SUBCUTANEOUS ADMINISTRATION OF FACTOR VIII
20230190881 · 2023-06-22
Inventors
Cpc classification
A61P7/04
HUMAN NECESSITIES
A61K9/0019
HUMAN NECESSITIES
C07K14/755
CHEMISTRY; METALLURGY
C07K2319/31
CHEMISTRY; METALLURGY
C07K2319/70
CHEMISTRY; METALLURGY
International classification
A61K9/00
HUMAN NECESSITIES
A61P7/04
HUMAN NECESSITIES
C07K14/755
CHEMISTRY; METALLURGY
Abstract
The present invention relates to the treatment of hemophilia A, in particular to means and methods for subcutaneous administration of Factor VIII (FVIII) proteins. More specifically, the invention relates to FVIII proteins comprising at least one albumin binding domain, which could be shown to have a high bioavailability after subcutaneous administration, in particular, for use in subcutaneous administration to a subject with hemophilia A. The invention also relates to the use of further agents enhancing the bioavailability of FVIII proteins comprising at least one albumin binding domain after subcutaneous administration of such FVIII proteins, in articular human albumin, hyaluronidase and derivatives thereof. The invention also relates to pharmaceutical compositions, combined administration, combined preparations, packages and kits.
Claims
1. A Factor VIII (FVIII) protein comprising at least one albumin binding domain, wherein the bioavailability of the Factor VIII protein after subcutaneous administration is at least 25% as measured in minipigs, for use in treatment of a subject having hemophilia A, wherein a dose of 1-1000 U/kg bodyweight is administered to the subject subcutaneously.
2. The Factor VIII protein for use of claim 1, wherein the bioavailability of the Factor VIII protein after subcutaneous administration is at least 30%, optionally, 30-60% as measured in minipigs.
3. The Factor VIII protein for use of claim 1, wherein the Factor VIII protein comprises at least two albumin-binding domains.
4. The Factor VIII protein for use of claim 1, wherein the FVIII protein is a single chain protein, wherein the FVIII protein preferably is at least partly B domain deleted.
5. The Factor VIII protein for use of claim 1, wherein the FVIII protein comprises a heavy chain portion and a light chain portion of Factor VIII, and wherein the albumin binding domain(s) is/are C-terminal to the heavy chain portion and/or C-terminal to the light chain portion, wherein, if the protein is a single chain protein, the albumin binding domain(s) is/are between the heavy chain portion and the light chain portion and/or C-terminal to the light chain portion.
6. The Factor VIII protein for use of claim 5, wherein at least one albumin binding domain is C-terminal to the heavy chain portion and, if the protein is a single chain protein, between the heavy chain portion and the light chain portion, and at least one albumin binding domain is C-terminal to the light chain portion, wherein, preferably, two albumin binding domains are C-terminal to the heavy chain portion and, if the protein is a single chain protein, between the heavy chain portion and the light chain portion, and two albumin binding domains are C-terminal to the light chain portion.
7. The Factor VIII protein for use of claim 1, wherein albumin binding domains are separated from the heavy chain portion and/or the light chain portion and/or other albumin-binding domains by a linker selected from the group comprising a) a linker comprising a thrombin-cleavable linker that optionally has the sequence of SEQ ID NO: 39, and b) a linker comprising a glycine-serine linker that optionally has the sequence of SEQ ID NO: 40 or SEQ ID NO: 41, and c) a linker comprising a thrombin-cleavable linker flanked on each side by a glycine-serine linker that optionally has the sequence of SEQ ID NO: 42 or SEQ ID NO: 43.
8. The Factor VIII protein for use of claim 1, wherein the albumin binding domain comprises a sequence according to SEQ ID NO: 44, wherein, preferably, the sequence is SEQ ID NO: 46.
9. The Factor VIII protein for use of claim 1, wherein the FVIII protein optionally is a single chain protein, wherein said protein comprises a heavy chain portion having at least 90% sequence identity to aa20-aa768 of SEQ ID NO: 16 and a light chain portion having at least 90% sequence identity to aa769-aa1445 of SEQ ID NO: 16.
10. The Factor VIII protein for use of claim 1, wherein the FVIII protein is a single chain protein comprising at least two albumin binding domains between the heavy chain portion and the light chain portion and at least two albumin binding domain C-terminal to the light chain portion, wherein the protein has at least 80% sequence identity to any of SEQ ID NO: 48, 49 or 51, wherein the protein preferably has at least 80% sequence identity to SEQ ID NO: 48, wherein the protein optionally comprises SEQ ID NO: 48.
11. The Factor VIII protein for use of claim 1, wherein the protein has at least 90% sequence identity to a Factor VIII protein of SEQ ID NO: 63, wherein only the A1, a1, A2, a2, a3, A3, C1 and C2 domains are considered for determination of sequence identity, wherein the protein optionally has SEQ ID NO: 114.
12. A pharmaceutical composition comprising the FVIII protein for use of claim 1, wherein the composition preferably is for human administration and optionally comprises a pharmaceutically acceptable excipient.
13. The pharmaceutical composition for use of claim 12, further comprising human albumin, wherein, preferably, the concentration of human albumin is 0.1-15% (w/v).
14. The pharmaceutical composition for use of claim 12, further comprising a hyaluronidase, preferably, a human hyaluronidase, wherein the dose of the hyaluronidase per injection optionally is 50-300 U.
15. A kit comprising a hyaluronidase, preferably, a human hyaluronidase, and a Factor VIII protein comprising at least one albumin binding domain(s), wherein, optionally, the Factor VIII protein comprises a heavy chain portion and a light chain portion of Factor VIII and the albumin binding domain(s) is/are C-terminal to the heavy chain portion and/or C-terminal to the light chain portion, wherein, if the protein is a single chain protein, the albumin binding domain(s) is/are between the heavy chain portion and the light chain portion and/or C-terminal to the light chain portion.
Description
FIGURE LEGENDS
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EXAMPLES
[0366] 1. Evaluation of Factor VIII Proteins Comprising Albumin-Binding Domains for Subcutaneous Treatment of Hemophilia A.
[0367] Material and Methods
[0368] Preparation of Constructs
[0369] Experiments were performed to find and develop a suitable backbone for integration of the albumin-binding domains. The experiments were done on the basis of a B-domain deleted version of FVIII and single chain variants of FVIII. The basic double chain construct was a codon-optimized sequence of ReFacto AF® (Pfizer), wherein for simplifying cloning, 6 restriction sites were added through silent mutations, but some of these restriction sites were again excluded due to codon-optimization. The basic double chain sequence is 6rs-REF (SEQ ID NO: 28). The basic single chain construct used was VO (SEQ ID NO: 16, EP19173440).
[0370] Firstly, the ABD protein sequence (Affibody AB, Solna, Sweden) was taken as a basis for design of the DNA sequence. If not mentioned otherwise, the ABD2 sequence was used. Moreover, codon optimized linkers were developed, which are partly cleavable by thrombin. If not otherwise stated, the glycine-serine linker was G1 and the thrombin-cleavable linker was L. Table 1 below demonstrates structures of fusion proteins with albumin-binding domains (ABD) for single chain molecules.
[0371] For the constructs encoding the FVIII of the invention and comparative constructs also analysed in this context, either the complete FVIII sequence or DNA regions carrying approx. 700-1200 bp from the FVIII a2 domain to the A3 domain were synthesized. The synthesized DNA was codon-optimized for the total target gene. The a2 to A3 DNA fragments were 5′ terminally flanked by an EcoRV restrictions site, and 3′ terminally flanked by an EcoRI restriction site, and these restriction sites were also present in the basic FVIII sequence used. For C-terminal fusion to the light chain, DNA fragments carrying approx. 1500-2100 bp were synthesized also in a codon-optimized form. Such DNA fragments were 5′ terminally flanked by an EcoRI restrictions site within the A3 domain, and 3′ terminally flanked by a NotI restriction site. Restriction of the DNA inserts and the FVIII backbone plasmid allowed for targeted ligation and generation of FVIII single chain plasmids. Completely synthesized FVIII DNA was 5′ terminally flanked by a HindIII restrictions site, and 3′ terminally flanked by a NotI restriction site.
[0372] By transformation of E. coli K12 with said plasmids, expansion of transformed bacteria under ampicillin selection and plasmid preparation, large amounts of the plasmids could be prepared. Genetic engineering work was carried out by Thermo Fisher Scientific after design with VectorNTI Software (Thermo Fisher Scientific, Massachusetts, USA).
[0373] Cultivation of CAP-T Cells
[0374] For analyzing the candidates for new recombinant FVIII molecules, the constructs, integrated in expression vectors, were transiently and stably expressed in human cell lines. The preferred cell lines are Hek293 and CAP cells, both of which originate from human amniocytes. Because of higher yields of active FVIII molecules CAP cells, in particular, CAP-T cells were chosen as the preferred expression system for transient transfection and CAP-Go cells for stable expression.
[0375] Transient transfection was performed with nucleofection programs. The supernatants were screened for FVIII activity and antigen. Purification of the recombinant proteins from CAP cells was done, including FVIII affinity chromatography.
[0376] In detail, CAP-T cells (Cevec Pharmaceuticals, Köln, Germany) were cultured in PEM medium supplemented with 4 mM GlutaMAX (Thermo Fisher Scientific, 35050038) and 5 μg/mL blasticidin (Thermo Fisher Scientific, R21001; complete PEM medium). In order to thaw the cells, the required amount of frozen vials were transferred to a 37° C. water bath. After thawing, each vial was transferred to 10 ml of chilled, complete PEM medium. The cell suspension was centrifuged at 150×g for 5 minutes. During this washing step the dimethyl sulfoxide (DMSO) used for cryopreservation was removed. The pellet was resuspended in 15 mL warm, complete PEM medium and transferred to a 125 mL shaker flask. The cells were incubated at 37° C. in a humidified incubator with an atmosphere containing 5% CO.sub.2. The flasks were set on a shaking platform, rotating at 185 rpm with an orbit of 50 mm.
[0377] Subculturing of the cells was performed every 3 to 4 days. The fresh culture was set to 0.5×10.sup.6 cells/ml by transferring the required amount of cultured cell suspension to a new flask and adding complete PEM medium. In the case that the transferred cell suspension would exceed 20% of the total volume, the suspension was centrifuged at 150×g for 5 minutes and the pellet was resuspended in fresh complete PEM medium. The volume of cell suspension per shaking flask was 20% of the total flask volume.
[0378] A minimum of three subcultures were performed after thawing before transfection experiments were performed.
[0379] Protein Expression in CAP-T Cells by Transient Transfection
[0380] The CAP-T cells were transfected using the 4D-Nucleofector™ (Lonza, Basel, Switzerland). For each transfection 10×10.sup.6 CAP-T cells were centrifuged at 150×g for 5 minutes in 15 mL conical tubes. The cells were resuspended in 95 μL supplemented SE Buffer, taking into account the volume of the pellet and the volume of the plasmid solution. Afterwards, 5 μg of the respective plasmid were added to the cell suspension followed by gentle mixing. The solution was transferred to 100 μL Nucleocuvettes. The used transfection program was ED-100. After the transfection, the cells from one Nucleocuvette were transferred to 125 ml shaker flasks, containing 12.5 mL complete PEM medium. The cells were cultivated for 4 days as described above. At day 4 the cells were harvested by centrifugation at 150×g for 5 minutes. Larger protein amounts could be produced by combining 12.5 mL approaches as described above.
[0381] Supernatants were screened for FVIII activity and antigen directly after harvest.
[0382] The recombinant Factor VIII protein was further analyzed. FVIII activity was measured by chromogenic activity assay and clotting activity FSL assay. The antigen was estimated by FVIII antigen ELISA. As a further assay for biological activity, the cleavage of the recombinant proteins by thrombin was analyzed. Moreover, chain distribution and appearance was tested by Western Blots. Further, vWF-binding and albumin binding were tested.
[0383] Protein Expression in CAP go Cells by Stable Cell Pools
[0384] In order to produce large material amounts to conduct minipig studies, stable CAP-Go pools expressing either AD2CD2_SC or AD2CD2-19M_SC were generated at Cevec Pharmaceuticals GmbH (Cologne, Germany). Therefore, the FVIII coding sequences were cloned into CEVEC's pStbl-bsd-MCS(−) plasmid using SgrD1 and Not1 restriction sites. The fragment was subsequently separated by agarose gel electrophoresis, purified by gel filtration and cloned into the pStbl-bsd-MCS(−), which was previously cut with SgrD1 and Not1 and treated with Calf-Intestine-Phosphatase (CIP). Inserts and vector were ligated using T4-DNA ligase and transformed into chemically competent E. coli cells (XL2-Blue). The plasmid DNA was purified using the Maxi Kit from Machery-Nagel. The whole cloning processes as well as the plasmid purifications were performed in a TSE-free production process.
[0385] Prior to nucleofection, circular plasmids were linearized with Scal. Therefore, 20-40 μg plasmid DNA were incubated 5-8 h with 50-200 U of the respective enzyme at 37° C. Subsequently, the DNA was purified by phenol-chloroform-isoamyl alcohol extraction and phenol was washed away with chloroform-isoamyl alcohol. To purify the DNA by ethanol precipitation, the DNA solution was supplemented with 1/10 volume of 3M NaOAc, pH=5.2 and 2 volumes of ethanol and incubated overnight at −20° C. The DNA precipitate was pelleted by centrifugation (30 min, 13 000 rpm, 4° C.), washed with 70% ethanol, centrifuged again, air-dried, and resuspended in TE buffer. The quality of the linearized DNA was assured by a DNA agarose gel analysis.
[0386] For nucleofection, CAP-Go cells were counted by Cedex XS (Roche Applied Science, Innovatis) and viable cell density and viability were determined. For each nucleofection reaction 1×10.sup.7 cells were harvested by centrifugation (150×g for 5 min). The cells were resuspended in 100 μL complete nucleofector solution V (Lonza) and mixed with 5 μg linearized plasmid of the respective construct. The DNA/cell suspension was transferred into a cuvette and the nucleofection was performed using the X.sub.001 program on the Nucleofector II (Lonza). After the pulse, cells were recovered by adding 500 μL prewarmed complete PEM medium (=supplemented with 4 mM L-alanyl-L-glutamine) to the cuvette and gently transferred into 11.5 mL complete PEM medium in a 125 mL shaking flask. The cuvette was washed once with 500 μL fresh medium to recover residual cells.
[0387] 72 h post-nucleofection the cell number and cell viability of the transfected cells were determined. The cells were harvested by centrifugation and resuspended in 20 mL complete PEM medium containing 5 μg/mL basticidin as selection marker. The cells were cultured at 37° C., 5% CO.sub.2 at 185 rpm with 5 cm amplitude in a Kühner shaking incubator. As soon as cells recovered from selection and could be expanded, cells from the stable pools were cryopreserved.
[0388] For batch production, the culture was inoculated at a viable cell density of 1×10.sup.6 cells/mL in either 800 mL complete PEM medium in a 2 L shake flask or, for larger productions runs, 4×2500 mL complete PEM medium each in a 5 L shake flask. The cells were incubated at 185 rpm (5 cm orbit), 37° C., 5% CO2 in a Kühner shaking incubator for 4 days. The cell supernatants containing FVIII were harvested by centrifugation and purified by affinity chromatography as described elsewhere in this document.
[0389] Protein Purification
[0390] FVIII-6rs and FVIII-19M was produced in CAP-T cells in up to 800 mL scales. Purification occurred directly from the cell culture supernatant by FPLC. The first step was either a tangential flow filtration or an ion exchange chromatography, using the strong anion exchange columns HiTrap Capto Q (GE Healthcare Europe GmbH, Freiburg). In this step the sample was concentrated, host cell proteins were lost and the buffer was exchanged. The fractions containing the eluted protein were determined according to the chromatogram. The second step was an affinity chromatography, using a column packed with the commercially available VIIISelect resin (GE Healthcare Europe GmbH, Freiburg). The fractions containing the eluted FVIII were determined according to the chromatogram. The last step was a buffer exchange to FVIII Formulation Buffer by size exclusion chromatography, using the HiTrap Desalting columns (GE Healthcare Europe GmbH, Freiburg). The fractions containing FVIII were determined according to a high UV peak and a stable conductivity peak in the chromatogram. After purification, the FVIII products were concentrated via spin columns (Merck Millipore, Darmstadt) with a molecular weight cut-off of 10 kDa. All columns were run under the conditions specified by the manufacturer.
[0391] FVIII Activity—Chromogenic Activity Assay
[0392] The activity of FVIII was determined by a chromogenic assay. In this two-step assay, FIXa and FVIIIa activate FX in the first step. In the second step, the activated FX hydrolyses a chromogenic substrate, resulting in a color change, which can be measured at 405 nm. Due to the fact that calcium and phospholipids are present in optimal amounts and an excess of FIXa and FX is available, the activation rate of FX is only dependent on the amount of active FVIII in the sample.
[0393] The reagents for this chromogenic FVIII activity assay were taken from the Coatest® SP FVIII Kit. The kit contained phospholipids, calcium chloride (CaCl.sub.2), trace amounts of thrombin, the substrate S-2765, a mixture of FIXa and FX and the thrombin inhibitor I-2581. The inhibitor was added in order to prevent hydrolysis of the substrate by thrombin, which was built during the reaction. All dilutions were performed in distilled water or Tris-BSA (TBSA) Buffer, containing 25 mM Tris, 150 mM sodium chloride (NaCl) and 1% Bovine serum albumin (BSA), set to pH 7.4. Each sample was diluted at least 1:2 with FVIII-depleted plasma. Further dilutions were performed using the TBSA Buffer.
[0394] The assay was performed using the BCS XP (Siemens Healthcare, Erlangen, Germany), a fully automated hemostasis analyzer. All reagents including water, TBSA Buffer and the samples were inserted into the analyzer. For each sample the analyzer mixed 34 μL calcium chloride, 20 μL TBSA Buffer, 10 μL sample, 40 μL water, 11 μL phospholipids and 56 μL FIXa-FX-mixture. This mixture was incubated for 300 seconds. Afterwards, 50 μL of S-2765+I-2581 were added to the reaction. Upon addition of the substrate, the absorption at 405 nm was measured for 200 seconds.
[0395] In order to calculate the amount of active FVIII, the software of the analyzer evaluated the slope of the measured kinetic between 30 seconds and 190 seconds after starting the reaction. This result was correlated to a calibration curve, generated with a biological reference preparation (BRP) of FVIII. The activity of the BRP is indicated in IU/mL. However, IU/mL can be assumed equivalent to U/mL. The results were indicated as “% of normal”. These results were converted to U/mL, as 100% of normal FVIII activity are equivalent to 1 U FVIII activity per mL.
[0396] Clotting Activity FSL
[0397] In addition to the two-stage chromogenic assay (see above), a one-stage clotting assay was also performed in order to determine the amount of active FVIII. During this assay, FVIII-depleted plasma, CaCl.sub.2, the activator Actin FSL and the FVIII-containing sample are mixed in one step. The activator leads to the generation of FXIa, which activates FIX. FVIIIa, FIXa and FX built the tenase complex and FX becomes activated. Further activation of prothrombin and fibrinogen finally leads to the formation of a fibrin clot. The time needed to form the clot, the activated partial thromboplastin time (aPTT), is measured. The aPTT varies, depending on the amount of FVIII.
[0398] The clotting assay was performed using the BCS XP. TBSA Buffer, FVIII-depleted plasma, Actin FSL, CaCl.sub.2) and the sample were inserted into the analyzer. The sample was diluted at least 1:2 with FVIII-depleted plasma. Further dilutions were performed using the TBSA Buffer. For each sample the analyzer mixed 45 μL TBSA Buffer, 5 μL sample, 50 μL FVIII-depleted plasma and 50 μL Actin FSL. The reaction was started by the addition of 50 μL CaCl.sub.2. The analyzer measured the time needed for clot formation.
[0399] In order to calculate the amount of active FVIII, the software of the analyzer evaluated a baseline extinction at 405 nm at the beginning of the reaction. All of the following extinction values, within a time of 200 seconds, were analysed regarding their difference to the baseline extinction. The first time point exceeding a defined threshold was determined as the clotting time. This result was correlated to a calibration curve, generated with a BRP of FVIII.
[0400] Thrombin Generation Assay (TGA)
[0401] In the Thrombin Generation Assay (TGA), the amount of generated thrombin is measured. The clotting cascade takes place, started via the extrinsic pathway by tissue factor. The thrombin finally generated cleaves a fluorogenic substrate which can be measured at 460 nm. The assay was performed with FVIII diluted in FVIII-deficient plasma. FVIII concentrations up to 0.25 U/ml were analyzed. TGA reagent C low and TGA substrate, both commercially available by Technoclone (Vienna), were added to each sample well referring to the manufacturer's protocol. TGA reagent low consist of low concentrations of phospholipid micelles containing recombinant human tissue factor, in order to initiate the clotting cascade. The substrate is the fluorogenic substrate finally cleaved by the generated thrombin. The reaction was performed at 37° C. in a plate reader and the development of the fluorogenic substrate was measured for two hours. In addition to the samples, a calibration curve was measured using the TGA Cal Set, also available by Technoclone (Vienna). The amount of generated thrombin was calculated based on the calibration curve. Additionally, the area under the curve and the time to maximum thrombin generation was calculated based on the first deviation of the generated curve.
[0402] FVIII Antigen ELISA
[0403] The amount of human FVIII antigen was determined using the Asserachrom® VIII:Ag ELISA (Diagnostica Stago, Asnières sur Seine Cedex, France). In this sandwich ELISA, the applied FVIII is bound by mouse monoclonal anti-human FVIII F(ab′).sub.2 fragments, which are coated to the plate by the manufacturer. The detection of the bound FVIII occurs via mouse monoclonal anti-human FVIII antibodies, which are coupled to a peroxidase. In the case that FVIII is present, the peroxidase-coupled antibody binds to FVIII and can be detected by the addition of a tetramethylbenzidine (TMB) solution. TMB turns from a clear to a blue-green solution upon reaction with peroxidase. After a short time, this reaction is stopped by the addition of sulfuric acid (H.sub.2SO.sub.4), which turns the solution yellow. The amount of bound FVIII correlates with the intensity of the yellow color, which can be measured at 450 nm. The final amounts of FVIII are calculated using a calibration curve generated by the measurement of at least five serial dilutions of a calibrator with a known antigen concentration.
[0404] The supplied calibrator and control were reconstituted with 500 μL of distilled water, 30 minutes before starting the ELISA. After this incubation time, the calibrator was diluted 1:10 in the supplied phosphate buffer. This represented the starting concentration. The calibrator was further serially diluted 1:2 up to a dilution of 1:64. As the concentration of the calibrator contained approximately 1 U/mL FVIII, depending on the batch, the starting concentration was equivalent to 0.1 U/mL FVIII whereas the last dilution contained approximately 0.0016 U/mL FVIII. The control was diluted 1:10 and 1:20 with the phosphate buffer. All samples were diluted with the phosphate buffer, depending on their previously determined activity (see above) with the aim to be in the middle of the calibration curve. After the dilution of FVIII samples, control and calibrator, 200 μL of each solution were applied per well in duplicates. In addition to that, two wells were filled with 200 μL of phosphate buffer as a blank control. The plate was incubated for 2 hours at room temperature covered with a film. During this time, the peroxidase-coupled anti-human FVIII antibodies were reconstituted with 8 ml phosphate buffer and incubated 30 minutes at room temperature. After the antigen immobilization, the wells were washed five times with the supplied washing solution, which was previously diluted 1:20 with distilled water. Immediately after the washing, 200 μL of the peroxidase-coupled anti-human FVIII antibodies were added to each well and incubated for 2 hours at room temperature covered by a film. Afterwards, the plate was washed five times as before. In order to reveal the amount of bound FVIII, 200 μL of TMB solution were added to each well and incubated for exactly 5 minutes at room temperature. This reaction was stopped by the addition of 50 μL 1 M H.sub.2SO.sub.4 to each well. After an incubation time of 15 minutes at room temperature, the absorbance of each well was measured at 450 nm using the POLARstar Omega plate reader (BMG LABTECH, Ortenberg, Germany).
[0405] The results of the ELISA were calculated using the MARS software (BMG Labtech). In a first step, all wells were blank corrected and the mean of the duplicates was calculated. Afterwards, a 4-parameter fit was applied, in order to calculate the concentrations from the calibration curve. According to this calibration curve the amount of FVIII antigen in each well was determined. In the last step, the values were corrected by the dilution factor, resulting in the FVIII antigen amount of each sample.
[0406] Adapted FVIII Antigen ELISA for Measuring Göttingen Minipig Samples
[0407] The supplied calibrator and control of the Asserachrom® VIII:Ag ELISA (Diagnostica Stago, Asnières sur Seine Cedex, France, Cat. No. 00280) were reconstituted with 500 μL of distilled water, 30 minutes before starting the ELISA. After this incubation time, the calibrator was diluted 1:5 (i.e. 1+4) in Göttingen minipig plasma resulting in the calibrator stock solution. Further, this calibrator stock solution was 6-times serially diluted 1:2 with minipig plasma. The calibrator stock solution as well as each serial dilution step was 1:2 diluted within the supplied phosphate buffer resulting in final calibrator concentrations of 96, 48, 24, 12, 6, 3, and 1.5 mU/mL. All samples and assay controls were diluted with minipig plasma, except for a last dilution step, which was performed 1:2 in the phosphate buffer. All dilutions aimed for the middle of the calibration curve. After the dilution of FVIII samples, controls, and calibrator, 100 μL of each solution were applied per well in duplicates (volume reduced by 50% in comparison to the manual). In addition, two wells were filled with 100 μL of phosphate buffer as a blank control. The plate was incubated for 2 hours at room temperature covered with a film. During this time, the peroxidase-coupled anti-human FVIII antibodies were reconstituted with 8 mL phosphate buffer and incubated 30 minutes at room temperature. After the antigen immobilization, the wells were washed five times with the supplied washing solution, which was previously diluted 1:20 with distilled water. Immediately after the washing, 200 μL of the peroxidase-coupled anti-human FVIII antibodies were added to each well and incubated for 2 hours at room temperature covered by a film. Afterwards, the plate was washed five times as before. In order to reveal the amount of bound FVIII, 200 μL of TMB solution were added to each well and incubated for exactly 5 minutes at room temperature. This reaction was stopped by the addition of 50 μL 1 M H.sub.2SO.sub.4 to each well. After an incubation time of 15 minutes at room temperature, the absorbance of each well was measured at 450 nm using the POLARstar Omega plate reader (BMG LABTECH, Ortenberg, Germany).
[0408] The results of the ELISA were calculated using the MARS software (BMG Labtech). In a first step, all wells were blank corrected and the mean of the duplicates was calculated. Afterwards, a 4-parameter fit was applied, in order to calculate the concentrations from the calibration curve. According to this calibration curve the amount of human FVIII antigen in each well was determined and the values were corrected by the dilution factor, resulting in the FVIII antigen amount of each sample. Since AD2CD2_SC and AD2CD2-19M_SC detection was reduced in the presence of albumin, a correction factor was determined by spiking the application solution into minipigs plasma for at least two or three different concentrations in the range of what was expected after i.v. administration in the plasma of model animals, e.g. 0.5 to 20 U/mL, herein 9.23 and 4.62 U/mL. A pre-correction factor for each test item group and each spiking concentration was calculated (=100/recovery in %). Correction factors for each test item group were calculated as mean of pre-correction factors of all spiking concentrations. The resulting correction factors were applied to calculate specific concentrations used for further pharmacokinetic evaluation.
[0409] Albumin Binding Capacity Assay
[0410] 20% human serum albumin (HSA) was diluted 1:4000 in PBS. 96-well ELISA plates were filled with 100 μL/well with diluted HSA solution and coated during a 2 h incubation at 37° C. and 400 rpm on a thermoshaker. ELISA plates were washed 3-times with 300 μL/well washing buffer. Standard control and FVIII samples either with or without Albumin pre-incubation were diluted with Tris/NaCl pH 7.4 to a concentration of 0.5 U/mL chromogenic activity and 100 μL/well were added as 7-step 1:2 serial dilution. Incubation was performed for 1 h at 37° C. covered on a thermoshaker. In the meantime, FIXa and FX were resolved together in 10 mL aqua dest., substrate (S-2765 and I-2581) was solved in 12 mL aqua dest. After FVIII incubation, plates were washed again 3 times with 300 μL/well washing buffer. Phospholipides and the FIXa/FX solution were mixed 1:5 and subsequently 50 μL/well of this solution were added and incubated for 5 min at 37° C. Without any washing step 25 μL CaCl.sub.2 was added to each well, followed by 5 min incubation at 37° C. Finally, 50 μL/well substrate were added and detection of activated FX-mediated substrate turnover was performed at 405 nm for 25 cycles followed by end point measurement using an ELISA reader.
[0411] vWF Binding Capacity Assay
[0412] 1 U/mL of each FVIII molecule was either pre-incubated or not with 40 mg/mL albumin for 30 min at RT to promote ABD-albumin binding and an assay for determining the vWF binding capacity was performed as follows:
[0413] Plasma purified vWF (Biotest AG) was diluted with 0.9% NaCl solution to a concentration of 0.1 U/mL. Coating onto 96-well ELISA plates was done by transferring 100 μL of this solution to each well followed by an 2 h incubation at 37° C. and 400 rpm. The wells were washed 3 times with 300 μL of washing buffer (8 mM sodium phosphate, 2 mM potassium phosphate, 0.14M NaCl, 10 mM KCl, 0.05% Tween-20, pH 7.4). FVIII standard (commercial rFVIII without vWF) and samples were pre-diluted with dilution buffer (25 mM Tris, 150 mM NaCl, pH 7.4) to a concentration of 0.25 U/mL according to chromogenic activity and transferred as a 7-step, serial 1:2 dilution into each plate well (100 μL/well). Incubation was carried out for 1 h at 37° C. and 400 rpm. In the meantime, FIXa and FX were resolved together in 10 mL aqua dest., substrate (S-2765 and 1-2581) was solved in 12 mL aqua dest. After FVIII incubation, plates were washed again 3 times with 300 μL/well washing buffer. Phospholipides and the FIXa/FX solution were mixed 1:5 and subsequently 50 μL/well of this solution were added and incubated for 5 min at 37° C. Without any washing step 25 μL CaCl.sub.2 was added to each well, followed by 5 min incubation at 37° C. Finally, 50 μL/well substrate were added and detection of activated FX-mediated substrate turnover was performed at 405 nm for 25 cycles followed by end point measurement using an ELISA reader.
[0414] Western Blot
[0415] Reducing Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
[0416] Cell supernatants, cell lysates or purified material of FVIII variants were appropriately diluted with 1×NuPAGE LDS Sample Buffer (4×, Thermo Fisher Scientific, NP0007) and further diluted 1:2 with reducing sample buffer. Reducing sample buffer was produced by combining 2.5 parts of NuPAGE LDS Sample Buffer with 1 part of NuPAGE Sample Reducing Agent (10×, Thermo Fisher Scientific, NP0004). 20 μL of each sample were mixed with 20 μL of reducing sample buffer in a 1.5 mL vial and heated for 10 min at 70° C. using a thermoshaker (Eppendorf). A NuPAGE 4-12% Bis-Tris Protein Gel (Thermo Fisher Scientific) was inserted into the XCell SureLock Mini-Cell Electrophoresis System (Thermo Fisher Scientific) and inner and outer chambers were filled with 1×NuPAGE MOPS SDS Running buffer (Thermo Fisher Scientific, NP0001). 500 μL of NuPAGE Antioxidant (Thermo Fisher Scientific) was added to the inner chamber. 10 μL of the each prepared sample and 4 μL of Precision Plus Protein All Blue Standard (Bio-Rad, 161-0373) diluted 1/10 in 1×LDS Sample Buffer were loaded onto the gel. The sample separation was achieved by running the gel at a constant voltage of 200 V for 50-60 min.
[0417] Non-Reducing SDS-PAGE
[0418] 20 μL of cell supernatants, cell lysates or purified material of FVIII variants were, either pre-diluted or not, appropriately diluted with 10 μL of NuPAGE LDS Sample Buffer (4×, Thermo Fisher Scientific, NP0007) and 10 μL aqua dest. Samples were heated for 10 min at 70° C. using a thermoshaker (Eppendorf). A NuPAGE™ 3-8% Tris-Acetate Protein Gel (Thermo Fisher Scientific) was inserted into the XCell SureLock Mini-Cell Electrophoresis System (Thermo Fisher Scientific) and inner and outer chambers were filled with 1×NuPAGE™ Tris-Acetate SDS Running Buffer (Thermo Fisher Scientific, LA0041). 10 μL of the each prepared sample and 4 μL of Precision Plus Protein All Blue Standard (Bio-Rad, 161-0373) diluted 1/10 in 1×LDS Sample Buffer were loaded onto the gel. The sample separation was achieved by running the gel at a constant voltage of 150 V for 55-70 min.
[0419] Western Blotting and Detection
[0420] To investigate the separated proteins by immunofluorescence detection, they were transferred onto an Odyssey nitrocellulose membrane (Li-Cor) or an Amersham Hybond Low Fluorescence 0.2 μm polyvinylidene fluoride (PVDF) membrane (GE Healthcare Life Sciences) by using the XCell II blot module (Thermo Fisher Scientific) for semi-wet protein transfer. The PVDF membrane was activated in methanol and then applied to the SDS gel, whereas the nitrocellulose membrane was directly applied to the SDS gel. The system was filled with NuPAGE transfer buffer (20×, Thermo Fisher Scientfic) according to the manufacturer's instructions. Protein blotting was performed for 1 h at 30 V. After protein transfer, the membrane was blocked over night at 4° C. in Odyssey blocking buffer (Li-Cor). Afterwards the membrane was incubated for 1 h at room temperature simultaneously with either a rabbit anti-coagulation factor VIII monoclonal antibody (Sino Biological, 13909-R226, 1:1000) and a mouse anti-human factor VIII monoclonal antibody (Merck, MAB038, 1:2500) or with 0.0004 μg/μL sheep anti-human factor VIII:C polyclonal antibody (Cedarlane, CL20035AP, 1.5000), each diluted in Odyssey Blocking buffer containing 0.05% Tween 20. After incubation, the membrane was washed 4-times for 5 min in 0.1% PBST. For detection of FVIII heavy and light chain the membrane was incubated with 0.067 μg/mL IRDye 800CW donkey anti-mouse (Li-Cor, 926-32212, 1:15000) and 0.067 μg/mL IRDye 680RD donkey anti-rabbit (Li-Cor, 926-68073, 1:15000) diluted in Odyssey blocking buffer containing 0.05% Tween 20 for 1 h at room temperature. Alternatively, the CF680 donkey anti-sheep IgG (H&L) antibody (Biotium, 20062-1) was used in a 1:5000 dilution in Odyssey blocking buffer for binding the respective primary antibody. Finally, the membrane was washed 4 times for 5 min in 0.1% PBST, 2 times for 5 min in PBS and rinsed in water. The membrane was visualized using the Licor Odyssey Imager.
[0421] Pharmacodynamic Studies
[0422] Coagulation factors were administered by a single intravenous tail vein injection into female haemophilia A mice with doses of up to 200 U/kg body weight or respective amounts of a control solution. After either 0.5, 4 or 20 h post dosing, a tail vein transection bleeding assay was performed as follows: The animals were anaesthetized with 5% isoflurane in 30% O2 and 70% N2O, and immediately placed in prone position on a heating pad at +37° C. Tail vein transection was performed as described by Johansen et al., 2016. Haemophilia 22(4):625-631.
[0423] Bleeding was monitored for 60 min and bleeding time was determined using a stop clock. Primary bleeding time was noted until first bleeding cessation. After the primary bleeding, the tail was put into a new centrifuge tube filled with pre-warmed saline. If the mouse was not bleeding at 15, 30 and 45 min post injury, the tail was lifted out of the saline and the wound was challenged by gently wiping it twice with a saline wetted gauze swab in the distal direction. Immediately after the challenge, the tail was re-submerged into the saline. The cumulative bleeding time of all following bleeds constitute the secondary bleeding time. The total bleeding time is defined as the sum of the primary and all secondary bleeding times.
[0424] Following the determination of bleeding time, the tubes were centrifuged at 4140 g at room temperature for 3 minutes. Apart from 1 mL, the supernatant was removed. The cell pellet was resuspended and hemoglobin content was determined by using a method similar to that described by Elm et al. (2012).
Results and Discussion
[0425] Generation and prescreening of several different FVIII-ABD fusion proteins covering FVIII double chain and single chain constructs was very promising in initial experiments and similar in both single chain and double chain backbones.
[0426] The formation of single chain FVIII molecule was increased compared to double chain forms when ABD was fused in between heavy chain and light chain (data not shown).
[0427] The FVIII proteins further developed and produced shown herein are listed in Table 1.
TABLE-US-00002 TABLE 1 Structure of exemplary variants of FVIII with ABD fusion. A = FVIII A1 + a1 + A2 + a2 + truncated B domain, C = FVIII a3 (optionally truncated) + A3 + C1 + C2 domains, L = Thrombin cleavable linker, G = flexible glycine-serine linker 1 (G1), D = ABD2, Construct name Structure AD2CD2_SC ALDGLGDLCLDGLGD_SC ADLCLD_SC ADLCLD_SC AbD2CD2_SC ALDGLGDLCLDGLGD_SC + Y1699F + Y1683F AD2CD_SC ALDGLGDLCLD_SC ACLD_SC ACLD_SC ADLC_SC ADLC_SC AD2C_SC AD2C_SC ACLDGLGD_SC ACLDGLGD_SC AD2CD2woLG_SC ADDCDD_SC AD2CD2woL_SC AGDGDGCGDGD_SC ACD4woLG_SC ACDDDD_SC ACL(GD)4_SC ACLGDGDGDGD_SC
[0428] Six single chain FVIII-ABD fusion molecules were generated in silico and respective DNA constructs were tested for their expression in either HEK293 or CAP-T cells (cf. Table 2). As all of those FVIII-ABD variants were expressed, secreted and functional, based on results of the chromogenic FVIII activity measurement, all molecules were produced in midi-scale CAP-T cell culture and successfully purified in larger amounts as needed for further characterizations and PK (pharmacokinetic) analysis.
TABLE-US-00003 TABLE 2 FVIII-ABD fusion proteins analyzed in supernatants of transfected HEK293 or CAP-T cells. Specific Chromo- chromo- Expres- genic Clotting genic sion activity activity FVIII:Ag activity Cell Construct [U/mL] [U/mL] [U/mL] [%] line ADLC_SC 0.98 0.9 0.47 209 HEK293 ACLD_SC 1.26 0.79 0.72 175 HEK293 ADLCLD_SC 1 0.45 n/a n/a HEK293 AD2CD2_SC 1.45 0.35 0.71 204 CAP-T AD2CD_SC 1.3 0.5 0.48 271 CAP-T AD2C_SC 2.0 1.0 n/a n/a CAP-T ADLC_SC 0.9 n/a n/a n/a CAP-T ADLCLD_SC 1.9 n/a n/a n/a CAP-T (n/a: not available)
[0429] All six purified FVIII-ABD fusion variants were extensively characterized by several methods including determination of FVIII antigen and chromogenic activity, Actin FSL clotting, heavy and light chain detection by western blotting (WB), thrombin-cleavage analysis and binding to vWF and albumin. Table 3 gives an overview of produced FVIII-ABD variants in terms of chromogenic and clotting activity as well as antigen levels in the final solutions. Measurement of these values indicated that FVIII-ABD fusion proteins are still capable of their biological function: Bridging factor IXa and factor X leading thereby to the activation of the latter one. Comparison of the specific chromogenic activity (chromogenic activity/antigen*100) demonstrates that ADLC_SC and ReFacto AF® are similar (109% vs 104%). However, the specific chromogenic activities of all other FVIII-ABDs are much better, ranging from 130% to 206%.
[0430] Interestingly, the results indicate that increasing numbers of ABD motifs within one FVIII molecule decrease the clotting activity and also the capability of vWF binding. The decrease in clotting activity may be caused by the setup of the assay, which is strictly time-dependent. This may not mirror in vivo clotting activity.
TABLE-US-00004 TABLE 3 Measurements of chromogenic FVIII activity, FSL clotting activity, and antigen levels. The indicated specific activity was calculated by the ratio of chromogenic activity and antigen. chromogenic FSL Specific FVIII-ABD activity clotting Antigen activity variant [U/ml] [U/ml] [U/ml] [%] ADLC_SC 147 101 135 109 ACLD_SC 150 100 101 149 ADLCLD_SC 128 80 62 206 AD2CD_SC 120 61 92 130 AD2CD2_SC 164 42 96 171 AD2C_SC 112 72 78 144 ReFacto AF ® 253 242 243 104 (Control)
[0431] Western blot was used to observe the heavy and light chain patterns of the different FVIII-ABD fusion molecules in comparison to ReFacto AF® (data not shown). The analysis demonstrated that, in contrast to ReFacto AF®, the FVIII-ABD variants were mostly expressed as single chain molecules.
[0432] Activation of FVIII-ABD variants was investigated by direct incubation with thrombin at 37° C. for 8 min and subsequent provision for reducing SDS-PAGE followed by western blotting. Band patterns of thrombin-activated or untreated FVIII-ABD molecules show that all FVIII-ABD molecules were activated by thrombin in a comparable manner as ReFacto AF® (data not shown).
[0433] Albumin binding of the ADLCLD_SC variant was tested by an assay in comparison to FVIII 6rs-Ref, demonstrating the capability of albumin binding (
[0434] The influence of ABD and linker modifications on the binding between FVIII and vWF were investigated in two settings: 1. directly, without the presence of albumin; 2. after a 30 min pre-incubation with physiological concentrations of albumin promoting the ABD-albumin binding. As demonstrated in
[0435] To investigate the impact of linkers on the production and functionality of FVIII-ABD variants, one preferred variant, AD2CD2_SC, was also produced (I) without any linkers between FVIII and ABD-Domains (AD2CD2woLG_SC) and (II) with G1 linkers but without thrombin-cleavable L linkers (AD2CD2woL_SC). These variants were compared to the double chain FVIII 6rs-Ref (ReFacto amino acid sequence), single chain FVIII backbone AC_SC and two FVIII-ABD variants having four C-terminal ABD domains either without any linkers (ACD4woLG_SC) or with one thrombin-cleavable linker followed by four ABD domains separated by G1 linkers (ACL(GD)4_SC).
[0436] Respective plasmids encoding the different FVIII variants were nucleofected into CAP-T cells and 4-day cell culture supernatants were tested for chromogenic FVIII activity, FVIII clotting activity and FVIII antigen levels according to the above-described methods. As shown in
[0437] A western blot analysis based on a non-reduced SDS-PAGE separation of these variants is demonstrated in
[0438] 3. Pharmacokinetic Experiments
[0439] Purification of FVIII ABD variants was performed for in vivo experiments, based on supernatants of transfected CAP-T cells, by strong anion exchange chromatography and affinity chromatography.
[0440] To investigate the half-life prolongation effect of ABD motifs introduced into the FVIII molecules, two pharmacokinetic (PK) studies were performed in hemophilia A mice. 12 mice per test item were used, 2 or 3 for each time point. All FVIII-ABD molecules were administered in a single dose of 200 U/kg body weight (6 ml/kg) into the tail vein by a single intravenous tail vein injection into female haemophilia A mice (B6, 12954-F8<tm1Kaz>/J). Plasma samples taken 0.5, 4, 8, 12, and 20 h (and 24 h) post injection were analyzed regarding FVIII chromogenic activity and antigen levels in citrate plasma which was subsequently extracted by centrifugation. Plasma samples were stored at −80° C. and analyzed for FVIII antigen and chromogenic activity. ReFacto AF® was tested as control beside the FVIII-ABD variants.
[0441] Results are shown in Table 4.
TABLE-US-00005 TABLE 4 Calculated t.sub.1/2 Of FVIII-ABD variants. FVIII-ABD variant Antigen: t.sub.1/2 Chromogenic activity: t.sub.1/2 Study 1 ACLD_SC 4.8 4.3 AD2CD_SC 4.3 5.0 AD2CD2_SC 14.5 9.4 ReFacto ® (control) 5.7 4.1 Study 2 ADLC_SC 7.8 6.8 ADLCLD_SC 10.4 10.5 AD2C_SC* 13.0 6.1 ReFacto ® (control) 7.0 6.8 *AD2C_SC data were highly variable. t.sub.1/2 is specified in hours
[0442] Thus, by intravenous pharmacokinetic studies in hemophilia A mice, preferred FVIII proteins of the invention were identified which show a half-life prolonged up to 2.5× (e.g., ADLCLD_SC—about 1.5×; AD2CD2_SC—about 2.5×). Pharmacokinetics of AbD2CD2_SC were tested in a separate study and were similar to AD2CD2_SC.
[0443] It is noted that the hemophilia A mouse model may even underestimate half-life extension due to the discrepancy of murine and human albumin (murine albumin only has a half-life of about two days). Nevertheless, the observed relative extended half-life of the FVIII proteins of the invention already allows a potential reduction of intravenous FVIII injection in hemophilia patients from 2-3 days to a once weekly dosing.
[0444] Moreover, a pharmacokinetic proof of concept study in albumin-deficient Tg32 mice having a knock-out of murine albumin and expressing human FcRn a-chain instead of the murine one (B6.Cg-Tg(FCGRT)32Dcr Alb.sup.em12Mvw FCgrt.sup.tm1Dcr/MvwJ, JAX Stock 025201) was performed. This mouse model (Alb.sup.−/mFcRn.sup.−/hFcRn.sup.+), compared to hemophilia A mice, reveals a situation closer to humans as injected human albumin has a half-life of approx. 20 days which is similar to the half-life in humans. Intravenous FVIII injection (AD2CD2_SC; ReFacto AF®) was done with 200 U/kg (based on chromogenic activity) plus 1% human albumin.
[0445] The results, shown in
[0446] In addition, an intravenous pharmacokinetic study was performed in Göttingen Minipigs. Three animals per group were injected with 30 U FVIII antigen/kg body weight with either (I) ReFacto AF®+1% human serum Albumin (HSA), (II) ReFacto AF®+10% HSA, (Ill) AD2CD2_SC+1% HSA or (IV) AD2CD2_SC+10% HSA via the ear vein. Blood samples were taken predose, 4, 12, 36, 48, and 120 h post administration and citrate plasma was isolated immediately by centrifugation. Bioanalytical sample measurement was performed by FVIII antigen ELISA, which is specific for human FVIII and does not detect any porcine FVIII. Evaluation by non-compartment analysis (
[0447] Moreover, pharmacodynamics studies have been performed. Hemophilia A mice (Jackson No. B6; 129S4-F8<tm1Kaz>/J) and control mice (Jackson No. C57BL/6NCrl) were intravenously injected with 200 U/kg (based on chromogenic FVIII activity) of each FVIII variant (ReFacto®, Eloctate®, AD2CD2_SC, ADLCLD_SC) or control solutions (Vehicle Control, 0.9% NaCl) and weight loss through bleeding, bleeding time and Hb amount by OD550 have been analyzed. Additional plasma sampling (0.5 h p.a. by retro-orbital withdraw, after experiment) have been done for analysis of FVIII activity. As shown in
[0448] 4. De-Immmunized FVIII-ABD Proteins
[0449] The 19 de-immunizing amino acid substitutions of FVIII-19M were incorporated into the FVIII-ABD fusion molecules on the DNA level. The DNA sequences was generated in silico using VectorNTI (Thermo Fisher Scientific, Massachusetts, USA), and afterwards the full FVIII sequence was synthesized and cloned into the target vector. By transformation of E. coli K12 with said plasmids, expansion of transformed bacteria under ampicillin selection and plasmid preparation, large amounts of the plasmids could be prepared. Genetic engineering work was carried out by Thermo Fisher Scientific.
[0450] Cultivation of CAP-T cells and expression of the FVIII encoding plasmids by transient transfection was done as described elsewhere in this document. In order to verify expression levels and functionality of de-immunized, FVIII proteins fused with albumin-binding domains, plasmids encoding the de-immunized FVIII-ABD variant AD2CD2-19M_SC, and the fusion molecule AD2CD2_SC, both including 4 albumin-binding domains, and the FVIII control 6rs-Ref (ReFacto sequence) were nucleofected into CAP-T cells. 4-day cell culture supernatants were tested for chromogenic FVIII activity and FVIII antigen levels according to the above-described methods. As shown in
[0451] First in vivo pharmacokinetic experiments were performed in hemophilia A mice (B6, 129S4-F8<tmlKaz>/J) with affinity chromatography-purified FVIII material to investigate the half-life prolongation effect of AD2CD2-19M_SC. 12 mice per test item were used, 3 for each time point. AD2CD2-19M_SC and ReFacto AF (control) were administered in a single dose of 200 U/kg body weight (7.14 ml/kg) into the tail vein by a single intravenous tail vein injection into female mice. Blood samples were taken 0.5, 4, 8, 12, and 20 h post injection and citrate plasma was extracted by centrifugation. Plasma samples were stored at −80° C. and analyzed for FVIII antigen and chromogenic activity as described. For pharmacokinetic evaluation, a non-compartmental analysis was performed using Phoenix WinNonlin (Certara USA Inc., USA). Mean values of the FVIII antigen levels over time are shown in
[0452] Furthermore, a pharmacokinetic study testing the AD2CD2-19M_SC molecule was performed in Göttingen Minipigs (Ellegaard, Dalmos, DK). Three animals per group were injected with 30 U FVIII antigen/kg bodyweight with either (I) ReFacto AF®+1% human serum Albumin (HSA), (II) ReFacto AF®+10% HSA, (Ill) AD2CD2_SC+1% HSA, (IV) AD2CD2_SC+10% HSA, (V) AD2CD2-19M_SC+1% HSA, (VI) AD2CD2-19M_SC+10% HSA via the ear vein. Blood samples were taken predose, 4, 12, 36, 48, and 120 h post administration and citrate plasma was isolated immediately by centrifugation. Bioanalytical sample measurement was performed by an adapted FVIII antigen ELISA as described above. Evaluation by non-compartment analysis (
[0453] AD2CD2-19M_SC was additionally tested for its in vivo functionality using a tail vein transection assay as described for pharmacodynamics studies. Hemophilia A mice (Jackson No. B6; 129S4-F8<tm1Kaz>/J) were intravenously injected with different doses of AD2CD2-19M_SC, covering 200 U/kg (group 1), 70 U/kg (group 2), 20 U/kg (group 3), 7 U/kg (group 4), and 2 U/kg (group 5) (all based on chromogenic FVIII activity) or formulation buffer (group 6) (n=10 mice per group). Non-hemophilia C57BL/6NCrl mice were used as control (group 7). The tail vein transection assay was performed 30 min post test item administration. Weight loss through bleeding, bleeding time and Hb amount by OD550 were analyzed as readouts. Additional plasma sampling (0.25 h p.a. by retro-orbital withdraw and after the experiment) have been done for analysis of FVIII activity. As shown in
[0454] In order to evaluate if de-immunized FVIII-ABD fusion proteins maintain a certain FVIII activity in the presence of inhibitory anti-FVIII antibodies originally raised against WT or B-domain truncated FVIII (bypassing activity), a modified Nijmegen-Bethesda assay was performed. The Bethesda assay is widely used to quantitate the concentration of a factor VIII inhibitor (inhibitory antibody). 1 Bethesda Unit (BU) is defined as the amount of an inhibitor that will neutralize 50% of 1 unit of FVIII activity in normal plasma after 120 minutes incubation at 37° C. Therefore, five different anti-FVIII antibodies (ESH-8, GMA-8009, GMA-8015, GMA-8026 and CL20035AP), all having inhibitory probabilities to human FVIII activity, were spiked 1:100 into imidazole buffer (Siemens Healthcare Diagnostics, Germany, #OQAA33), which served as stock solutions. Recombinant FVIII variants ReFacto AF®, AD2CD2_SC, and AD2CD2-19M_SC were spiked to a final concentration of 1 U/mL into FVIII-depleted plasma (Siemens Healthcare Diagnostics, Germany, #OTXW17). Standard human serum (Siemens Healthcare Diagnostics, Germany, #ORKL17) was reconstituted in imidazole buffer resulting in a FVIII activity of 1 U/mL serving as further control. Anti-FVIII antibody stocks were diluted 1:2 up to 1:1024 (1:2 serial dilutions) in FVIII-depleted plasma containing the FVIII products. Additionally, each FVIII product diluted 1:2 with FVIII-depleted plasma was determined as baseline FVIII activity (should result in approx. 0.5 U/mL). A FVIII-inhibitor plasma standard (Technoclone, Austria, #5159008, 16.0 BU/ml) diluted 1:2 to 1:128 (1:2 serial dilution series) with FVIII-depleted plasma was used as positive control. All samples were incubated for 2 h at 37° C. and the activity was determined by chromogenic FVIII activity measurements. The remaining FVIII activity within each samples was calculated by the following formula:
[0455] Chromogenic FVIII activity sample [U/mL]/chromogenic FVIII activity baseline [U/mL]*100
[0456] Subsequently, Bethesda units were calculated in remaining activity ranges of 25-75% using the following formula:
2−Log(remaining FVIII activity)/0.30103*dilution factor
[0457] Afterwards, the Bethesda units of each sample were divided by the Bethesda units of the positive control of each run for a more stringent comparison.
[0458] FVIII bypassing activity results of AD2CD2_SC and AD2CD2-19M_SC in comparison to a standard human plasma (SHP) and ReFacto AF® against the five inhibitory anti-FVIII antibodies ESH-8, GMA-8009, GMA-8015, GMA-8026, and CL20035AP are shown in
[0459] 5. Subcutaneous Administration
[0460] For a proof of concept study for subcutaneous administration, FVIII proteins incorporating at least one albumin binding domain were tested in hemophilia A-mice and in minipig in comparison to ReFacto® AF (Pfizer) which is one of the most common B-domain deleted FVIII products.
[0461] Because the albumin binding domains as integrated into the FVIII fusion protein, bind human albumin with a higher affinity compared to murine and porcine albumin (binding affinities are about 1:10 or 1:100), the FVIII protein incorporating at least one albumin binding domain was administered in the presence of human albumin. Co-administered albumin might also have additional effects of stabilization in terms of shielding the FVIII-ABD fusion polypeptide from cellular and enzymatic degradation, and increase the bioavailability by albumin-mediated transport pathways. Further, the compound hyaluronidase, which is known to increase availability especially in subcutaneous administration, was tested in combination with the FVIII with at least one albumin binding domain. The formulation of hyaluronidase as available already contains addition of human albumin (0.1%).
[0462] For all tests shown herein, the FVIII single chain construct AD2CD2_SC (38_ALDGLGDLCLDGLGD_SC, also designated AD2CD2, SEQ ID NO: 48) or, for the minipigs, single chain AD2CD2-19M_SC (SEQ ID NO: 114) was used, wherein two albumin binding domains sequences (D) are inserted between A2 and A3 domain. Two further are located at the C-terminus.
[0463] 5.1 Pharmacokinetic Studies of Recombinant FVIII Molecule AD2CD2 SC by Subcutaneous Administration in Hemophilia a Mice
[0464] 5.1.1 Subcutaneous administration of the recombinant FVIII molecule AD2CD2 in Comparison to ReFacto AF® in a PK Study Using Hemophilia a Mice
[0465] The aim of this study was to investigate the feasibility of subcutaneous (s.c.) administration of AD2CD2_SC in the presence of either 1% human albumin or Hylenex® (recombinant human hyaluronidase, vorhyaluronidase alfa). It was compared to the commercially available rFVIII product ReFacto AF® co-administered with Hylenex®. Coagulation factors were administered by a single s.c. bolus injection into the back region of female haemophilia A mice. Blood sampling was performed 1, 4, and 20 h post treatment and citrate plasma was subsequently extracted by centrifugation. Plasma samples were further analyzed for chromogenic FVIII activity.
TABLE-US-00006 Hemophilia A mice (B6; 129S-F8.sup.tm1Kaz/J), 15 female animals (five per group) Test items a) ReFacto AF ® b) AD2CD2_SC c) Hylenex ® d) HSA (Albiomin ®, Biotest AG, Dreieich) 20% (200 g/L) Vehicle FVIII Formulation Buffer Route of administration Subcutaneous bolus injection into the back region Administration volume 6.67 mL/kg b.w. Injection speed Dose/Approx. 15 sec
[0466] Frequency of administration Single dosing on test day 1 [0467] Group 1: 400 U ReFacto®/kg b.w.+400 U Hylenex/kg b.w. [0468] Group 2: 400 U AD2CD2_SC/kg b.w.+400 U Hylenex/kg b.w. [0469] Group 3: 400 U AD2CD2_SC/kg b.w.+1% albumin (FVIII formulation buffer)
[0470] Hylenex® (Halozyme Therapeutics, Inc): human Hyaluronidase, 150 U/ml, 8.5 mg/ml sodium chloride, 1.4 mg/ml dibasic sodium phosphate, 1 mg/ml human albumin, 1.5 mg/ml L-methionine, 0.2 mg/ml polysorbate 80
[0471] Thus, the concentration of HSA in application solutions of group 1 and group 2 was 0.4 mg/ml, while the HSA concentration of group 3 was 10 mg/ml.
[0472] Tests showed that albumin and Hylenex® itself have no influence on chromogenic FVIII activity (incubation 60 min at room temperature).
[0473] Results are shown in
[0474] 5.1.2 Subcutaneous Administration and Bioavailability of the Recombinant FVIII Molecule AD2CD2_SC in Comparison to ReFacto AF® in a PK Study Using Hemophilia A mice
[0475] The aim of this study was to investigate the bioavailability and pharmacokinetics of AD2CD2_SC in the presence of Hylenex® (recombinant human hyaluronidase) after subcutaneous (s.c.) administration. It was compared to the commercially available recombinant FVIII product ReFacto AF® co-administered with Hylenex®. This study extended the previously performed s.c. PK study, while lowering the administered dose to 200 U/kg b.w. comparable to previous i.v. injections. Coagulation factors were administered by a single s.c. bolus injection into the back region of female hemophilia A mice. Blood sampling was performed 4, 12, 24, 36, 48 and 60 h post treatment (using satellite mice) and citrate plasma was subsequently extracted by centrifugation. Plasma samples were further analyzed for chromogenic FVIII activity and FVIII antigen concentrations.
[0476] Hemophilia A mice (B6; 129S-F8.sup.tm1Kaz/J), 20 female animals, 10 mice per group
TABLE-US-00007 Test items a) ReFacto AF ® b) AD2CD2_SC c) Hylenex ® d) HAS (Albiomin ®, Biotest AG, Dreieich) 20%
[0477] The test items were diluted with FVIII formulation buffer to a final concentration of 33.33 U/mL. The administration volume was 6 mL/kg b.w.
[0478] Group 1: 200 U ReFacto AF®/kg b.w.+200 U Hylenex®/kg b.w.
[0479] Group 2: 200 U AD2CD2/kg b.w.+200 U Hylenex®/kg b.w.
[0480] Thus, the concentration of HSA in application solutions of each group was 0.333 mg/ml.
[0481] The mean results are shown in
TABLE-US-00008 TABLE A Half-life T.sub.max C.sub.max AUC.sub.last Test item [h] [h] [U/mL] [U/mL*h] Mean ReFacto AF ® + — 4.00 0.02 0.14 Hylenex ® AD2CD2_SC + 8.73 12.00 0.26 6.67 Hylenex ® Median ReFacto AF ® + — — 0.00 0.00 Hylenex ® AD2CD2_SC + 22.76 12.00 0.26 6.13 Hylenex ®
TABLE-US-00009 TABLE B Half-life T.sub.max C.sub.max AUC.sub.last Test item [h] [h] [U/mL] [U/mL*h] Mean ReFacto AF ® + — — 0.00 0.00 Hylenex ® AD2CD2_SC + 8.38 24.00 0.24 7.75 Hylenex ® Median ReFacto AF ® + — — 0.00 0.00 Hylenex ® AD2CD2_SC + 10.45 24.00 0.19 7.07 Hylenex ®
[0482] Analysis of bioavailability is based on mean and median activities for AD2CD2_SC plus Hylenex® in subcutaneous and intravenous administration from prior studies. The bioavailability is calculated according to the following formula:
[0483] Where
[0484] AUC.sub.0-inf is the AUC from dosing time extrapolated to infinity, based on the last observed concentration (C.sub.last), i.e., the elimination rate constant λ.sub.z is used to estimate the AUC.sub.t-inf (C.sub.last/λ.sub.z) from the last observed concentration until the time point of concentration zero is reached, which is added to the AUC.sub.0-t, calculated for the period from predose, which is at maximum 2 h before injection, over the maximum observed blood concentration possible until the lower limit of quantification (LLOQ), but at least until a concentration of 0.01 U/mL is reached:
[0485] Table C compares the bioavailability of AD2CD2_SC upon s.c. administration with or without Hylenex®.
TABLE-US-00010 TABLE C Half-life C.sub.max AUC.sub.0-inf Test item [h] T.sub.max [h] [U/mL] [U/mL*h] F [%] Mean s.c. AD2CD2_SC + 8.73 12.00 0.26 6.79 15.3 Hylenex ® i.v. AD2CD2_SC 12.00 0.50 3.06 44.47 Median s.c. AD2CD2_SC + 22.76 12.00 0.26 7.87 18.6 Hylenex ® i.v. AD2CD2_SC 10.33 0.50 3.28 42.39
[0486] In this study AD2CD2_SC plus Hylenex® and ReFacto AF® plus Hylenex® (each 200 U/kg b.w.) are compared after subcutaneous administration in hemophilia A mice for up to 60 hours. FVIII activity (chromogenic activity) and human FVIII antigen (ELISA) have been analysed. ReFacto AF® co-administered with Hylenex® did not demonstrate relevant FVIII plasma levels after s.c. injections. In comparison, AD2CD2_SC co-administered with Hylenex® resulted in considerable FVIII plasma levels even up to 60 hours. The combined (s.c. depot+plasma) half-life of AD2CD2_SC was 8.73 h (mean) and 22.76 h (median) based on the chromogenic activity, whereas ReFacto® was not really detectable at all after subcutaneous administration. It is noted that, due to the relatively low numbers of animals, outliers have a higher influence on the mean than on the median. The bioavailability of AD2CD2_SC in the mice was approx. 18%. This was calculated by comparing the area under the curve (AUC) with the AUC of identical i.v. doses from previous studies.
[0487] 5.2 Pharmacokinetic Study of Recombinant FVIII Molecule AD2CD2 SC in Minipigs by a Comparative Study with Intravenous and Subcutaneous Administration
[0488] To further investigate the FVIII proteins comprising at least one albumin binding domain after subcutaneous administration, the minipig, in particular, the Göttingen minipig (Ellegaard, Dalmos, DK) was chosen as a relevant model based on the similarity of porcine and human dermal tissue. In this study, a FVIII-ABD fusion molecule having 19 deimmunizing amino acid substitutions within the FVIII regions incorporated to prevent human FVIII inhibitor development or potentially enable a certain bypassing activity in case of present inhibitory anti-FVIII antibodies was also tested. This molecule is designated as AD2CD2-19M_SC.
[0489] 5.2.1 Comparative Study with Intravenous Administration in Minipigs
[0490] To compare the observed effect of the construct AD2CD2_SC after subcutaneous administration, firstly the construct was investigated for its pharmacokinetic properties and to determine a baseline level after intravenous administration in the minipig model. Therefore, 18 male naiive Göttingen minipigs (3 per group) were intravenously treated with 30 U/kg FVIII:Ag of either ReFacto AF® or AD2CD2_SC or AD2CD2-19M_SC co-formulated with either 1 or 10% human albumin (Albiomin® 20%, Biotest). Blood samples (˜1.8 mL) were taken at the timepoints: predose, 0.5, 4, 12, 24, 36, 48, 72, 96, 120 and 144 h post dose. Sodium citrate was used as anti-coagulant. Plasma was directly isolated by centrifugation.
[0491] Plasma samples were measured by FVIII antigen ELISA. As albumin-binding to AD2CD2_SC and AD2CD2-19M_SC had an influence on antibody-binding, a correction factor was determined (see below in method section).
[0492] In order to evaluate if measurements of chromogenic FVIII activity, clotting FVIII activity and FVIII antigen ELISA are applicable with the Göttingen minipig plasma matrix, preceding tests were performed by spiking ReFacto AF® and AD2CD2_SC in minipig plasma and determining the recovery. As the minipigs are not hemophilia minipigs, they have endogenous FVIII activity. Thus, for chromogenic FVIII activity and clotting FVIII activity, high background levels of approx. 4 to 7.5 U/ml were found. Thus, low concentrations of recombinant FVIII, e.g. at the terminal phase of a pharmacokinetic study, disappear in this high background. In comparison, the FVIII antigen ELISA was found to be specific for human FVIII and it does not detect porcine FVIII. However, interactions of ReFacto AF® and especially AD2CD2_SC and AD2CD2-19M_SC with porcine von-Willbrand Factor (vWF) and porcine and human albumin demonstrate influences on the measurements. Therefore, the FVIII Antigen ELISA was adapted as described in the method section.
[0493] Mean FVIII plasma concentrations based on FVIII:Ag measurements after intravenous injection of 30 U FVIII:Ag/kg bodyweight are shown in
TABLE-US-00011 TABLE D Mean Median HL- HL- t.sub.1/2 extension t.sub.1/2 extension Component [h] [x-times] [h] [x-times] ReFacto AF ® + 1% HSA 7.07 1 6.28 1 ReFacto AF ® + 10% HSA 6.44 1 5.96 1 AD2CD2_SC + 1% HSA 18.63 2.64 18.62 2.96 AD2CD2_SC + 10% HSA 20.68 3.21 20.82 3.49 AD2CD2-19M_SC + 1% 19.15 2.71 18.70 2.98 HSA AD2CD2-19M_SC + 10% 21.00 3.26 20.52 3.44 HSA
[0494] In a comparative study 18 minipigs were intravenously treated with 30 U/kg (based on FVIII:Ag) of either ReFacto AF®, AD2CD2_SC or AD2CD2-19M_SC in combination with human serum albumin (HSA) at 1% and 10%. Plasma samples were taken at several time points, analyzed by FVIII:Ag ELISA and raw values were evaluated by performing a non-compartmental analysis (NCA) using Phoenix WinNonlin. ReFacto AF® resulted in a terminal half-life of approx. 7 h, while half-lives for AD2CD2_SC and AD2CD2-19M_SC resulted in approx. 19 h in the presence of 1% HSA or in approx. 21 h in the presence of 10% HSA (mean values). Thus, incorporation of four albumin binding domains led to a half-life (HL) extension in intravenous administration of up to 3.3-times in this model compared to ReFacto AF®.
[0495] 5.2.2 Pharmacokinetic Study with Subcutaneous Administration in Minipigs
[0496] The aim of this study was to investigate the bioavailability and pharmacokinetics of AD2CD2-19M_SC in the presence of Hylenex® (recombinant human hyaluronidase) plus 1% human albumin or in the presence of 3% albumin after subcutaneous (s.c.) administration in minipigs (twelve male Göttingen minipigs).
[0497] AD2CD2-19M_SC was compared to the commercially available recombinant FVIII product ReFacto AF® co-administered with Hylenex® plus 1% human albumin. The administered dose for AD2CD2-19M_SC respectively ReFacto AF® was 300 U FVIII:Ag/kg b.w., one group obtained a dose for AD2CD2-19M_SC of 150 U/kg b.w. The dose for co-administered Hylenex® was 16.13 U/kg b.w. Coagulation factors were administered by a single s.c. injection of the minipigs behind the ear. 1 h prior to FVIII administration, all animals were dosed via intravenous (i.v.) injection (saphenous vein) with 1.25 mL Albiomin® 20% (a 200 mg/mL HSA solution, Biotest AG, Dreieich)/kg bodyweight. Blood samples were collected from the vena cava into commercial vacuum blood collection tubes containing sodium citrate at the time points: pre-dose, 0.5, 4, 12, 24, 36, 48, 72, 96, 120, 144, 192 and 240 h post dose. Plasma samples were further analyzed for human FVIII antigen (ELISA) adapted to minipig matrix as described above.
[0498] The dose formulation preparation and the dosing regime was is specified in Table E.
TABLE-US-00012 TABLE E Dose Formulation Preparation Each formulation was prepared immediately prior to dose administration based on the method supplied by the sponsor. The details of each preparation are shown in the table below: Volume Volume of Volume of of Volume of Total Volume Test Item Albumin Hylenex Formulation of Formulation Added Added Added Buffer Formulation Concentration Group Test Item Vehicle (mL) (mL) (mL) Added (mL) (mL) (U/mL) 1 ReFacto ® 1% HSA// 8.4 0.6 3 N/A 12 700 Hylenex 2 AD2CD2- 3% HSA 8.4 1.8 N/A 1.8 12 700 19M_SC 3 AD2CD2- 1% HSA// 8.4 0.6 3 N/A 12 700 19M_SC Hylenex 4 AD2CD2- 1% HSA// 4.2 0.6 3 4.2 12 350 19M_SC Hylenex Dosing Regimen Animals were dosed via intravenous (IV) injection with 1.25 mL/kg Albiomin ® 20% (a 200 mg/ml HSA solution) ca 1 h before dosing test items. Animals were dosed subcutaneously (SC) according to the following design: Dose Dose Level Concentration Dose Volume Group Test Item/Dose Vehicle Route (U/kg b.w.) (U/mL) (mL/kg b.w.) 1 ReFacto ®//1% HSA//Hylenex ® SC 300 700 0.43 2 AD2CD2-19M_SC//3% HSA 3 AD2CD2-19M_SC//1% HSA//Hylenex ® 4 AD2CD2-19M_SC//1% HSA//Hylenex ® 150 350
[0499] Mean FVIII plasma concentrations based on FVIII:Ag measurements after subcutaneous injection of 300 U/kg or 150 U/kg FVIII are shown in
TABLE-US-00013 TABLE F FVIII Half- C.sub.max AUC.sub.last Bioavail- Construct dose life (h) (U/ml) (U/ml*h) HLE* ability (%) ReFacto AF ®//1% 300 U/kg 8.55 0.65 10.70 1 20 HSA//Hylenex ® AD2CD2-19M_SC// 300 U/kg 19.54 1.67 81.17 2.3 38 3% HSA AD2CD2-19M_SC// 300 U/kg 20.33 2.47 105.39 2.4 50 1% HSA//Hylenex ® AD2CD2-19M_SC// 150 U/kg 23.47 0.63 35.61 2.75 34 1% HSA//Hylenex ® *HLE = Half-life extension
[0500] These results show that in minipigs, the pharmaceutical compositions of the invention have an even better bioavailability after s.c. administration than in mice, with a relative increase of at least 70% with a reduced dose of FVIII-ABD compared to higher dose of ReFacto®, while with the same dose of FVIII-ABD, there was an relative increase in bioavailability of at least 150%. Even without Hylenex®, but with an increased amount of human serum albumin, the bioavailability was 90% increased compared to ReFacto®.
[0501] The study was performed in 12 Göttingen minipigs. 300 U/kg or 150 U/kg of either ReFacto AF® or AD2CD2-19M_SC were injected subcutaneously in the presence of human serum albumin (HSA) and in the presence or absence of Hylene®. Bioavailability of up to 50% were observed for AD2CD2-19M_SC at doses of 300 U/kg in the presence of 1% HSA and Hylenex in comparison to the intravenous treatment of the comparative study. In comparison, ReFacto AF® resulted in 20% bioavailability. In the absence of Hylenex® and in the presence of 3% HSA, bioavailability of AD2CD2-19M_SC was only slightly lower with 38%. Regarding the time-vs-concentration areas under the curve until the last measured concentration (AUC.sub.last), ReFacto AF® resulted in approx. 11 U/ml*h, while for AD2CD2-19M_SC AUC.sub.last values of 81 (with 3% HSA) and 105 (with 1% HSA and Hylene®) were observed. The results of all studies demonstrate a clear benefit of FVIII-ABD (with or without 19M) in plasma half-life, but especially in the opportunity to dose FVIII subcutaneously.