IMMUNOADSORPTION

Abstract

Upon administration of rAAV vectors the humoral immune response (neutralizing antibodies) is the first barrier that needs to be overcome. Surprisingly it was found that by using immunoadsorption for depletion of immunoglobulins from the blood (plasma), subjects can be highly efficiently treated with rAAV vectors, i.e. obtain highly efficient transduction after rAAV vector administration, in spite of the presence of high levels of nAb.

Claims

1-20. (canceled)

21. A method of administering a recombinant adeno-associated virus (rAAV) to a subject, the method comprising (a) depleting a subject's immunoglobulins from (i) cerebrospinal fluid (CSF) by contacting the subject's CSF with an extracorporeal device for immunoadsorption, the device comprising a binding moiety attached to a matrix, wherein the binding moiety binds immunoglobulins; or (ii) CSF and blood by contacting the subject's CSF with an extracorporeal device for immunoadsorption, the device comprising a binding moiety attached to a matrix, wherein the binding moiety binds immunoglobulins; and by contacting the subject's blood with an extracorporeal device for immunoadsorption, the device comprising a binding moiety attached to a matrix, wherein the binding moiety binds immunoglobulins; and (b) subsequently administering an rAAV to the subject.

22. The method according to claim 21, wherein the rAAV is administered intravenously.

23. The method according to claim 21, wherein the rAAV is administered to the central nervous system.

24. The method according to claim 21, wherein depleting the immunoglobulins comprises: (a) utilizing a binding moiety that binds immunoglobulins, and/or (b) utilizing a binding moiety that binds anti-rAAV immunoglobulins.

25. The method according to claim 21, wherein the immunoglobulins in the subject's CSF or CSF and blood are depleted by at least 90%.

26. The method according to claim 21, wherein (a) is performed at least two times before (b) is performed.

27. The method according to claim 26, wherein (a) is performed at least 5 times.

28. The method according to claim 21, further comprising subjecting the subject to plasma exchange before (b) is performed.

29. The method according to claim 21, further comprising administering an immune suppressing pharmaceutical.

30. A method of decreasing a humoral immune response to a recombinant adeno-associated virus (rAAV) gene therapy in a subject, the method comprising: (a) contacting the subject's: (i) cerebrospinal fluid (CSF) with an extracorporeal device comprising a binding moiety that selectively binds anti-AAV immunoglobulins, wherein the binding moiety is attached to a matrix, thereby decreasing the humoral immune response to the rAAV gene therapy, or (ii) CSF with an extracorporeal device comprising a binding moiety that selectively binds anti-AAV immunoglobulins, wherein the binding moiety is attached to a matrix, and blood with an extracorporeal device comprising a binding moiety that selectively binds anti-AAV immunoglobulins, wherein the binding moiety is attached to a matrix, thereby decreasing the humoral immune response to the rAAV gene therapy; (b) subsequently administering rAAV therapy to the subject.

31. A method for reducing an anti-rAAV immunoglobulin concentration in the cerebrospinal fluid (CSF) of a subject, the method comprising: (a) obtaining CSF from the subject, wherein the CSF comprises neutralizing antibodies that bind to a rAAV as a result of previously receiving a treatment comprising an rAAV vector, (b) contacting the CSF with an extracorporeal immunoadsorption device, the device comprising a binding moiety attached to a matrix, wherein the binding moiety comprises an AAV epitope that is known to generate anti-AAV antibodies to selectively bind anti-rAAV immunoglobulins, (c) administering the CSF subjected to immunoadsorption to the subject, thereby reducing the anti-rAAV immunoglobulin concentration in the subject's CSF.

32. The method according to 31, further comprising: (a) obtaining blood from the subject, wherein the blood comprises neutralizing antibodies that bind to the rAAV as a result of previously receiving a treatment comprising an rAAV vector, (b) separating the blood into plasma components and cellular components, (c) contacting the plasma components with the extracorporeal device, (d) reconstituting the blood by combining the cellular components with the plasma components that were subjected to immunoadsorption, and (e) administering the reconstituted blood to the subject, thereby reducing the anti-rAAV immunoglobulin concentration in the subject's blood.

33. The method according to claim 31, further comprising administering an rAAV vector to the subject.

34. The method according to claim 33, wherein the rAAV vector is administered to the subject's central nervous system.

35. The method according to claim 32, wherein the rAAV is administered intravenously.

36. The method according to claim 31, wherein the anti-rAAV immunoglobulin concentration in the CSF is depleted by at least 90%.

37. The method according to claim 32, wherein the anti-rAAV immunoglobulin concentration in the CSF and the blood is depleted by at least 90%.

38. The method according to claim 32, further comprising subjecting the subject to plasma exchange.

39. The method according to claim 31, further comprising administering an immune suppressing pharmaceutical.

40. The method according to claim 31, wherein the binding moiety is selected from the group consisting of AAV capsids, AAV VP1, AAV VP2, AAV VP3, and AAV capsid peptides.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0069] FIG. 1A: Immunoadsorption set up. From a (human) subject blood is continuously withdrawn. Optionally, an anticoagulant (heparin) may be added to the withdrawn blood. The blood is subsequently processed by a separating device (A) separating the blood into plasma (c) and cellular constituents (b). The plasma subsequently flows through an immunoadsorption column (C). The immunoadsorption set up can comprise two adsorption columns (B and C) that may be used in an alternating fashion such as in the examples. For example, while one is used for plasma processing, the other column may be cleaned. This way, the binding capacity of the columns may be used optimally. Alternatively, the columns B and C may be different types of columns that are used in an alternating fashion. The processed plasma (d) is subsequently recombined with the cellular constituents (b) and infused back (e) to the (human) subject.

[0070] FIG. 1B: Immunoadsorption set up. From a (human) subject blood is continuously withdrawn. Optionally, an anticoagulant (heparin) may be added to the withdrawn blood. The blood is subsequently processed by a separating device (A) separating the blood into plasma (c) and cellular constituents (b). The plasma subsequently flows through an immunoadsorption column (C). The immunoadsorption set up can comprise two adsorption columns (B and C) used in an alternating fashion wherein when one is used for plasma processing, the other column is being cleaned. The processed plasma (d) is subsequently passed through a second column (D) which is different from columns B and C, e.g. an immunoadsorption specific/selective for anti-AAV immunoglobulins. The processed plasma (f) is than recombined with the cellular constituents (b) and infused back (e) to the (human) subject.

[0071] FIG. 1C: Immunoadsorption set up. From a (human) subject blood is continuously withdrawn. Optionally, an anticoagulant (heparin) may be added to the withdrawn blood. The blood is subsequently processed by a separating device (A) separating the blood into plasma (c) and cellular constituents (b). The plasma subsequently flows through an immunoadsorption column (C). The immunoadsorption set up can comprise several adsorption columns (B, C and D) used in an alternating fashion wherein when one is used for plasma processing, the other columns may be cleaned. The columns used may be different or the same. For example, columns B and C may be used in an alternating fashion as shown in the examples. Plasma processing may be switched to column D after or before one or more cycles of processing by the B/C columns. The processed plasma (d) is subsequently recombined with the cellular constituents (b) and infused back (e) to the (human) subject.

[0072] FIGS. 2A and 2B. Neutralizing antibody titer reduction during immunoadsorption. The neutralizing antibody titer was determined in plasma obtained from non-human primates (0108, 7028, 7310) at different time points. Each timepoint indicating one cycle of immunoadsorption. FIGS. 2A and 2B shows results from different neutralizing antibody titration assays using PLC/PRF/5 cells or HEK293 cells, respectively.

[0073] FIG. 3. SEAP expression in NHP. AAV5 SEAP was administered at day 0. AAV5 hFIX was administered at day 49.

[0074] FIG. 4. human FIX expression in NHP. AAV5 hFIX was administered at day 49. No hFIX expression was detected in non-human primates that were not subjected to immunoadsorption. Initial hFIX expression levels were at the same level as observed in naïve non-human primates.

EXAMPLES

[0075] AAV Vectors

[0076] Two different AAV vectors of the serotype 5 were used (AAV5). A first vector encoded the secreted embryonic alkaline phosphates (SEAP) (AAV5-SEAP) and the second vector encoded human coagulation factor IX (hFIX) (AAV5-hFIX). Non-human primates (Macaca fascicularis, NHP, 3 animals per group) tested negative for the presence of anti-AAV serotype 5 neutralizing antibodies were used. The first administration of AAV5-SEAP (1×10.sup.13 gc/kg) was at day 0. The second administration of AAV5-hFIX (1×10.sup.13 gc/kg) was at day 49. At most 24 hours before the second administration immunoadsorption was performed.

[0077] Immunoadsorption

[0078] The immunoadsorption process was as follows. Blood was previously collected from the NHP in lithium-heparin tubes and stored at 4° C. for processing. After centrifugation at 3500 rpm for 5 min plasma was discarded and 1 mL of SAG-mannitol per 4 mL of whole blood was added. The blood concentrate was used to reconstitute blood to fill the LIFE 18 equipment volume for the immunoadsorption process (i.e. tubing, separator devices, immunoadsorption devices). The extracorporeal volume was about 80 ml and the blood volume of the animals was estimated to be about 8% of the bodyweight, i.e. for an animal of about 3 kg this was assessed to be about 300 ml. Animals had received heparin prior to the immunoadsorption process. Animals were cannulated at a vein and an artery and connected to the LIFE 18 equipment. Cellular components were separated from blood plasma using a filtration device LIFE 18 Disk Separator (item nr. 330-000-038, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The plasma was subsequently passed through a Therasorb Ig flex column/device from Miltenyi (item nr. 330-000-462 Miltenyi Biotex, Bergisch Gladbach, Germany) which uses polyclonal sheep anti-human Ig with Sepharose CL-4B as a matrix. The plasma that was subjected to immunoadsorption was recombined with the cellular components. Heparin, CaCl2 and plasmalyte was added and reconstituted blood infused back to the animal.

[0079] Transgene Expression

[0080] The analysis of SEAP expression was performed with the SEAP Reporter Gene Assay, chemiluminescent (Ref: 11 779 842 001) from Sigma. This assay allows the quantitation of SEAP in the serum samples. In each plate, a negative (water) and a positive control (different concentrations serving as standard) was included. The SEAP concentration was calculated extrapolating from the SEAP standard curve. hFIX expression was measured using ELISA plates (Nunc MaxiSorp plate. Ref: 456537, Thermo Scientific) coated with 50 μL of anti-hFIX (AHIX-5041, HTI) diluted 1:3000 in carbonate buffer overnight at 4° C. The next day plates were washed three times with PBS Tween20 (PBSt) and blocked with blocking solution (PBSt+6% BSA) for one hour at RT. Plates were washed three times with 200 μL PBSt; All sera samples and all FIX standards were diluted 1:100 in incubation buffer (2% BSA in PBSt). The dilutions were added at a final volume of 100 μL and incubated for one hour at RT. All samples were tested in duplicate. All samples were tested in duplicate. Plates were washed three times with PBSt, 100 μL of HRPO-conjugated anti-hFIX were added (Ref: CL20040APHP, Cedarlane Laboratories, diluted 1:2000) and plates incubated for one hour at RT. They were then washed three times with PBSt and the reaction was revealed with TMB substrate. It was stopped after 30 min by addition of H2504 2N. The absorbance was read at 450 nm in a microplate reader. The total antibody titre was calculated as the serum dilution with an absorbance five-fold higher than the negative control.

[0081] Neutralizing Antibody (NAbs) Titer

[0082] The measurement of NAbs in serum was based on an in vitro assay using AAV5 carrying the transgene luciferase (AAV5-luc) and the hepatic cell line PLC/PRF/5 (ATCC CRL-8024) or HEK293 cells. Transgene expression is revealed by addition of luciferin. Cells were seeded into a 96-well plate at a density of 10.sup.4 cells/well. NHP sera were prepared in DMEM/2% FBS in a total volume of 100 μL, beginning with a 1:4 dilution followed by a dilution series of 1:2. Cells were infected with 10.sup.6 AAV5 particles and wild type (wt) adenovirus was added at an MOI of 1. AAV was diluted in 100 μL DMEM/2% FBS and incubated with serial serum dilutions for 2 h at 37°. The mixture was then used to infect cells. Each serum dilution was tested in duplicate. Negative controls of non-infected cells as well as positive controls of infected cells without NHP serum were included in each plate. The infected cells were incubated for 48 h and luciferase activity was quantified. Light emission from each well was measured in photons/cm2/seg.

[0083] Anti-AAV Antibody Titer

[0084] The quantification of total Abs against AAV5 was based on an ELISA assay using the specific capsid to coat the plate. The presence of total Abs specific against each capsid is revealed using Protein A Peroxidase. ELISA plates (Nunc MaxiSorp plate. Ref: 456537, Thermo Scientific) were coated with antigen (AAV5 cap) at 100 ng/well in carbonate buffer overnight to 4° C. The next day plates were washed three times with PBS tween-20 (PBSt) to eliminate the rest of the antigen and blocked with blocking solution (PBS+3% FBS) to prevent unspecific binding. After washing three times with 200 μL PBSt, serum dilutions in PBSt were added, starting with 1:9 followed by a dilution series of 1:3 in a final volume of 100 μL. All samples were tested in duplicate. Negative controls without serum were included in each plate. The serum dilutions were incubated for 2 h at 37°. After this, the serum was removed, the plate was washed three times with PBSt, and 100 μL of protein A peroxidase diluted 1:10,000 in blocking solution were added for one hour. The plate was washed three times with PB St and the reaction was revealed with TMB substrate and stopped 30 min later with H2504 2N. The absorbance was read at 450 nm in a microplate reader. The total antibody titre was calculated as the serum dilution which had an absorbance five-fold higher than the negative control. The total antibody titer reduction observed was about 9-fold, which was in line with the reduction observed in the Nab assay.

[0085] Ig, IgM, IgG1 Measurements

[0086] In order to facilitate the completion of an AAV re-administration procedure in the clinical setting, the total Immunoglobulin, IgM and IgG1 concentrations were measured before and after the immunoadsorption procedure. Measurements were carried out using assays developed for Macaca fascicularis. Reductions in general immunoglobulins concentrations observed were compatible with the Tab assay.

[0087] Results and Discussion

[0088] AAV neutralizing antibody titer and AAV binding antibody titer was measured in the NHP throughout the entire experiment. The AAV neutralizing antibody titer and AAV binding antibody titer were in the range as observed in naïve subjects having AAV administered. The immunoadsorption process strongly reduced the neutralizing antibody titer (FIGS. 2A-2B) in plasma. Importantly, after the second vector administration of AAV hFIX the animals that had received immunoadsorption showed high levels of hFIX expression around 7 days post administration. hFIX declined 20 days post administration and remained stable thereafter (FIG. 4). Such an expression profile in time has been described in previous NHP studies, also without prior exposure to AAV vectors and is independent of the serotype used. In contrast, the animals that did not receive immunoadsorption completely lacked any detectable hFIX expression at any time point. After the second administration a secondary response against AAV5 was observed by an increase in neutralizing antibody titre and AAV binding antibody titre. Combined, the results show that immunoadsorption resulted in a strongly reduced AAV neutralizing antibody titer in serum which provided for a time window that allows for highly efficient rAAV transduction.

[0089] Furthermore, measurements of Ig, IgM and IgG1 in the NHP related to NAb and TAb assays. This confirms that commercially available assays that are available for human Ig, IgM and/or IgG1 measurements may be used in a clinical setting to control the immunoadsorption and re-administration procedure. Such assays allow to determine the number of cycles required (by measuring immunoglobulin level before the immunoadsorption procedure), and/or allow to determine whether the desired reduction in immunoglobulin concentration has been achieved (by measuring the concentration shortly after the immunoadsorption procedure). Hence, the immunoadsorption procedure can be adapted (e.g. determining the number of cycles required) depending on immunoglobulin measurements in human subjects to ensure successful re-administration. This may allow for a well-controlled procedure wherein both the immunoadsorption and re-administration are performed on the same day.