a schematic representation of the study design of the repeat-dose clinical trials for rFVIIIFc- VWF-XTEN.

[0059] Figures 3A and 3B are graphical representations of FVIII activity over time following the first dose for the single-dose (Fig. 3A) clinical trial study or the repeat-dose (Fig. 3B) clinical trial study as measured by activated Partial Thromboplastin Time (aPTT) assay.

[0060] Figures 4A-D are graphical representations of model predicted results versus observed clinical trial results for Individual Patient results (Fig. 4A-B) and population results (Fig. 4C-D) on both a linear scale (Fig. 4A; Fig. 4C) and a log scale (Fig. 4B; Fig. 4D), with goodness of fit diagnostics applied. Dots represent observed data. Straight line is the loess smooth curve of FVIII activity observed and predicted by the model.

[0061] Figures 5A and 5B provide a visual comparison of simulated FVIII activity (Fig. 5A) to observed phase 1 repeat-dose clinical trial FVIII activity (Fig. 5B). Florizontal lines indicate 5, 10, 15, 100, and 150 IU/dL; solid colored lines indicate the mean activity; shaded regions indicate the 90% prediction interval for week 4 only. [0062] Figures 6A and 6B provide a visual representation of predicted steady-state FVIII activity peaks and troughs using the model. Results of these predictions are provided as mean (shaded area = 90% prediction interval).

[0063] Figure 7 is a visual representation of the body weight distribution used for the clinical trial simulations, using the weighted average of observed body weight distribution from rFVIIIFc-VWF-XTEN clinical trials.

[0064] Figures 8A and 8B are graphical representations of Cmax (Fig. 8A) and Ctrough (Fig. 8B) for virtual subject responses in clinical trial simulations at a simulated dose of 50 lU/kg or 65 lU/kg. [A] Median (5th— 95th percentile) Cmax FVIII activity was 159 (112-223) IU/dL for 50 lU/kg dose and 204 (146-297) for 65 lU/kg [B] Median (5th— 95th percentile). Ctrough FVIII activity was 9.59 (3.96-19.3) for 50 lU/kg and 12.6 (5.83-26.1) for 65 lU/kg

[0065] Figure 9A is a cartoon of a rFVIIIFc-VWF-XTEN for illustrative purposes. Figure 9B is an illustrative cartoon showing the domain structure and post-translational modifications of a rFVIIIFc-VWF-XTEN that is also known as BIVV001. In some embodiments, the rFVIIIFc-VWF-XTEN is generated by coexpressing two polypeptide chains in HEK293 cells, a human cell line. In some embodiments, a D1D2 (propeptide) is removed during intracellular processing. In the cartoon, the two chains are held together by disulfide bonds in the Fc region. Additional details regarding BIVV001 may be found in Chhabra et al. (2020) Blood, 135 (17): 1484-1496, the entire content of which is incorporated herein by reference.

[0066] Figure 10 is a visual representation of a software-based system that can be used in methods disclosed herein.

[0067] Figure 11 is a visual representation of an exemplary network-based system that can be used according to the methods disclosed herein. The exemplary network-based system can be used for obtaining an estimated subject individualized dosing information, subject individualized PK information, and subject median PK information.

[0068] Figure 12 shows a schematic diagram of an example computing system 500.

DETAILED DESCRIPTION

[0069] With the emergence of extended half-life replacement products, treatment goals are now expanding beyond targeting low annualized bleed rate (ABR) to include long-term outcomes associated with higher plasma FVIII activity levels, such as long-term joint protection. Achieving these goals requires sustained, high plasma FVIII levels over long periods of time. rFVIIIFc-VWF-XTEN (e.g., the rFVIIIFc-VWF-XTEN also known as BIVV001 and efanesoctocog alfa) represents a new class of FVIII replacement, circulates independently of endogenous von Willebrand factor (VWF), and provides high sustained FVIII activity (see, e.g., Chhabra, et al. Blood. 2020;135(17):1484-1496, the entire content of which is incorporated herein by reference for all purposes). However, convenient and accurate methods are needed for determining individual subject dosing information for FVIII replacement therapy to help subjects achieve treatment goals.

[0070] The present disclosure provides, inter alia, methods of treatment and a software-based system for estimating individual subject PK of a rFVIIIFc-VWF-XTEN for treatment of hemophilia A or a method of estimating individual subject PK of a rFVIIIFc-VWF-XTEN using the software-based system. The software-based system applies a novel rFVIIIFc-VWF-XTEN population PK model [A] for estimating dose information for subjects receiving rFVIIIFc-VWF-XTEN as FVIII replacement treatment.

Definitions

[0071] 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" is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower).

[0072] The term "polypeptide," "peptide" and "protein" are used interchangeably and refer to a polymeric compound comprised of covalently linked amino acid residues.

[0073] The term "polynucleotide" and "nucleic acid" are used interchangeably and refer to a polymeric compound comprised of covalently linked nucleotide residues.

[0074] The term "administering," as used herein, means to or prescribe or give a therapeutic agent (i.e. rFVIIIFc-VWF-XTEN) to a subject via an intravenous route, e.g., intravenous injection and intravenous infusion, e.g., via central venous access. The therapeutic agent can be administered as part of a pharmaceutical composition comprising at least one excipient.

[0075] The term "dosing interval," as used herein, means the amount of time that elapses between multiple doses being administered to a subject. Dosing interval can thus be indicated as ranges. A more detailed discussion is presented below regarding the disclosed methods and subject dosing interval.

[0076] The term "dosing frequency" as used herein refers to the frequency of administering doses of rFVIIIFc- VWF-XTEN in a given time. Dosing frequency can be indicated as the number of doses per a given time, e.g., once a week or once in two weeks. A more detailed discussion is presented herein regarding the disclosed methods and subject dosing frequency.

[0077] The term "bleeding episode" as used herein is given a standardized definition: A bleeding episode starts from the first sign of a bleed, and ends 72 hours after the last treatment for the bleeding, within which any symptoms of bleeding at the same location, or injections less than or equal to 72 hours apart, is considered the same bleeding episode. See Blanchette V. (2006) Haemophilia 12:124-7. As used herein, any injection to treat the bleeding episode, taken more than 72 hours after the preceding one, is considered the first injection to treat a new bleeding episode at the same location. Likewise, any bleeding at a different location is considered a separate bleeding episode regardless of time from the last injection.

[0078] The term "prophylaxis of one or more bleeding episode" or "prophylactic treatment" as used herein means administering rFVIIIFc-VWF-XTEN in multiple doses to a subject over a course of time to increase the level of FVIII activity in a subject's plasma. In some embodiments, "prophylaxis of one or more bleeding episode" indicates use of rFVIIIFc-VWF-XTEN to prevent or inhibit occurrence of one or more spontaneous or uncontrollable bleeding or bleeding episodes or to reduce the frequency of one or more spontaneous or uncontrollable bleeding or bleeding episodes. In some embodiments, the increased FVIII activity level is sufficient to decrease the incidence of spontaneous bleeding or to prevent bleeding in the event of an unforeseen injury. Prophylactic treatment decreases or prevents bleeding episodes. Prophylactic treatment can be individualized, for example, to compensate for inter-subject variability with a FVIII replacement therapy.

[0079] The term "on-demand treatment," as used herein, means treatment that is intended to take place over a short course of time and is in response to an existing condition, such as a bleeding episode, or a perceived short term need such as planned surgery. The term "on-demand treatment" is used interchangeably with "episodic" treatment. In some embodiments, the on-demand regimen is for perioperative management of bleeding.

[0080] The methods provided herein can be applied to a subject in need of prophylactic treatment or episodic/on-demand treatment. In some embodiments, the subject in need of prophylactic treatment or episodic/on-demand treatment suffers from hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas sheath. In some embodiments, the subject is in need of treatment for surgery, including, e.g., surgical prophylaxis or peri-operative management. In some embodiments, the surgery is minor surgery or major surgery. Exemplary surgical procedures include tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, joint replacement surgery (e.g., total knee replacement, hip replacement, and the like), heart surgery, and caesarean section.

[0081] The term "treat", "treatment", or "treating", as used herein refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition, or the prophylaxis of one or more symptoms associated with a disease or condition. In some embodiments, "treatment of" or "treating" hemophilia A includes prevention of one or more symptoms of hemophilia A (such as spontaneous bleeding). In some embodiments, "treatment of" or "treating" hemophilia A includes reducing the likelihood of a bleeding episode or reducing the severity of a bleeding episode. In some embodiments, treatment is prophylactic treatment. In some embodiments, treatment is on-demand treatment. In some embodiments, the term "treatment" or "treating" means FVIII replacement therapy with a rFVIIIFc-VWF-XTEN. In some embodiments, "treatment" or "treating" means reduction of the frequency of one or more symptoms of hemophilia A, e.g., spontaneous or uncontrollable bleeding episodes.

[0082] The term "perioperative management" as used herein means use of rFVIIIFc-VWF-XTEN before, concurrently with, or after an operative procedure, e.g., a surgical operation. The use for "perioperative management" of one or more bleeding episode includes surgical prophylaxis before (i.e., preoperative), during (i.e., intraoperative), or after (i.e., postoperative) a surgery to prevent one or more bleeding or bleeding episode or reducing or inhibiting spontaneous and/or uncontrollable bleeding episodes before, during, and after a surgery.

[0083] Pharmacokinetic (PK) parameters include the terms above and the following terms, which have their ordinary meaning in the art, unless otherwise indicated. Some of the terms are explained in more detail in the Examples. PK parameters can be based on FVIII antigen level (often denoted parenthetically herein as "antigen") or FVIII activity level (often denoted parenthetically herein as "activity"). In the literature, PK parameters are often based on FVIII activity level due to the presence in the plasma of some subjects of endogenous, inactive FVIII, which interferes with the ability to measure administered (i.e., exogenous) FVIII using antibody against FVIII, respectively. Flowever, when FVIII is administered as part of a chimeric protein or hybrid protein containing a heterologous polypeptide such as an FcRn BP, administered (i.e., exogenous) FVIII antigen can be accurately measured using antibody to the heterologous polypeptide. In addition, certain PK parameters can be based on model predicted data (often denoted parenthetically herein as "model predicted") or on observed data (often denoted parenthetically herein as "observed").

[0084] "Baseline," as used herein, is the lowest measured plasma FVIII level in a subject prior to administering a dose. The FVIII plasma levels can be measured at two time points prior to dosing: at a screening visit and immediately prior to dosing. Alternatively, (a) the baseline in subjects whose pretreatment FVIII activity is <1%, who have no detectable FVIII antigen, and have nonsense genotypes can be defined as 0%, (b) the baseline for subjects with pretreatment FVIII activity <1% and who have detectable FVIII antigen can be set at 0.5%, (c) the baseline for subjects whose pretreatment FVIII activity is between 1 - 2% is Cmin (the lowest activity throughout the PK study), and (d) the baseline for subjects whose pretreatment FVIII activity is >2% can be set at 2%. Activity above the baseline pre-dosing can be considered residue drug from prior treatment, and can be decayed to baseline and subtracted from the PK data following rFVIIIFc-VWF-XTEN dosing.

[0085] " T _1/2 β," or "T beta" or "Beta FIL," as used herein, is half-life associated with elimination phase, T _1/2 β=(ln2)/el imination rate constant associated with the terminal phase. The T _1/2 beta can be measured by FVIII activity or by FVIII antigen level in plasma. The T _1/2 beta based on activity is shown as T _1/2 beta (activity), and the T _1/2 beta based on the FVIII antigen level can be shown as T _1/2 beta (antigen). Both T _1/2 beta (activity) and T _1/2 beta (antigen) can be shown as ranges or a geometric mean.

[0086] As used herein, "trough" or "trough level" is the lowest plasma FVIII activity level reached after administering a dose of FVIII molecule, e.g. rFVIIIFc-VWF-XTEN, and before the next dose is administered, if any. Depending on context, trough may be used interchangeably herein with "threshold." Baseline FVIII levels are subtracted from measured FVIII levels to calculate the trough level.

[0087] The term "annualized bleeding rate" ("ABR") as used herein refers to the number of bleeding episodes (including spontaneous and traumatic bleeds) experienced by a subject during a defined time period, extrapolated to 1 year. For example, two bleeds in six months would indicate an ABR of four. The median ABR provides a single number to describe all subjects, indicating that half of the subjects had individual ABRs less than or equal to the median and half had ABRs greater than or equal to the median.

[0088] The term "patient" and "subject" are used interchangeably herein and refers to a human. Subject as used herein includes an individual who has been diagnosed with hemophilia A, who are susceptible to uncontrolled bleeding episodes, e.g., hemophilia, or any combinations thereof. Subjects can also include an individual who is in danger of one or more uncontrollable bleeding episodes prior to a certain activity, e.g., a surgery, a sport activity, or any strenuous activities. In some embodiments, the subject can have a baseline FVIII activity less than 1%, less than 0.5%, less than 2%, less than 2.5%, less than 3%, or less than 4%. In some embodiments, the subject has severe hemophilia A, defined as <1 IU/dL (<1%) endogenous FVIII activity. In some embodiments, the subject has no other coagulation disorder in addition to hemophilia A.

[0089] As used herein "Extended Length Polypeptide" or "Extended Length Polypeptide sequence" refers to certain polypeptides with non-naturally occurring, substantially non-repetitive sequences 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. Extended Length Polypeptides include XTEN ^® sequences (Amunix Pharmaceuticals, Inc.). As a chimeric polypeptide partner, Extended Length Polypeptides can serve as a carrier, conferring certain desirable pharmacokinetic, physicochemical and pharmaceutical properties when linked to a VWF protein or a FVIII sequence of the disclosure to create a chimeric polypeptide. Such desirable properties include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics. As used herein, "Extended Length Polypeptide" specifically excludes antibodies or antibody fragments such as single chain antibodies or Fc fragments of a light chain or a heavy chain.

[0090] As used herein "software-based system" refers to an algorithm or set of algorithms capable of being implemented by a processing device. The software-based System may be embodied in software which includes but is not limited to firmware, resident software, microcode, etc. and may take the form of a computer program product accessible from a computer-usable or computer-readable medium providing program code for use by or in connection with a computer or any instruction execution system. For the purposes of this description, a computer-usable or computer readable medium can be any apparatus that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device. The medium can be an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system (or apparatus or device) or a propagation medium. Examples of a computer-readable medium include a semiconductor or solid state memory, magnetic tape, a removable computer diskette, a random access memory (RAM), a read-only memory (ROM), a rigid magnetic disk, and an optical disk, including compact disc-read only memory (CD-ROM), compact disc-read/write (CD-R/W) and DVD. Non-limiting examples of software-based systems include network-based systems and web-based systems.

[0091] As used herein "Bayesian estimation program" refers to programming which implements the Bayesian general inference framework. Generally, in the estimation process, the Bayesian method employs both the evidence contained in the observation signal and the accumulated prior probability of the process. In certain embodiments, the Bayesian estimation program implements a Bayesian estimator. Bayesian statistics, models, and estimators are well known in the art. See, e.g., Jaynes, E.T. (1985). Bayesian Methods: General Background. Maximum-Entropy and Bayesian Methods in Applied Statistics. Cambridge University Press; pp. 1-25; Bessiere, P. et al. (2013). Bayesian Programming. CRC Press. ISBN 978-1-4398-8032-6.

[0092] As used herein the term "processing device" refers to a data processing system suitable for storing and/or executing program code to implement the software-based system and may include at least one processor coupled directly or indirectly to memory elements through a system bus. The processor(s), the electronic circuitry that executes instructions that make up the program code, may be instantiated by a microprocessor, microcontroller, multi-core processor, array of processors, or vector processors. The memory elements can include local memory employed during actual execution of the program code, bulk storage, and cache memories which provide temporary storage of at least some program code in order to reduce the number of times code must be retrieved from bulk storage during execution. Input/output or I/O devices (including but not limited to keyboards, displays, pointing devices, etc.) can be coupled to the system either directly or through intervening I/O controllers. Network adapters may also be coupled to the system to enable the processing device to become coupled to other processing devices or remote printers or storage devices through intervening private or public networks. Modems, cable modems and Ethernet cards are just a few of the currently available types of network adapters. The processing device may also be a shared data processing system such as a network-based (e.g., web-based) server system, accessible via a network such as the Internet, that is capable of accessing and executing program code to implement the software-based system.

Description of rFVIIIFc-VWF-XTEN

[0093] Recombinant coagulation FVIII Fc- von Willebrand factor-XTEN fusion protein ("rFVIIIFc-VWF-XTEN"), is a long-acting, fully recombinant fusion protein consisting of single chain B-domain deleted (BDD) human FVIII (FVIII), the Fc domain of human immunoglobulin G1 (IgGl), the FVIII-binding D'D3 domain of human von Willebrand factor (VWF), and 2 Extended Length Polypeptide sequences. In some embodiments, the rFVIIIFc- VWF-XTEN comprises a first polypeptide and a second polypeptide that are covalently bound to each other via disulfide bonds. In some embodiments, the rFVIIIFc-VWF-XTEN comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 3 covalently bound to a second polypeptide comprising the amino acid sequence of SEQ ID NO: 6, wherein the first polypeptide and the second polypeptide are covalently bound to each other via disulfide bonds. Such rFVIIIFc-VWF-XTEN is also known as efanesoctocog alfa and BIVVOOl. Schematic representations of rFVIIIFc-VWF-XTEN are presented in Figure 9A and 9B. In some embodiments, rFVIIIFc-VWF-XTEN is produced by recombinant DNA technology in a human embryonic kidney (HEK) cell line, HEK293F. In some embodiments, the cell line expresses an rFVIIIFc-XTEN polypeptide (SEQ ID NO: 1), an rVWFFc-XTEN polypeptide (SEQ ID NO: 4), and a soluble PACE enzyme. Non-limiting examples of nucleotide sequences encoding the rFVIIIFc-XTEN polypeptide (SEQ ID NO: 2) and the rVWFFc-XTEN polypeptide (SEQ ID NO: 5) can be found in Table 5, below. Amino acid sequences for the rFVIIIFc-XTEN polypeptide without a signal peptide (SEQ ID NO: 3) and rVWFFc-XTEN polypeptide without a signal peptide or D1D2 portion of VWF (SEQ ID NO: 6) can be found in Table 5, below. Additional details regarding the rFVIIIFc-VWF-XTEN that is also known as BIVVOOl can be found in Chhabra, et al. Blood. 2020;135(17):1484-1496, the entire content of which is incorporated herein by reference.

[0094] As with all FVIII replacement treatments, the clinical response to rFVIIIFc-VWF-XTEN can vary from subject to subject. Examples 1 and 2 summarize two Phase I clinical studies which tested the safety, tolerability, and PK of rFVIIIFc-VWF-XTEN in human subjects with hemophilia A. As such, specific dose recommendations, including initial recommended dose and dosing intervals, based only on data from subject groups can be inaccurate. In some embodiments, the methods disclosed herein provide individualized dose information and thus avoid the disadvantages and inaccuracies in making dosing decisions.

[0095] In some embodiments for subjects receiving prophylactic treatment with rFVIIIFc-VWF-XTEN, if a subject's plasma FVIII level fails to increase as expected and/or fails to achieve treatment goals after rFVIIIFc- VWF-XTEN administration at an initial recommended dose and dose interval, the methods disclosed herein can be used to determine individual subject dose information (i.e., plasma FVIII levels). Based on this individual subject dose information, the dose and/or dose interval of rFVIIIFc-VWF-XTEN can be adjusted to achieve treatment goals, such as a minimum plasma FVIII level (e.g., trough).

[0096] In some embodiments, for subjects receiving on-demand treatment with rFVIIIFc-VWF-XTEN, if the subject's bleeding is not controlled after rFVIIIFc-VWF-XTEN administration at an initial recommended dose and dose interval, the methods disclosed herein can be used to determine individual subject dose information. Based on this individual subject dose information, the dose and/or dose interval of rFVIIIFc-VWF-XTEN can be adjusted to achieve satisfactory bleed control. [0097] In some embodiments, the methods disclosed herein can be used to estimate a minimum trough level for a subject.

[0098] Subject's plasma can be monitored for FVIII activity levels, e.g., the one-stage clotting assay, to confirm adequate FVIII levels have been achieved and maintained, when clinically indicated. FVIII activity can be measured by any known methods in the art. A number of tests are available to assess the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assay, ROTEM assay, prothrombin time (PT) test (also used to determine INR), fibrinogen testing (often by the Clauss method), platelet count, platelet function testing (often by PFA-100), TCT, bleeding time, mixing test (whether an abnormality corrects if the subject's plasma is mixed with normal plasma), coagulation factor assays, antiphospholipid antibodies, D-dimer, genetic tests (e.g., factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRVVT), miscellaneous platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM ^® , e.g., ROTEM ^® ), or euglobulin lysis time (ELT).

[0099] The aPTT test is a performance indicator measuring 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. It is typically used in conjunction with prothrombin time (PT), which measures the extrinsic pathway. (See, e.g., Kamal et al., Mayo Clin Proc., 82(7):864-873 (2007)). In some embodiments, 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).

[0100] In some embodiments, the aPTT assay may also be used for assessing the potency of a chimeric polypeptide prior to administration to a subject. (Hubbard AR, et al. J Thromb Haemost 11: 988-9 (2013)). In some embodiments, 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 subject.

[0101] ROTEM analysis provides information on the whole kinetics of hemostasis: clotting time, clot formation, clot stability and lysis. The different parameters in thromboelastometry are dependent on the activity of the plasmatic coagulation system, platelet function, fibrinolysis, or many factors which influence these interactions. This assay can provide a complete view of secondary hemostasis.

[0102] 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 one embodiment, the chromogenic assay is the BIOPHEN FVIII:C assay (Hyphen Biomed, Neurville sur Oise, France) [0103] The chromogenic assay is recommended by the FVIII and Factor IX Subcommittee of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Flemostatsis (ISTH). Since 1994, the chromogenic assay has also been the reference method of the European Pharmacopoeia for the assignment of FVIII concentrate potency. Thus, in some embodiments, the chimeric polypeptide comprising a FVIII polypeptide has FVIII activity comparable to a chimeric polypeptide comprising mature FVIII polypeptide or a BDD FVIII polypeptide (e.g., RECOMBINATE ^® , KOGENATE FS ^® , HELIXATE FS ^® , XYNTHA/REFACTO AB ^® , HEMOFIL-M ^® , MONARC-M ^® , MONOCLATE-P ^® , HUMATE-P ^® , ALPHANATE ^® , KOATE-DVI ^® , AFSTYLA ^® , AND HYATE:C ^® ).

[0104] In some embodiments, a chromogenic assay may also be used for assessing the potency of a chimeric polypeptide prior to administration to a subject. (Hubbard AR, et al. J Thromb Flaemost 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 subject.

Subject Dosing Information

[0105] For most currently available FVIII replacement therapies, the required FVIII dose for each subject is calculated using the following formula:

Number of factor VIII units required (IU) = Body Weight (kg) x (B)

Desired FVIII Increase (IU/dL or % of normal) x 0.5(IU/kg per IU/dL))

[0106] This calculation provides a general estimation of the dosing requirements of a subject based on body weight as a subject specific variable and the desired FVIII activity level increase.

[0107] Disclosed herein is a more sophisticated model [A] for determining or estimating dosing information for hemophilia A subjects receiving FVIII replacement therapy with rFVIIIFc-VWF-XTEN. The rFVIIIFc-VWF-XTEN popPK model [A] is represented as follows:

0108] Model Abbreviations are: A _central , amount of drug; clearance; clearance from the individual; CLTV, the typical value; c _plasma , concentration in the plasma; magnitude of SD of proportional residual error; magn[tude of SD of proportional additive error; Hct, hematocrit; V _central,i, is the post-hoc individual variability for in subject i; η _CL,ί, post-hoc individual variability for in subject i; is the allometric exponent of WT on volume of the central compartment; V _central,i, volume of the central compartment in subject, i; TV, typical value for volume of the central compartment; W, weight of the current point (total SD of WT, body weight; is the model-estimated plasma concentration; e is residual error.

[0109] Investigation into covariates that affect FVIII activity has historically focused on VWF concentration. Flowever, VWF concentration was not found to be a covariate in the rFVIIIFc-VWF-XTEN popPK data and model disclosed herein. Without being bound by any scientific theory, it is noted that rFVIIIFc-VWF-XTEN clearance is independent of endogenous VWF.

[0110] In the rFVIIIFc-VWF-XTEN model [A], the major covariates for FVIII activity identified as body weight on clearance and body weight and hematocrit on central volume (allometric exponents -0.568 and 0.703, respectively; Table 2).

[0111] Flematocrit (Fit or HCT) is the volume percentage of red blood cells (RBC) in total blood volume. Hematocrit effect on population pharmacokinetic models has been examined previously (see, e.g., WO2015/085276; Nestorov, et al. Clin Pharm Drug Dev. (2015): 163-174). The hematocrit test, also known as a packed-cell volume (PCV) test, is a simple and well-known blood test. It is usually part of a complete blood count (CBC) test. The conventional means to measure hematocrit in clinical medicine is to puncture the skin, draw blood from a vein or capillary into a small-diameter tube, and measure the solid (packed-cell) fraction that remains after centrifugation of the blood. In some embodiments, blood is collected by a health care professional, such as a physician or nurse, e.g. in a medical office. However, alternative methods for non- invasive measurement of hematocrit have been developed and may be used in some embodiments. (See, e.g. US 6,606,509). For example, hematocrit can be calculated by an automated analyzer or by optical methods such as spectrophotometry.

[0112] Provided herein is a combination comprising a kit or tool for estimating hematrocrit levels and software that is or is part of a software-based system that uses a rFVIIIFc-VWF-XTEN model (e.g., rFVIIIFc-VWF-XTEN model [A]) to provide dosing or PK information in accordance with one or more of the methods disclosed herein.

[0113] In some embodiments, a haemophilia A subject's hematocrit is measured and the rFVIIIFc-VWF-XTEN model (such as rFVIIIFc-VWF-XTEN model [A]) disclosed herein is applied to determine or estimate rFVIIIFc- VWF-XTEN dosing information for the individual subject. In some embodiments, the subject's hematocrit is measured and/or determined by the subject. In some embodiments, the subject's hematocrit is measured and/or determined by a health care professional. In some embodiments, the subject's hematocrit is combined with other subject characteristics, such as clearance, central volume, body weight on clearance, body weight on clearance, and the like, and applied using the model disclosed herein to determine or estimate rFVIIIFc- VWF-XTEN dosing information for the individual subject.

[0114] In some embodiments, hematocrit is measured prior to estimating the dose of chimeric protein. In some embodiments, the subject information entered is hematocrit. In some embodiments, measurement of hematocrit is performed by a healthcare professional. In some embodiments, measurement of hematocrit is performed by the subject. In some embodiments, hematocrit is measured by the same device into which subject information is entered. In some embodiments, hematocrit is measured by a device into which additional subject information is not entered. In some embodiments, hematocrit is measured by an implantable device. In some embodiments, hematocrit is measured by an injectable device.

[0115] Some embodiments comprise administering a dose of rFVIIIFc-VWF-XTEN to a human subject in need thereof at a dosing interval, wherein the dose and/or the dosing interval is identified using the subject's hematocrit and body weight, but not the subject's VWF level. The present disclosure provides methods of administering a dose of rFVIIIFc-VWF-XTEN to a human subject in need thereof at a dosing interval, wherein the dose and/or the dosing interval is identified by applying the rFVIIIFc-VWF-XTEN model [A] disclosed herein.

[0116] The rFVIIIFc-VWF-XTEN model [A] disclosed herein can be used to determine individual subject dosing information in order to evaluate and/or confirm subject treatment goals. This individual subject dosing information can be used to determine dose and/or dosing interval of rFVIIIFc-VWF-XTEN in future treatments. In some embodiments, subject treatment goals comprise high plasma FVIII levels over long periods of time.

[0117] In some embodiments, the therapeutically effective dose of rFVIIIFc-VWF-XTEN is between about 20IU/kg and about 90IU/kg. In some embodiments, the therapeutically effective dose is 20-30 lU/kg, 30-40 lU/kg, 40-50 IU/kg, 50-60 lU/kg, 60-70 lU/kg, 70-80 lU/kg, or 80-90 lU/kg. In some embodiments, the subject is administered a dose of about 50 IU/kg.

[0118] In some embodiments, the methods disclosed herein are applied to determine a subject's individualized interval prophylaxis. The term "individualized interval prophylaxis" as used herein means use of rFVIIIFc-VWF-XTEN for an individualized dose and/or dosing interval or frequency to prevent or inhibit occurrence of one or more spontaneous and/or uncontrollable bleeding or bleeding episodes or to reduce the frequency of one or more spontaneous and/or uncontrollable bleeding or bleeding episodes.

[0119] In some embodiments, administration of rFVIIIFc-VWF-XTEN is for individualized (tailored) prophylaxis and results in an annualized bleed rate (ABR) of less than about 5.5, less than about 5.4, less than about 5.3, less than about 5.2, less than about 5.1, less than about 5.0, less than about 4.9, less than about 4.8, less than about 4.7, less than about 4.6, or less than about 4.5.

[0120] In some embodiments, administration of a rFVIIIFc-VWF-XTEN is for weekly prophylaxis and results in an ABR of less than about 9.0, less than about 8.9, less than about 8.8, less than about 8.7, less than about 8.6, less than about 8.5, less than about 8.4, less than about 5.5, less than about 5.4, less than about 5.3, less than about 5.2, less than about 5.1, less than about 5.0, less than about 4.9, less than about 4.8, less than about 4.7, less than about 4.6, or less than about 4.5.

[0121] In some embodiments, administration of a rFVIIIFc-VWF-XTEN is for episodic or on-demand treatment and results in an ABR of less than about 55, less than about 54, less than about 53, less than about 52, less than about 51, less than about 50, less than about 49, less than about 48, or less than about 47.

[0122] In some embodiments, the rFVIIIFc-VWF-XTEN dosing interval can be at least about every three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days or longer.

[0123] In some embodiments, subject treatment goals include achieving high FVIII plasma activity levels and/or high trough levels. As used herein, a "trough level" in a hemophilia subject is the measurement of the lowest concentration reached by a factor therapy, e.g., rFVIIIFc-VWF-XTEN therapy, before the next dose is administered. The methods disclosed herein can be used to determine subject dosing information in order to achieve specific FVIII plasma activity levels and/or trough levels. Administration of rFVIIIFc-VWF-XTEN has been shown to successfully achieve high FVIII plasma activity levels and/or high trough levels in hemophilia A subjects. In some embodiments, administration of rFVIIIFc-VWF-XTEN results in a FVIII trough level of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%. In some embodiments, administration of rFVIIIFc-VWF-XTEN (i.e., immediately after injection) results in a FVIII plasma activity level of at least about 10%, at least about 15%, at least about 20%, at least about 40%, or at least about 50%. In some embodiments, administration of rFVIIIFc-VWF-XTEN results in a FVIII plasma activity level (i.e., immediately after injection) of greater than 150%. In some embodiments, rFVIIIFc-VWF-XTEN is administered to maintain a FVIII trough level of at least about 5%, between about 1 and about 20 lll/dL, between about 2 and about 20 lll/dL, between about 3 and about 20 lll/dL, between about 4 and about 20 lll/dL, between about 5 5 and about 20 IU/dL, between about 6 and about 20 IU/dL, between about 7 and about 20 IU/dL, between about 8 and about 20 lll/dL, between about 9 and about 20 IU/dL, or between about 10 and about 20 IU/dL during a dosing interval. As used herein, plasma activity level is expressed as a percent (%). Alternatively, plasma activity can be expressed in IU/dL units, wherein 1% is equal to 1 IU/dL.

[0124] In some embodiments, administration of rFVIIIFc-VWF-XTEN results in at least a near-normal level of FVIII activity in the subject for about 1, 2, 3, or 4 days. In some embodiments, near-normal FVIII activity is defined as 40% or greater. In some embodiments, administration of rFVIIIFc-VWF-XTEN results in at least a normal level of FVIII activity in the subject for about 1, 2, 3, or 4 days. In some embodiments, normal FVIII activity is defined as 50% or greater. In some embodiments, administration of rFVIIIFc-VWF-XTEN results in at least a near-normal level of FVIII activity in the subject for at least three days. In some embodiments, administration of rFVIIIFc-VWF-XTEN results in at least a near-normal level of FVIII activity in the subject for about 4 days. In some embodiments, the rFVIIIFc-VWF-XTEN model [A] is used to determine individual subject dosing information in order to achieve at least near-normal FVIII activity in the subject for marabout 1, 2, 3 or 4 days.

Method, System, and Storage Medium for Estimating Subject Individualized Dosing Information, Subject Individualized PK Information, and Subject Median PK Information

[0125] Included herein is a method (e.g., a computer-implemented method) of estimating rFVIIIFc-VWF-XTEN dosing information individualized for a subject, the method comprising: (a) receiving subject information and/or desired treatment outcome information by a software-based system containing a rFVIIIFc-VWF-XTEN popPK model (e.g., rFVIIIFc-VWF-XTEN popPK model [A]), b) calculating, by the software-based system, individualized dosing information using the rFVIIIFc-VWF-XTEN popPK model and the received information, and c) outputting, by the software-based system, the individualized dosing information. Further disclosed is the method as described herein, further comprising selecting a dosing regimen based on the output individualized dosing information of (c) and administering the rFVIIIFc-VWF-XTEN to the subject according to the selected dosing regimen. In some embodiments, the software-based system of the methods described herein also contains a Bayesian estimation program.

[0126] In some embodiments (a) further comprises receiving, by the software-based system, subject information.

[0127] In some embodiments the subject information is age, hematocrit, and/or body weight. Additional subject information further includes diagnostic (baseline) FVIII level, PK determinations, time of PK sampling, dosing history if PK samples were taken from multiple doses, actual dose, FVIII activity level, etc. [0128] In some embodiments, output information is, e.g., PK curve, PK parameter such as incremental recovery (Cmax/dose), mean residence time, terminal tl/2, clearance, Vss, AUC/dose, doses and associated troughs, and intervals and associated troughs.

[0129] For example, for assessing individualized subject PK, the system can recommend that the user input 2- 3 optimized PK sampling time points. In this case, system output can include PK curve and one or more selected PK parameters.

[0130] As additional examples, to select an individualized dosing regimen using the output individual PK parameters discussed in the preceding paragraph, (i) the dose selected for acute treatment can be based on user input of the desired rise in plasma FVIII activity level following the dose, (ii) the dose selected for prophylaxis can be based on user input of the desired dosing interval, or (iii) the selected interval for prophylaxis can be based on user input for the desired dose. In the second case, system output can be a table of doses and associated troughs, e.g., x lU/kg, 1% trough, y lU/kg, 2% trough, etc. In the third case, system output can be a table of intervals and associated troughs, e.g., x days, 1% trough, y lU/kg, 2% trough, etc.

[0131] In some embodiments, the user may wish to use the system without inputting any individualized PK data. In this case, the dosing output would be based on the population median rather than being individualized for the particular subject. In this way, the user inputs, e.g., body weight and/or hematocrit, and (i) the desired rise in plasma FVIII activity level following the dose, (ii) the desired dose interval for prophylaxis, or (iii) the desired dose for prophylaxis. In the first case, the system can output the dose. In the second case, the system can output the dose and associated trough. In the third case, the system can output the interval and associated trough.

[0132] In some embodiments, the system is compliant with patient privacy laws. In some embodiments, the system is encrypted, e.g., with SSL. In some embodiments, input subject information is made anonymous.

[0133] In some embodiments, the system includes a user help function.

[0134] In some embodiments, the method can be carried out by, e.g., a subject, a physician, a nurse, or another healthcare practitioner. In some embodiments, the method is carried out by the subject.

[0135] Additional embodiments include a computer readable storage medium having instructions stored thereon that, when executed by a processor, cause the processor to perform any of the above methods.

[0136] Additional embodiments include a system comprising a processor and a memory, the memory having instructions stored thereon that, when executed by the processor, cause the processor to perform any of the above methods.

[0137] The user of the system or computer readable storage medium, can be, e.g., a subject, a physician, a nurse, or another healthcare practitioner. [0138] In some embodiments, the subject information entered into the system includes body weight. In some embodiments, the subject information entered into the system is hematocrit. In some embodiments, the desired treatment outcome information is desired rise in plasma FVIII activity level following dosing and the output information is dose for acute treatment. In some embodiments, the desired treatment outcome information is desired dosing interval and the output information is dose for prophylaxis. In some embodiments, the desired treatment outcome information is desired dose and the output information is interval for prophylaxis.

[0139] The present disclosure also includes a software-based method of estimating individual subject rFVIIIFc- VWF-XTEN PK, the method comprising: (a) receiving, by one or more electronic devices, individual rFVIIIFc- VWF-XTEN PK information, (b) transmitting, by a processing device, the individual rFVIIIFc-VWF-XTEN PK information to a software-based application program accessible through a web server, wherein the application is programmed to implement a rFVIIIFc-VWF-XTEN population pharmacokinetic (popPK) model, such as the model disclosed in the Examples herein, and, optionally, a Bayesian estimation program, (c) receiving from the software-based server and program, individualized subject rFVIIIFc-VWF-XTEN PK information calculated using the popPK model, the optional Bayesian estimation program, and the transmitted information of (b) and (d) outputting, by the one or more electronic devices, the calculated subject PK information. In some embodiments, the method also comprises selecting a dosing regimen based on the output calculated subject PK information of (d) and administering the rFVIIIFc-VWF-XTEN to the subject according to the selected dosing regimen.

[0140] In some embodiments, the individual rFVIIIFc-VWF-XTEN PK information includes 2-3 PK sampling time points. In some embodiments, the individual rFVIIIFc-VWF-XTEN PK information includes one or more of subject body weight, diagnostic (baseline) factor level, dosing history if PK samples were taken from multiple doses, actual dose, actual time of PK sampling, factor activity level, subject body weight, and/or subject hematocrit.

[0141] In some embodiments the output individualized subject PK includes a PK curve or a PK parameter selected from incremental recovery (Cmax/Dose), mean residence time, terminal tl/2, clearance, Vss and AUC/Dose. In some embodiments, the desired treatment outcome information based on the individual subject's PK is desired rise in plasma FVIII activity level following dosing and the output information is dose for acute treatment.

[0142] In some embodiments, the methods disclosed herein include an electronic device. An electronic device can include, but is not limited to, a device having a processor and memory for executing and storing instructions. The electronic device may also include a display and one or more computer input devices such as a keyboard, a mouse and/or a joystick. In some embodiments, the electronic device is a general-purpose computing and data communication device such as digital pen, a smart phone, a tablet computer, a personal digital assistant, a handheld computer, a laptop computer, a point-of-sale transaction device, a scanner, a camera, and a fax machine. The electronic device may also have multiple processors and multiple shared or separate memory components. For example, the electronic device may be a clustered computing environment or server farm.

[0143] Alternatively, the electronic device can be a specialized data collection, computing and communications device such as, for example, a point-of-care (POC) device capable of receiving subject demographic information including age, vital signs including body weight, and/or blood characterizing values including hematocrit. The blood characterizing values may be received by the electronic device via a data communications channel, manual entry, and/or by diagnostic processes performed by the electronic device. Diagnostic processes performed on subject blood samples within the device may include ultrasound measurements, impedance measurements, conductivity measurements, and/or optical measurements. The electronic device may be further configured to receive, detect, record and/or communicate additional subject information including diagnostic (baseline) FVIII level, PK determinations, time of PK sampling, dosing history if PK samples were taken from multiple doses, actual dose, FVIII activity level. The electronic device communicates with one or more network-based (e.g., web-based) application programs over one or more networks, such as the Internet. Similar to the electronic device, the network-based (e.g., web-based) application program can be implemented using any general-purpose computer capable of serving data to the electronic device. The electronic device can receive individualized subject rFVIIIFc-VWF-XTEN PK information from a network-based (e.g., web-based) server and program. In some embodiments, the electronic device can assist in selecting a dosing regimen based on the output calculated subject PK information.

[0144] The methods and systems described herein may be implemented in or via a mobile device. Mobile devices include navigation devices, mobile phones, mobile phones, mobile personal digital information processing terminals, laptops, palmtops, netbooks, pagers, electronic book terminals, music players, and the like. These devices, apart from other components, may comprise a storage medium such as flash memory, buffers, RAM, ROM and one or more computing devices. A computing device associated with the mobile device may be adapted to execute program code, methods, and instructions stored thereon. As another example, a mobile device may be configured to execute instructions in cooperation with other devices. The mobile device may communicate with a base station that is connected to the server and configured to execute the program code. Mobile devices can also communicate over peer-to-peer networks, mesh networks, or other communication networks. The program code may be stored in a storage medium associated with the server and executed by a computing device embedded in the server. The base station may comprise a computing device and a storage medium. The storage medium may store program code and instructions that are executed by a computing device associated with the base station.

[0145] In some embodiments, the methods and systems described herein are directed to a kit for collecting subject information. Although different embodiments of the kit may include different components, an exemplary kit includes a diagnostic device such as a processing element and/or a calculation element for acquiring information from the subject, and a transmission element that transmits the subject information to a computer device through a wired or wireless connection. The transmitting element in the kit may be configured to transmit subject information in real time when the device is in use, or the diagnostic information may be transmitted with receipt of instructions from a user or provider. Any of the components of the kit, such as the body, can be configured as a hands-free unit during use or as a handheld unit during use. In some embodiments, the kit includes a device for measuring the subject's hematocrit. In other embodiments, a point- of-care device may be used to measure hematocrit.

Exemplary Computinfi Environments for the Disclosed Methods and Systems

[0146] Various modeling techniques, dosage calculations, and estimations described herein can be implemented by software, firmware, hardware, or a combination thereof. Figure 9 illustrates an example computer system 1900 in which the embodiments, or portions thereof, can be implemented as computer- readable code. In another embodiment, for rFVIIIFc-VWF-XTEN, the modeling disclosed in the Examples herein can be implemented in system 1900.

[0147] Computer system 1900 includes one or more processors, such as processor 1904. Processor 1904 is connected to a communication infrastructure 1906 (for example, a bus or network).

[0148] Computer system 1900 also includes a main memory 1908, preferably random access memory (RAM), and may also include a secondary memory 1910. In accordance with implementations, user interface data may be stored, for example and without limitation, in main memory 1908. Main memory 1908 may include, for example, cache, and/or static and/or dynamic RAM. Secondary memory 1910 may include, for example, a hard disk drive and/or a removable storage drive. Removable storage drive 1914 may include a floppy disk drive, a magnetic tape drive, an optical disk drive, a flash memory, or the like. The removable storage drive 1914 reads from and/or writes to removable storage unit 1916 in a well-known manner. Removable storage unit 1916 may include a floppy disk, magnetic tape, optical disk, etc. which is read by and written to by removable storage drive 1914. As will be appreciated by persons skilled in the relevant art(s), removable storage unit 1916 includes a computer readable storage medium having stored therein computer software and/or data.

[0149] Computer system 1900 may also include a display interface 1902. Display interface 1902 may be adapted to communicate with display unit 1930. Display unit 1930 may include a computer monitor or similar means for displaying graphics, text, and other data received from main memory 1908 via communication infrastructure 1906. In alternative implementations, secondary memory 1910 may include other similar means for allowing computer programs or other instructions to be loaded into computer system 1900. Such means may include, for example, a removable storage unit 1922 and an interface 1920. Examples of such means may include a program cartridge and cartridge interface (such as that found in video game devices), a removable memory chip (such as an EPROM, or PROM) and associated socket, and other removable storage units 1922 and interfaces 1920 which allow software and data to be transferred from the removable storage unit 1922 to computer system 1900. [0150] Computer system 1900 may also include a communications interface 1924. Communications interface 1924 allows software and data to be transferred between computer system 1900 and external devices. Communications interface 1924 may include a modem, a network interface (such as an Ethernet card), a communications port, a PCMCIA slot and card, or the like. Software and data transferred via communications interface 1924 are in the form of signals which may be electronic, electromagnetic, optical, or other signals capable of being received by communications interface 1924. These signals are provided to communications interface 1924 via a communications path 1926. Communications path 1926 carries signals and may be implemented using wire or cable, fiber optics, a phone line, a cellular phone link, an RF link or other communications channels.

[0151] In this document, the term "computer readable storage medium" is used to generally refer to non- transitory storage media such as removable storage unit 1916, removable storage unit 1922, and a hard disk installed in hard disk drive 1912. Computer readable storage medium can also refer to one or more memories, such as main memory 1908 and secondary memory 1910, which can be memory semiconductors (e.g. DRAMs, etc.). These computer program products are means for providing software to computer system 1900.

[0152] Computer programs (also called computer control logic) are stored in main memory 1908 and/or secondary memory 1910. Computer programs may also be received via communications interface 1924 and stored on main memory 1908 and/or secondary memory 1910. Such computer programs, when executed, enable computer system 1900 to implement embodiments as discussed herein. In particular, the computer programs, when executed, enable processor 1904 to implement processes of the present disclosure, such as certain methods discussed above. Accordingly, such computer programs represent controllers of the computer system 1900. Where embodiments use software, the software may be stored in a computer program product and loaded into computer system 1900 using removable storage drive 1914, interface 1920, or hard drive 1912.

[0153] Embodiments may be directed to computer program products comprising software stored on any computer readable medium. Such software, when executed in one or more processing devices, causes a processing device to operate as described herein. Embodiments may employ any computer useable or readable medium. Examples of computer readable storage media include, but are not limited to, non-transitory primary storage devices (e.g., any type of random access memory), and non-transitory secondary storage devices (e.g., hard drives, floppy disks, CD ROMS, ZIP disks, tapes, magnetic storage devices, and optical storage devices, MEMS, nano-technological storage device, etc.). Other computer readable media include communication mediums (e.g., wired and wireless communications networks, local area networks, wide area networks, intranets, etc.).

[0154] Non-limiting examples of software-based systems include network-based systems and web-based systems. [0155] Figure 10 illustrates an example of a network-based system 2000 for a rFVIIIFc-VWF-XTEN, in which the embodiments, or portions thereof, can be implemented as computer-readable code. In some embodiments, for rFVIIIFc-VWF-XTEN, the modeling disclosed in the Examples herein can be implemented in system 2000.

[0156] Network-based system 2000 includes network 2004 that can be any network or combination of networks that can carry data communication, and may be referred to herein as a computer network. Such network 2004 can include, but is not limited to, a local area network, medium area network, and/or wide area network such as the Internet. Network 2004 can support protocols and technology including, but not limited to, World Wide Web protocols and/or services. Intermediate web servers, gateways, or other servers may be provided between components of system 2000 depending upon a particular application or environment.

[0157] Figure 10 shows a block diagram of an exemplary network-based system 2000 for obtaining an estimated subject individualized dosing information, subject individualized PK information, and subject median PK information. System 2000 includes an electronic device 2008 that can communicate over network 2004. Electronic device 2008 includes a transmitting engine 2012, and receiving engine 2016. Transmitting engine 2012 may transmit messages over network 2004. For example, transmitting engine 2012 may transmit information associated with dosing information individualized for a subject. Receiving engine 2016 may receive messages over network 2004 (e.g., from server 2020). For example, receiving engine 2016 may receive a response associated with individualized calculated dosing information transmitted over network 2004 by server 2020.

[0158] Electronic device 2008 can include computer system 1900 and can include, but is not limited to, a personal computer, mobile device such as a mobile phone, workstation, embedded system, game console, television, set-top box, or any other computing device. Further, electronic device 2008 can include, but is not limited to, a device having a processor and memory 2017 for executing and storing instructions.

[0159] Server 2020 includes a receiving engine 2026 and a communications interface 2028. Receiving engine 2026 may receive messages over network 2004 (e.g., from electronic device 2008) and communicate the received message to application program 2032. In one embodiment, application program 2032 is programmed to implement a rFVIIIFc-VWF-XTEN population pharmacokinetic (popPK) model such as the model disclosed in the Examples herein, and, optionally, a Bayesian estimation program. Output of application program 2032 may be communicated by communications interface 2028 over network 2004. For example, transmitting engine 2030 may transmit output information associated with dosing information individualized for a subject over network 2004 to electronic device 2008.

[0160] Figure 11 shows a schematic diagram of an example computing system 500. The system 500 can be used for the operations described in association with the implementations described herein. For example, the system 500 may be included in any or all of the external devices discussed herein. The system 500 includes a processor 510, a memory 520, a storage device 530, and an input/output device 540. Each of the components 510, 520, 530, and 540 are interconnected using a system bus 550. The processor 510 is capable of processing instructions for execution within the system 500. In one implementation, the processor 510 is a single-threaded processor. In another implementation, the processor 510 is a multi-threaded processor. The processor 510 is capable of processing instructions stored in the memory 520 or on the storage device 530 to display graphical information for a user interface on the input/output device 540.

[0161] The memory 520 stores information within the system 500. In one implementation, the memory 520 is a computer-readable medium. In one implementation, the memory 520 is a volatile memory unit. In another implementation, the memory 520 is a non-volatile memory unit. The storage device 530 is capable of providing mass storage for the system 500. In one implementation, the storage device 530 is a computer-readable medium. In various different implementations, the storage device 530 may be a floppy disk device, a hard disk device, an optical disk device, or a tape device. The input/output device 540 provides input/output operations for the system 500. In one implementation, the input/output device 540 includes a keyboard and/or pointing device. In another implementation, the input/output device 540 includes a display unit for displaying graphical user interfaces that enable a user to access data related to an item that is collected, stored and queried.

[0162] The features described can be implemented in digital electronic circuitry, or in computer hardware, firmware, software, or in combinations of them. The apparatus can be implemented in a computer program product tangibly embodied in an information carrier, e.g., in a machine-readable storage device, for execution by a programmable processor; and method steps can be performed by a programmable processor executing a program of instructions to perform functions of the described implementations by operating on input data and generating output. The described features can be implemented advantageously in one or more computer programs that are executable on a programmable system including at least one programmable processor coupled to receive data and instructions from, and to transmit data and instructions to, a data storage system, at least one input device, and at least one output device. A computer program is a set of instructions that can be used, directly or indirectly, in a computer to perform a certain activity or bring about a certain result. A computer program can be written in any form of programming language, including compiled or interpreted languages, and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment.

[0163] Suitable processors for the execution of a program of instructions include, by way of example, both general and special purpose microprocessors, and the sole processor or one of multiple processors of any kind of computer. Generally, a processor will receive instructions and data from a read-only memory or a random access memory or both. The essential elements of a computer are a processor for executing instructions and one or more memories for storing instructions and data. Generally, a computer will also include, or be operatively coupled to communicate with, one or more mass storage devices for storing data files; such devices include magnetic disks, such as internal hard disks and removable disks; magneto-optical disks; and optical disks. Storage devices suitable for tangibly embodying computer program instructions and data include all forms of non-volatile memory, including by way of example semiconductor memory devices, such as EPROM, EEPROM, and flash memory devices; magnetic disks such as internal hard disks and removable disks; magneto optical disks; and CD-ROM and DVD-ROM disks. The processor and the memory can be supplemented by, or incorporated in, ASICs (application-specific integrated circuits).

[0164] To provide for interaction with a user, the features can be implemented on a computer having a display device such as a CRT (cathode ray tube) or LCD (liquid crystal display) monitor for displaying information to the user and a keyboard and a pointing device such as a mouse or a trackball by which the user can provide input to the computer.

[0165] The features can be implemented in a computer system that includes a back-end component, such as a data server, or that includes a middleware component, such as an application server or an Internet server, or that includes a front-end component, such as a client computer having a graphical user interface or an Internet browser, or any combination of them. The components of the system can be connected by any form or medium of digital data communication such as a communication network. Examples of communication networks include, e.g., a LAN, a WAN, and the computers and networks forming the Internet.

[0166] The computer system can include clients and servers. A client and server are generally remote from each other and typically interact through a network, such as the described one. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

[0167] In addition, the logic flows depicted in the figures do not require the particular order shown, or sequential order, to achieve desirable results. In addition, other steps may be provided, or steps may be eliminated, from the described flows, and other components may be added to, or removed from, the described systems. Accordingly, other implementations are within the scope of the following claims.

[0168] Having now described the present invention 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 invention. All patents and publications referred to herein are expressly incorporated by reference.

EXAMPLES

Example 1. Single Dose rFVIIIFc-VWF-XTEN Treatment in Previously Treated Adults with Severe Hemophilia A (EXTEN-A)

[0169] Study 242HA101 (ClinicalTrials.gov Identifier: NCT03205163) was a first-in-human, Phase l/2a open- label, dose-escalation, multicenter study to assess the safety, tolerability, and PK of a single IV dose of rFVIIIFc-VWF-XTEN in adult male previously treated patients (PTPs) with severe hemophilia A. Participants received a single IV dose of 25 lU/kg or 65 lU/kg rFVIII comparator (Advate ^® ) in the low and high dose cohorts, respectively, followed by a washout period and a single IV dose of 25 lU/kg or 65 lU/kg of rFVIIIFc-VWF-XTEN in the low and high dose cohorts, respectively. Of the 16 participants enrolled in the study, all received a single dose of rFVIII (25 lU/kg, n=7, 65 lU/kg, n=9) and 15 received a single dose of rFVIIIFc-VWF-XTEN (25 lU/kg, n=6, 65 lU/kg, n=9). Overall, rFVIIIFc-VWF-XTEN was well-tolerated.

[0170] Pharmacokinetic results

[0171] In the low-dose (25 lU/kg) cohort, 6 participants had evaluable PK data for rFVIIIFc-VWF-XTEN treatment. The average FVIII activity level as measured by the one-stage aPTT assay in the low-dose (25 lU/kg) cohort was 26.2% after 72 hours for rFVIIIFc-VWF-XTEN as compared to 0.7% for rFVIII. The extended PK profile for rFVIIIFc-VWF-XTEN showed average FVIII activity levels of 12.2% at 5 days, 5.3% at 7 days, and 1.3% at 10 days, based on the one-stage aPTT assay. The geometric mean of half-life was 37.6 hours for rFVIIIFc-VWF-XTEN compared with 9.1 hours for rFVIII based on FVIII activity measured by the one-stage aPTT clotting assay [GMR=4.1 (95% Cl: 2.9, 5.8); p<0.001] in the low-dose (25 lU/kg) cohort.

[0172] In the high-dose (65 lU/kg) cohort, 8 participants had evaluable PK data for rFVIIIFc-VWF-XTEN treatment. The average FVIII activity level as measured by the one-stage aPTT assay in the high-dose (65 lU/kg) cohort, was 78.2% after 72 hours for rFVIIIFc-VWF-XTEN as compared to 2.3% for rFVIII. The extended PK profile for rFVIIIFc-VWF-XTEN showed average FVIII activity levels of 37.8% at 5 days, 17.0% at 7 days and 1.1% at 14 days, based on the one-stage aPTT assay. The geometric mean of half-life was 42.5 hours for rFVIIIFc-VWF-XTEN compared with 13.2 hours for rFVIII based on FVIII activity measured by the one-stage aPTT clotting assay [geometric means ration (GMR)=3.2 (95% Cl: 2.8, 3.8); p<0.001] in the high-dose (65 lU/kg) cohort.

Example 2. Repeat Dose rFVIIIFc-VWF-XTEN Treatment in Previously Treated Adults with Severe Hemophilia A

[0173] Study 242HA102 was a Phase 1, open-label, single-site study to assess the safety, tolerability, and PK of repeat-dose rFVIIIFc-VWF-XTEN in adult male PTPs with severe hemophilia A. After a brief washout period, subjects were administered a total of 4-once weekly doses of BIVV001 IV at 50 lU/kg (Cohort 1) or 65 lU/kg (Cohort 2) on Days 1, 8, 15, and 22. A predose PK sample was taken on Day 1. Multiple PK samples were taken after dosing on Days 1 and 22, and a trough (168h) sample was taken prior to dosing on Days 8, 15, and 22. Overall, rFVIIIFc-VWF-XTEN was well-tolerated.

[0174] Pharmacokinetic results

[0175] The PK analysis set (PKAS) included 9 subjects from the 50 lU/kg cohort and 14 subjects from 65 lU/kg, who had adequate PK sample collections following BIVV001 administration. [0176] Cohort 1 (50 lU/kg): Following BIVV001 dosing of 50 lU/kg on Day 1, mean FVIII activities based on one- stage and chromogenic assays at 72 h were 45.53% and 29.37%, at 120 h were 21.56% and 13.49%, and at 168 h were 7.91% and 5.97%, respectively (Table 2 and Table 3). On Day 22 with QW dosing, mean FVIII activities based on one-stage and chromogenic assays at 72 h were 46.28% and 30.46%, at 120 h were 22.30% and 14.48%, and at 168 h were 9.83% and 6.74%, respectively.

[0177] Cohort 2 (65 lU/kg): Following BIVV001 dosing of 65 lU/kg on Day 1, mean FVIII activities based on one- stage and chromogenic assays at 72 h were 65.65% and 35.09%, at 120 h were 26.88% and 14.32%, and at 168 h were 10.54% and 6.49%, respectively. On Day 22 with QW dosing, mean FVIII activities based on one-stage and chromogenic assays at 72 h were 69.31% and 37.63%, at 120 h were 27.21% and 16.51%, and at 168 h were 11.81% and 7.64%, respectively.

Example 3. Population Pharmacokinetic Analysis of rFVIIIFc-VWF-XTEIM in Subjects with Severe Hemophilia A

[0178] The objective of this study is was to develop a population model that characterizes rFVIIIFc-VWF-XTEN PK and to identify factors that determine rFVIIIFc-VWF-XTEN PK variability in adults with severe hemophilia A.

[0179] Methods: Design of the two Phase 1 clinical studies for rFVIIIFc-VWF-XTEN (EXTEN-A, repeat dosing) are described in Examples 2 and 3 (FIGs. 1 and 2). In the two Phase 1 studies, FVIII activity was measured as a marker for rFVIIIFc-VWF-XTEN PK via a one-stage activated partial thromboplastin time (aPTT) clotting assay using a product specific-standard (single-dose study: N=15; repeat-dose study: N=24).

[0180] For the single dose, EXTEN-A clinical trial (NCT03205163), PK data was sampled at the following timepoints: Pre-dose, 0.17, 0.5, 1, 3, 6, 9, 24, 48, 72, 96, 120, 168, and 240 hours post-dose for both the 25 lU/kg and 65 lU/kg cohort. In addition to these timepoints, PK data was sampled 336 hours post-dose in the 65 lU/kg cohort.

[0181] For the repeat dose study, PK data was sampled at slightly different time points depending on which dose had been administered. Dose 1 was sampled pre-dose and 0.5, 3, 24, 48, 72, and 120 hours post-dose. Dose 2 and 3 were sampled pre-dose only. Dose 4 was sampled pre-dose and 24, 48, 72, 120, 168, 240, 336, and 672 hours post-dose.

[0182] Population PK analysis: Population characteristics of FVIII activity were evaluated using mixed-effects modeling with maximal likelihood parameter estimation methods. Models were estimated using NONMEM version 7.4.2. Covariate effect was assessed using a forward inclusion, backward elimination process. Goodness of fit assessments, diagnostic plots, minimum value of the objective function, and the evaluation of shrinkage were performed to evaluate population PK models. The best model was selected based on objective function value improvement measure and goodness of fit figures. [0183] Simulations: Simulations using weight-based dosing were performed first (70 kg fixed reference body weight, simulated 5-100 lU/kg doses) to predict steady-state FVIII activity peaks and troughs in different dose groups, and the 50 lU/kg and 65 lU/kg activity-time profiles. Clinical trial simulations were performed next (included 600 simulated individuals and empirical body weight distribution with a weighted-average from rFVIIIFc-VWF-XTEN clinical trials). Time above FVIII activity thresholds, Cmax and Ctrough FVIII activity, and proportion of simulated individuals were predicted using the model.

[0184] Results:

[0185] The demographics of the subjects enrolled in the single-dose (EXTEN-A) and repeat-dose study and used here in developing the Phase I PK model are shown in Table 1.

Table 1.

SD, standard deviation; VWF, von Willebrand Factor.

[0186] Determination of Final Model

[0187] In both Phase 1 studies, single- and repeat-dose rFVIIIFc-VWF-XTEN provided high and sustained FVIII activity for up to 7 days post dose (Figure 3). Based on the population PK analysis described above, the final population PK model for rFVIIIFc-VWF-XTEN was a one-compartment model, with population parameter estimates shown in Table 2. The major covariates for FVIII activity identified as body weight on clearance and body weight and hematocrit on central volume (allometric exponents -0.568 and 0.703, respectively; Table 2). VWF concentration was not a covariate, consistent with rFVIIIFc-VWF-XTEN clearance being independent of endogenous VWF.

Table 2. aaPTT, activated partial thromboplastin time; Cl, confidence interval; PK, pharmacokinetic; RSE, relative standard error. [0188] The final population PK model for rFVIIIFc-VWF-XTEN is represented below as [A]:

[0189] Model Abbreviations are: A _central , amount of drug; CL, clearance; CL,, clearance from the individual; CLT _V , the typical value; C _plasma , concentration in the plasma; E _prop , magnitude of SD of proportional residual error; E _add , magnitude of SD of proportional additive error; Hct, hematocrit; nV _central,i , is the post-hoc individual variability for V _central in subject i; η _CL , _ί, post-hoc individual variability for CL in subject i; is the allometric exponent of WT on CL; V _central , volume of the central compartment; V _central ,i, volume of the central compartment in subject, i; V _central , typical value for volume of the central compartment; W, weight of the current point (total SD of RSE); WT, body weight; C _plasma is the model-estimated plasma concentration; e is residual error. [0190] Validation: Goodness of fit diagnostics demonstrate that the model adequately describes the data when model predicted results were compared with observed results. (Figure 4). The model was therefore deemed appropriate for use in rFVIIIFc-VWF-XTEN simulations.

Example 4. Clinical trial simulations using the population pharmacokinetic model of rFVIIIFc- VWF-XTEN

[0191] The PK model for rFVIIIFc-VWF-XTEN described in the previous example was used to simulate or estimate various subject dosing scenarios. Weight-based dosing simulations of FVIII activity (70 kg fixed body weight, simulated 5-100 lU/kg doses) produced results similar to those observed in the Phase 1 repeat dose trial (Figure 5). Steady-state FVIII activity peaks and troughs were also predicted using the model (Figure 6). [0192] Clinical trial simulations were also performed, which included 600 simulated individuals with a body weight distribution from rFVIIIFc-VWF-XTEN clinical trials (Figure 7). At a dose of 50 lU/kg per week, the population PK model predicted mean time above 40% (normal or near-normal FVIII activity levels) and 10% of 3.25 days and 6.90 days, respectively, representing high sustained FVIII activity. Meanwhile, predicted mean time for FVIII activity above 150% was short (0.77 hours). Median (5th to 95th percentile) Ctrough was 9.6% (4.0% to 19.3%).

FVIII, factor VIII; QW; per week. ^a Based on the one-stage (aPTT) clotting assay. ^b Values are mean (SD).

[0193] Clinical trial simulation C _max and C _{trou h}

[0194] The PK model was used to estimate the C _max and C _trough levels of the 600 virtual subjects. At 50 lU/kg and 65 lU/kg dosing, the percentage of virtual subjects with a C _max >150 lU/kg (the upper limit of normal) were 62% and 94%, respectively (FIG. 7A). Higher and at the 65 lU/kg dose is consistent with the predicted dose-proportionality of rFVIIIFc-VWF-XTEN PK (Table 3).

[0195] Percentage of simulated individuals with FVIII above threshold levels

[0196] The PK model was used to estimate the percentage of simulated individuals with FVIII above threshold levels. The 600 virtual subjects underwent clinical trial simulations at doses of 50 lU/kg and 65 lU/kg, and were assessed for FVIII activity above specific thresholds (3 lU/kg, 5 lU/kg, 10 lU/kg, and 15 lU/kg) for the entirety of the dosing regimen. Activity above these thresholds was predicted using a non-compartmental analysis. Results are shown in Table 4.

Table 4.

[0197] Conclusions: The population PK model developed for rFVIIIFc-VWF-XTEN demonstrated suitability for evaluating FVIII activity in simulated representative populations.

Table 5. rFVIIIFc-VWF-XTEN Sequence Information