Regimen for vector-based therapies

Abstract

The invention relates to a method or regimen comprising the administration of IgG cysteine protease to a subject in order to improve the benefit of a subsequent vector-based therapy, where the vector-based therapy treats a disease or condition in said subject. Accordingly, the invention also relates to the treatment of diseases or conditions with a vector-based therapy. The method comprises administering to the subject at least two doses of an IgG cysteine protease; and subsequently administering said vector-based therapy. Methods of the invention are particularly useful for subjects who have pre-existing immunity or pre-existing neutralizing antibodies against the vector-based therapy.

Description

[0001] REGIMEN FOR VECTOR-BASED THERAPIES

[0002] Field of the Invention

[0003] The present invention relates to a method or regimen comprising the administration of IgG cysteine protease to a subject in order to improve the benefit of a subsequent vector-based therapy. The invention also relates to in vitro methods for determining whether a subject is in need of a method or regimen described herein.

[0004] Background of the Invention

[0005] There is an ever-expanding list of vector-based therapies which continue to be developed, including: cell therapies such as stem cell therapies and vector-carrying autologous or allogeneic cell therapies: viral vector based therapies such as viral vector gene therapies and oncolytic viral therapies; and even non-viral vector based approaches to gene therapies including the use of lipid and / or protein based nanoparticles.

[0006] However, there is a common challenge faced by all these therapies which is the specific targeting of the vectors themselves by the subject’s own adaptive immune system. Depending on the strength of the immune response, this can lead to a significant dampening or even failure of the vector-based therapy.

[0007] The host immune response is known to be particularly problematic for therapies involving viral vectors such as adenovirus and adeno-associated virus (AAV). This is because humans are routinely exposed to these viruses and many people have preexisting antibodies which can neutralize these viral vectors. Moreover, when any vector-based therapy is administered, this will also elicit an immune response and the development of immunological memory, and this can inhibit future redosing with the same or cross reactive vectors.

[0008] WO 2020 / 016318 considered the administration of IgG cleaving enzymes such as IgG cysteine proteases prior to a gene therapy in order to reduce the immune response against said gene therapy. However, there are instances where this may not reduce the neutralizing titer of a subject enough so that they can receive a gene therapy. Therefore, there is a need for regimens when administering IgG cysteine proteases to subjects before a vector-based therapy, particularly for subjects who have pre-existing neutralizing antibodies against said vector-based therapy.

[0009] Summary of the Invention

[0010] IgG cysteine proteases are well known, and have been studied extensively for use as a conditioning treatment prior to a transplant. For example such enzymes have been used (again typically before transplant) to inactivate DSA in sensitized solid organ transplant recipients (see, e.g. Jordan et al, Transplantation; Aug 2021, 105(8): 1808-1817). The intended purpose is the same: to improve the likelihood of a successful transplant.

[0011] The present inventors have determined that for vector-based therapies, subjects who have pre-existing immunity or pre-existing neutralizing antibodies (e.g. from a previous treatment or from environmental exposure) require further consideration. In these contexts, there are subjects where the F(ab’)2 fragments derived from said pre-existing neutralizing antibodies remain neutralizing (z.e. the neutralizing activity of the antibodies are not mediated by the Fc-portion of the antibody). This can be a problem when using IgG cysteine proteases in any conditioning regimen prior to a vector-based therapy because the by-products of IgG cleavage by such an enzyme are two fragments - the F(ab’)2 fragment and the Fc fragment. If the F(ab’)2 fragments remain neutralizing, then the cleavage itself may not reduce the overall neutralizing titer. One option may be to wait for the clearance of F(ab’)2 fragments from circulation for neutralizing activity against the vector-based therapy (F(ab’)2 fragments are known to have a much shorter half-life than intact IgG). However, during that time the serum IgG levels will begin to recover and there may not be a window where the neutralizing titer has been reduced to a level which will allow for the vector-based therapy to be administered.

[0012] In one aspect, the present invention provides a method of treating a subject in need of a vector-based therapy, the method comprising:

[0013] (a) administering to the subject a first dose of an IgG cysteine protease;

[0014] (b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and (c) subsequent to step (b), administering the vector-based therapy; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a).

[0015] In another aspect, the present invention provides a method of improving the benefit to a subject of a vector-based therapy, the method comprising:

[0016] (a) administering to the subject a first dose of an IgG cysteine protease;

[0017] (b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and

[0018] (c) subsequent to step (b), administering to the subject the vector-based therapy; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a).

[0019] In another aspect, the present invention provides a method of treating a disease in a subject, the method comprising:

[0020] (a) administering to the subject a first dose of an IgG cysteine protease;

[0021] (b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and

[0022] (c) subsequent to step (b), administering a vector-based therapy which treats the disease; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a).

[0023] In another aspect, the present invention provides a method for reducing or inhibiting an immune response against a vector-based therapy, the method comprising:

[0024] (a) administering to the subject a first dose of an IgG cysteine protease;

[0025] (b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and

[0026] (c) subsequent to step (b), administering the vector-based therapy; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a).

[0027] In another aspect, the present invention provides a method for increasing or improving transduction of a transgene into a cell in a subject, the transgene delivered by way of a viral or non-viral vector, the method comprising:

[0028] (a) administering to the subject a first dose of an IgG cysteine protease;

[0029] (b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and

[0030] (c) subsequent to step (b), administering the viral or non-viral vector comprising the transgene; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a).

[0031] In another aspect, the present invention provides a conditioning regimen for administering a vector-based therapy to a subject, comprising:

[0032] (a) administering to the subject a first dose of an IgG cysteine protease;

[0033] (b) subsequent to step (a), optionally administering to the subject at least one further dose of an IgG cysteine protease; and

[0034] (c) subsequent to step (b), administering the vector-based therapy;

[0035] (d) subsequent to step (c) administering to the subject one further dose of an IgG cysteine protease, optionally wherein the interval between step (c) and step (d) is no more than 3 days, more optionally no more than 2 days or 1 day wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of steps (a), (b) and (d) are the same.

[0036] In accordance with the method or conditioning regimen of the invention, the subject has pre-existing neutralizing IgG antibodies against the vector-based therapy, wherein said pre-existing neutralizing IgG antibodies remain neutralizing when digested to F(ab’)2 fragments.

[0037] Brief Description of the Figures

[0038] Figure 1: The pharmacokinetics for five different dose cohorts of N240 and one dose cohort of imlifidase as reference, all data is from healthy individuals. Dose levels are according to legend.

[0039] Figure 2: The pharmacodynamic (PD) effect of N240, imlifidase and Placebo in healthy individuals. PD is determined by determining the total concentration of intact IgG and scIgG, thus the detection is Fc dependent. PD in the figure is presented as change from baseline (pre-dose). A) From pre-dose until end of trial, at 2 months. B) enlarged figure over the first two weeks following treatment. Shades of grey and dose level are according to legend.

[0040] Figure 3: ADA titres following the first month after treatment with N240 in healthy subjects. Median titres are presented with error bars as the inter quantile range. All subjects treated with N240 (regardless of dose) included in analysis.

[0041] Figure 4: The ratio of N240 treated subjects where in vitro treatment (i.e. re-dosing) would have an effective (10% of baseline) treatment. Only subjects treated with doses with full effect (0.18, 0.50, 1.00 mg / kg) included in analysis. In vitro treatment concentrations correspond approx. Cmax of 0.5 mg / kg, 1.0 mg / kg and 2 mg / kg, respectively, for 10 pg / mL, 21 pg / mL and 40 pg / mL.

[0042] Figure 5: SDS-PAGE evaluation of F(ab’)2 presence and re-appearance of scIgG / intact IgG over the first 11 days.

[0043] Figure 6: The effect of N240 and imlifidase on depending on method used for assessing IgG. The MSD / ECL method is determining the total concentration of intact IgG and scIgG. The nephelometry method determines total concentration of Intact IgG, scIgG and F(ab’)2. IgG in the figure is presented as change from baseline for the first month following treatment. Shades of grey and dose level are according to legend.

[0044] Figure 7: Depicted is a stratification algorithm detailing the assay steps a patient has to pass to qualify for the appropriate preconditioning regimen for vector-based therapies (with oncolytic virus (OV) and gene therapy shown as examples of vector-based therapies). Sero-negative individuals can proceed directly to their vector-based therapy. Sero-positive individuals will be further stratified with the help of in vitro neutralizing antibody assays including an IgG-degrading enzymes step to cleave IgGs into their Fc and F(ab’)2 parts. If the neutralizing serum activity is sufficiently overcome by cleaving the Nabs into F(ab’)2 fragments, then the patient requires only one preconditioning treatment with IgG-cleaving enzymes to move on to their vector-based therapy. In contrast, if the in vitro cleavage with IgG-cleaving enzymes does not convert the serum Nabs sufficiently into non-neutralizing serum, then this patient group will need to undergo additional treatment rounds with IgG-cleaving enzymes before vector-based therapies, allowing for the in vivo elimination of F(ab’)2 fragments while cleaving in parallel de novo produced IgG.

[0045] Figure 8: Pharmacodynamics of imlifidase treatment in a patient who was seropositive for anti-AAV8 antibodies. The MSD / ECL method is determining the total concentration of intact IgG and scIgG. The nephelometry method determines total concentration of Intact IgG, scIgG and F(ab’)2.

[0046] Figure 9: Results of cell-based NAb assay to evaluation of neutralizing F(ab’)2 in a patient who was seropositive for anti-AAV8 antibodies treated with imlifidase, before and 48 hours after treatment.

[0047] Brief Description of the Sequences

[0048] SEQ ID NO: 1 is the full sequence of IdeS including N terminal methionine and signal sequence. It is also available as NCBI Reference sequence no. WP_010922160.1 SEQ ID NO: 2 is the mature sequence of IdeS, lacking the N terminal methionine and signal sequence. It is also available as Genbank accession no. ADF13949.1 SEQ ID NO: 3 is the full sequence of IdeZ including N terminal methionine and signal sequence. It is also available as NCBI Reference sequence no. WP 014622780.1.

[0049] SEQ ID NO: 4 is the mature sequence of IdeZ, lacking the N terminal methionine and signal sequence.

[0050] SEQ ID NO: 5 is the sequence of a hybrid IdeS / Z. The N terminus is based on IdeZ lacking the N terminal methionine and signal sequence.

[0051] SEQ ID NOs: 6 to 25 are the sequences of exemplary proteases for use in the methods of the invention.

[0052] SEQ ID NO: 26 is the sequence of an IdeS polypeptide. Comprises the sequence of SEQ ID NO: 2 with an additional N terminal methionine and a histidine tag (internal reference pCART124).

[0053] SEQ ID NO: 27 is the sequence of an IdeZ polypeptide. Comprises the sequence of SEQ ID NO: 4 with an additional N terminal methionine and a histidine tag (internal reference pCART144).

[0054] SEQ ID NO: 28 is the sequence of an IdeS / Z polypeptide. Comprises the sequence of SEQ ID NO: 5 with an additional N terminal methionine and a histidine tag (internal reference pCART145).

[0055] SEQ ID NO: 29 is the contiguous sequence PLTPEQFRYNN, which corresponds to positions 63-73 of SEQ ID NO: 3.

[0056] SEQ ID NO: 30 is the contiguous sequence PPANFTQG, which corresponds to positions 58-65 of SEQ ID NO: 1.

[0057] SEQ ID NO: 31 is the contiguous sequence DDYQRNATEAYAKEVPHQIT, which corresponds to positions 35-54 of SEQ ID NO: 3.

[0058] SEQ ID NO: 32 is the contiguous sequence DSFSANQEIRYSEVTPYHVT, which corresponds to positions 30-49 of SEQ ID NO: 1.

[0059] SEQ ID NOs: 33 to 55 are nucleotide sequences encoding proteases set out above.

[0060] SEQ ID NOs: 56 to 69 are the sequences of exemplary proteases for use in the methods of the invention.

[0061] SEQ ID NO: 70 is the contiguous sequence NQTN, which corresponds to positions 336- 339 of SEQ ID NO: 1.

[0062] SEQ ID NO: 71 is the contiguous sequence DSFSANQEIR YSEVTPYHVT, which corresponds to positions 30-49 of SEQ ID NO: 1. SEQ ID NOs: 72 to 86 are nucleotide sequences encoding polypeptides disclosed herein.

[0063] SEQ ID NO: 87 is the sequence SFSANQEIRY SEVTPYHVT, which corresponds to positions 31-49 of SEQ ID NO: 1.

[0064] SEQ ID NO: 88 is the sequence DYQRNATEAY AKEVPHQIT, which corresponds to positions 36-54 of the IdeZ polypeptide NCBI Reference Sequence no WP_014622780.1.

[0065] SEQ ID NO: 89 is the sequence DDYQRNATEA YAKEVPHQIT, which may be present at the N terminus of a polypeptide of the invention.

[0066] SEQ ID NO: 90, which corresponds to the enzyme N240, and SEQ ID NO: 91 are further exemplary proteases for use in the methods of the invention. SEQ ID NO: 91 is the same as SEQ ID NO: 90, except it lacks the first twenty residues at the N terminus of SEQ ID NO: 90 consisting of the contiguous sequence DDYQRNATEAY AKEVPHQIT.

[0067] SEQ ID NO: 92 is an IdeE sequence from Streptococcus equi ssp. equi. and is another IgG cysteine protease that may be used in the methods of the invention.

[0068] SEQ ID NO: 93 is a sequence of another IgG cysteine protease that may be used in the methods of the present invention.

[0069] SEQ ID NO: 94 is an IgdE sequence from Streptococcus suis. and is another IgG cysteine protease that may be used in the methods of the invention.

[0070] SEQ ID NOs: 95-97 are IdeE variant IgG cysteine proteases which may be used in the methods of the invention.

[0071] SEQ ID NOs: 98, 100, 102, 104, 111 to 2688 are further variant cysteine proteases which may be used in the methods of the invention. In particular, SEQ ID NOs: 99, 101 and 103 are examples where cysteine proteases have been included as part of a fusion protein.

[0072] SEQ ID NO: 105 is an exemplary Human Serum Albumin polypeptide sequence.

[0073] SEQ ID NO: 106 is an exemplary Fc domain sequence.

[0074] SEQ ID NO: 107-110 are exemplary linker sequences.

[0075] SEQ ID NO: 99 is an exemplary fusion protein comprising the IgG cysteine protease of SEQ ID NO: 98, a GS linker, and the HSA domain of SEQ ID NO: 105. SEQ ID NO: 101 is an exemplary fusion protein comprising the IgG cysteine protease of SEQ ID NO: 100, a GS linker, and the HSA domain of SEQ ID NO: 105.

[0076] SEQ ID NO: 103 is an exemplary fusion protein comprising the Fc domain of SEQ ID NO: 106, the linker sequence of SEQ ID NO: 110, and the IgG cysteine protease of SEQ ID NO: 102.

[0077] Detailed Description of the Invention

[0078] It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0079] In addition, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a polypeptide” includes “polypeptides”, and the like.

[0080] A “polypeptide” is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The term “polypeptide” thus includes short peptide sequences and also longer polypeptides and proteins. As used herein, the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including both D or L optical isomers, and amino acid analogs and peptidomimetics.

[0081] The terms “patient” and “subject” are used interchangeably and typically refer to a human. References to IgG typically refer to human IgG unless otherwise stated.

[0082] Amino acid identity as discussed above may be calculated using any suitable algorithm. For example the PILEUP and BLAST algorithms can be used to calculate identity or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0083] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873- 5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Alternatively, the UWGCG Package provides the BESTFIT program which can be used to calculate identity (for example used on its default settings) (Devereux et al ( 1984) Nucleic Acids Research 12, 387-395).

[0084] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Methods and conditioning regimen

[0085] The present invention relates generally to a conditioning regimen for administering a vector-based therapy to a subject, comprising:

[0086] (a) administering to the subject a first dose of an IgG cysteine protease;

[0087] (b) subsequent to step (a), optionally administering to the subject at least one further dose of an IgG cysteine protease;

[0088] (c) subsequent to step (b), administering the vector-based therapy; and

[0089] (d) subsequent to step (c) optionally administering to the subject one further dose of an IgG cysteine protease, optionally wherein the interval between step (c) and step (d) is no more than 3 days, more optionally no more than 2 days or 1 day.

[0090] Typically, the regimens and methods disclosed herein utilise at least two doses of an IgG cysteine protease. In such methods, the interval between doses of an IgG cysteine protease is at least 2 days and no more than 14 days, preferably no more than 9 days.

[0091] In some embodiments, the IgG cysteine protease that is administered at different steps of the method of regimen (such as steps (a), (b) and (d) above) are the same.

[0092] As such, a regimen described herein may also be described as a method.

[0093] Therefore, the present invention also provides a method of treating a subject in need of a vector-based therapy, the method comprising:

[0094] (a) administering to the subject a first dose of an IgG cysteine protease;

[0095] (b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and

[0096] (c) subsequent to step (b), administering the vector-based therapy; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a). In another aspect, the present invention provides a method of improving the benefit to a subject of a vector-based therapy, the method comprising:

[0097] (a) administering to the subject a first dose of an IgG cysteine protease;

[0098] (b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and

[0099] (c) subsequent to step (b), administering to the subject the vector-based therapy; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a).

[0100] In another aspect, the present invention provides a method of treating a disease in a subject, the method comprising:

[0101] (a) administering to the subject a first dose of an IgG cysteine protease;

[0102] (b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and

[0103] (c) subsequent to step (b), administering a vector-based therapy which treats the disease; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a).

[0104] In another aspect, the present invention provides a method for reducing or inhibiting an immune response against a vector-based therapy, the method comprising:

[0105] (a) administering to the subject a first dose of an IgG cysteine protease;

[0106] (b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and

[0107] (c) subsequent to step (b), administering the vector-based therapy; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a). In another aspect, the present invention provides a method for increasing or improving transduction of a transgene into a cell in a subject, the transgene delivered by way of a viral or non-viral vector, the method comprising:

[0108] (a) administering to the subject a first dose of an IgG cysteine protease;

[0109] (b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and

[0110] (c) subsequent to step (b), administering the viral or non-viral vector comprising the transgene; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a).

[0111] Any of the methods may comprise a further step (d) comprising: subsequent to step (c) administering to the subject one further dose of an IgG cysteine protease, optionally wherein the interval between step (c) and step (d) is no more than 3 days, more optionally no more than 2 days or 1 day. The IgG cysteine protease of step (d) may be the same as the IgG cysteine protease in step (a) and / or (b).

[0112] In some embodiments, the interval between doses of an IgG cysteine protease is 2, 3, 4, 5, 6, 7, 8 or 9 days, such as 4, 5, or 6 days. In some embodiments the interval between doses of an IgG cysteine protease is 4 or 5 days.

[0113] In some embodiments, the interval between the first and final doses of an IgG cysteine protease is no more than 14 days, preferably no more than 9 days.

[0114] In some embodiments, the first dose of an IgG cysteine protease is administered at a dosage of 0.1 to 2 mg / kg BW. In some embodiments, the first dose of an IgG cysteine protease is administered at a dosage of 0.2 to 2 mg / kg BW, optionally at a dosage of 0.2 to 1 mg / kg BW, 0.2 to 0.5 mg / kg BW, 0.5 to 1 mg / kg BW, 0.5 to 2 mg / kg BW, or 1 to 2 mg / kg BW. In some embodiments, the first dose of an IgG cysteine protease is administered at about 0.25 mg / kg BW, about 0.5 mg / kg BW, about 1 mg / kg BW, about 1.5 mg / kg BW or about 2 mg / kg BW. In some embodiments, at least one further dose of an IgG cysteine protease is administered at a dosage of 0.1 to 2 mg / kg BW. In some embodiments, at least one further dose of an IgG cysteine protease is administered at a dosage of 0.2 to 2 mg / kg BW, optionally at a dosage of 0.2 to 1 mg / kg BW, 0.2 to 0.5 mg / kg BW, 0.5 to 1 mg / kg BW, 0.5 to 2 mg / kg BW, or 1 to 2 mg / kg BW. In some embodiments, at least one further dose of an IgG cysteine protease is administered at about 0.25 mg / kg BW, about 0.5 mg / kg BW, about 1 mg / kg BW, about 1.5 mg / kg BW or about 2 mg / kg BW.

[0115] In some embodiments, the interval between steps (b) and (c) is no more than 4 days.

[0116] T G cysteine protease

[0117] The amount of an IgG cysteine protease that is administered (such as in steps (a), (b) and (d)) is preferably sufficient to inactivate all or substantially all IgG molecules present in the serum of the subject.

[0118] The term “serum IgG molecule(s)” or “IgG molecule(s) present in the serum” refers to any gamma immunoglobulin (IgGl, IgG2, IgG3 and IgG4) molecule which is present in human tissue or in circulation prior to a method of the invention being carried out. Such IgG molecules may have been produced endogenously from an individual’s B-cells or may be exogenous gamma immunoglobulins which have been administered to a subject prior to the method of the invention being carried out - including any therapeutic IgG molecule of any origin. Inactivation of serum IgG typically means a reduction in the Fc receptor interaction of IgG molecules. The term “Fc receptor” refers to Fc gamma immunoglobulin receptors (FcyRs) which are present on cells. In humans, FcyR refers to one, some, or all of the family of receptors comprising FcyRI (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIC (CD32C), FcyRIIIA (CD 16a) and FcyRIIIB (CD16b). As used herein, the term FcyR includes naturally occurring polymorphisms of FcyRI (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIC (CD32C), FcyRIIIA (CD 16a) and FcyRIIIB (CD 16b). Inactivation of IgG can also reduce complement activation, which is also fundamentally dependent on interactions through an intact IgG molecule. IgG cysteine proteases cleaves IgG such that the antigen binding domains (F(ab’)2) and Fc interacting domains are separated from each other. In such cases, Fc receptor interaction of serum IgG molecules is reduced because the quantity of intact IgG molecules in the serum is reduced.

[0119] The enzyme is preferably administered by intravenous infusion, but may be administered by any suitable route including, for example, intradermal, subcutaneous, percutaneous, intramuscular, intra-arterial, intraperitoneal, intraarticular, intraosseous or other appropriate administration routes. The amount of the enzyme that is administered may be between 0.2 to 2 mg / kg BW (body weight), optionally at a dosage of 0.2 to 1 mg / kg BW, 0.2 to 0.5 mg / kg BW, 0.5 to 1 mg / kg BW, 0.5 to 2 mg / kg BW, or 1 to 2 mg / kg BW. A particularly preferred dose is approximately 0.25 mg / kg BW, about 0.5 mg / kg BW, about 1 mg / kg BW, about 1.5 mg / kg BW or about 2 mg / kg BW. Under certain circumstances (as outlined below) a higher preferred dose may be adopted, particularly for an initial dose of enzyme.

[0120] The IgG cysteine protease for use with the invention is specific for IgG. The IgG cysteine protease may be from a Streptococcus bacterium, such as Streptococcus pyogenes, Streptococcus equi. Streptococcus suis, or Streptococcus zooepidemicus . The IgG cysteine protease may be a IdeS, MAC2, SpeB, IdeZ, IdeS / Z, IgdE or IdeE polypeptide. In preferred embodiments, the IgG cysteine protease for use in the methods of the invention is IdeS (Immunoglobulin G-degrading enzyme of S. pyogenes'), otherwise known as imlifidase, or a homologue or variant thereof. IdeS is an extracellular cysteine protease produced by the human pathogen S. pyogenes.

[0121] IdeS was originally isolated from a group A Streptococcus strain of serotype Ml, but the ides gene has now been identified in all tested group A Streptococcus strains. IdeS has an extraordinarily high degree of substrate specificity, with its only identified substrate being IgG. IdeS catalyses a single proteolytic cleavage in the lower hinge region of the heavy chains of all subclasses of human IgG. IdeS also catalyses an equivalent cleavage of the heavy chains of some subclasses of IgG in various animals. IdeS efficiently cleaves IgG to Fc and F(ab’)2 fragments via a two-stage mechanism. In the first stage, one (first) heavy chain of IgG is cleaved to generate a single cleaved IgG (scIgG) molecule with a non-covalently bound Fc / 2 molecule. The scIgG molecule is effectively an intermediate product which retains the remaining (second) heavy chain of the original IgG molecule. In the second stage of the mechanism this second heavy chain is cleaved by IdeS to release a F(ab’)2 fragment and a homodimeric Fc fragment. These are the products generally observed under physiological conditions. Under reducing conditions the F(ab’)2 fragment may dissociate to two Fab’ fragments and the homodimeric Fc may dissociate into its component monomers. IdeS has been shown to be particularly effective at cleaving IgG in humans. The entire plasma IgG-pool is cleaved within minutes of dosing with IdeS, and IgG levels in blood remain low for more than a week until newly synthesized IgG appeared in plasma. This demonstrates that the entire extracellular IgG pool and not only the plasma pool (i.e. serum IgG molecules) is cleaved by IdeS (Winstedt et al; PloS One 2015; 10(7): e0132011).

[0122] SEQ ID NO: 1 is the full sequence of IdeS including the N terminal methionine and signal sequence. It is also available as NCBI Reference sequence no.

[0123] WP 010922160.1. SEQ ID NO: 2 is the mature sequence of IdeS, lacking the N terminal methionine and signal sequence. It is also available as Genbank accession no. ADF13949.1. A variant of IdeS is the protein MAC2, the full sequence of which is available as Genbank Accession no. AFC67907.1. This protein (with or without signal sequence) is also suitable for use in the methods described herein.

[0124] In alternative embodiments, the IgG cysteine protease for use in the methods of the invention is IdeZ, which is an IgG cysteine protease produced by Streptococcus equi ssp. Zooepidemicus. a bacterium predominantly found in horses. SEQ ID NO: 3 is the full sequence of IdeZ including N terminal methionine and signal sequence. It is also available as NCBI Reference sequence no. WP 014622780.1. SEQ ID NO: 4 is the mature sequence of IdeZ, lacking the N terminal methionine and signal sequence. In alternative embodiments, the IgG cysteine protease for use in the methods of the invention is a hybrid IdeS / Z, such as that of SEQ ID NO: 5, which also lacks the N- terminal methionine and signal sequence.

[0125] In preferred embodiments the IgG cysteine protease has the sequence of SEQ ID NO: 90. This IgG cysteine protease is an engineered variant protease which has been demonstrated to have improved efficacy. This IgG cysteine protease may be referred to as N240. SEQ ID NO: 90 is also presented without the N-terminal methionine and without the signal sequence. A further variant protease derived from SEQ ID NO: 90 is SEQ ID NO: 91, which comprises a complete deletion of the first 20 amino acids of SEQ ID NO: 90 (DDYQRNATEAY AKEVPHQIT).

[0126] In preferred embodiments, IgG cysteine protease for use in the invention may comprise or consist of SEQ ID NO: 2, 4, 5, 90 or 91. Proteases for use in the invention may comprise an additional methionine (M) residue at the N terminus and / or a tag at the C terminus to assist with expression in and isolation from standard bacterial expression systems. Suitable tags include a histidine tag which may be joined directly to the C terminus of a polypeptide or joined indirectly by any suitable linker sequence, such as 3, 4 or 5 glycine residues. The histidine tag typically consists of six histidine residues, although it can be longer than this, typically up to 7, 8, 9, 10 or 20 amino acids or shorter, for example 5, 4, 3, 2 or 1 amino acids.

[0127] In further preferred embodiments, the IgG cysteine protease for use in the invention may comprise, consist essentially, or consist of the sequence of any one of SEQ ID NOs: 6 to 25, 56 to 69. These sequences represent IgG cysteine protease polypeptides with increased protease activity and / or reduced immunogenicity. Each of SEQ ID NOs: 6 to 25, 56 to 69 may optionally include an additional methionine at the N terminus and / or a histidine tag at the C terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C terminus by a linker of 3x glycine or 5x glycine residues. In further preferred embodiments, the IgG cysteine protease for use in the invention may comprise, consist essentially, or consist of the sequence of any one of SEQ ID NOs: 92 to 97, or a variant thereof having at least 80%, at least 85%, preferably at least 90%, at least 95%, at least 98% or at least 99% identical to the sequence of SEQ ID NOs: 92 to 97. Each of SEQ ID NOs: 92 to 97 (or any variant thereof) may optionally include an additional methionine at the N terminus and / or a histidine tag at the C terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C terminus by a linker of 3x glycine or 5x glycine residues.

[0128] In further preferred embodiments, the IgG cysteine protease for use in the invention may comprise, consist essentially, or consist of the sequence of any one of SEQ ID NOs: 98, 100, 102, 104, 111 to 2688 or a variant thereof having at least 80%, at least 85%, preferably at least 90%, at least 95%, at least 98% or at least 99% identical to the sequence of SEQ ID NOs: 98, 100, 102, 104, 111 to 2688. Each of SEQ ID NOs: 98, 100, 102, 104, 111 to 2688 (or any variant thereof) may optionally include an additional methionine at the N terminus and / or a histidine tag at the C terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C terminus by a linker of 3x glycine or 5x glycine residues.

[0129] In further preferred embodiments, the IgG cysteine protease for use in the invention may comprise, consist essentially, or consist of the sequence of any one of SEQ ID NOs: 6 to 25, 56 to 69, optionally with up to 3 (such as 1, 2 or 3) amino acid substitutions. Each of SEQ ID NOs: 6 to 25, 56 to 69 and variants thereof may optionally include an additional methionine at the N terminus and / or a histidine tag at the C terminus.

[0130] In the context of IgG cysteine proteases for use in the invention, they may be part of a fusion protein, wherein the IgG cysteine protease is linked to another secondary polypeptide moiety via a polypeptide linker or by a peptide bond (i.e. without a linker polypeptide sequence). In such a fusion protein, the IgG cysteine protease may be presented N-terminal or C-terminal to the secondary polypeptide. Examples of secondary polypeptides in IgG cysteine protease fusion proteins may be an Fc domain (such as SEQ ID NO: 106) or an HSA domain (such as SEQ ID NO: 105). Exemplary linker sequences may be GS or any one of SEQ ID NOs: 107-110. SEQ ID NOs: 99, 101 and 103 are exemplary fusion peptides.

[0131] The IgG cysteine protease polypeptide for use in the invention is typically at least 100, 150, 200, 250, 260, 270, 280, 290, 300 or 310 amino acids in length. The polypeptide of the invention is typically no larger than 400, 350, 340, 330, 320 or 315 amino acids in length. It will be appreciated that any of the above listed lower limits may be combined with any of the above listed upper limits to provide a range for the length the polypeptide of the invention. For example, the polypeptide may be 100 to 400 amino acids in length, or 250 to 350 amino acids in length. The polypeptide is preferably 290 to 320 amino acids in length, most preferably 300 to 315 amino acids in length.

[0132] The sequence of an IgG cysteine protease for use in the invention may comprise a variant of the amino acid sequence of SEQ ID NO: 2, 4, 5 or 90, which is at least 80% identical to the amino acid sequence of SEQ ID NO: 2, 4, 5 or 90. The variant sequence may be at least 80%, at least 85%, preferably at least 90%, at least 95%, at least 98% or at least 99% identical to the sequence of SEQ ID NO: 2, 4, 5 or 90. The variant may be identical to the sequence of SEQ ID NO: 2, 4, 5 or 90 apart from the inclusion of one or more of the specific modifications identified in WO2016 / 128558, WO2016 / 128559 or WO2021 / 233911. Identity relative to the sequence of SEQ ID NO: 2, 4, 5 or 90 can be measured over a region of at least 50, at least 100, at least 200, at least 300 or more contiguous amino acids of the sequence shown in SEQ ID NO: 2, 4, 5 or 90, or more preferably over the full length of SEQ ID NO: 2, 4, 5 or 90.

[0133] The IgG cysteine protease for use in the invention may be a polypeptide that comprises a variant of the amino acid sequence of SEQ ID NO: 2, 4, 5 or 90 that has cysteine protease activity, in which modifications, such as amino acid additions, deletions or substitutions are made relative to the sequence of SEQ ID NO: 2, 4, 5 or 90. Such modifications are preferably conservative amino acid substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art.

[0134] IgG cysteine protease activity may be assessed by any suitable method, for example by incubating a polypeptide with a sample containing IgG and determining the presence of IgG cleavage products. Suitable methods are described in the WO2016 / 128559. Suitable assays include an ELISA-based assay, such as that which is described in WO2016 / 128559. In such an assay, the wells of an assay plate will typically be coated with an antibody target, such as bovine serum albumin (BSA). Samples of the polypeptide to be tested are then added to the wells, followed by samples of targetspecific antibody that is antibody specific for BSA in this example. The polypeptide and antibody are allowed to interact under conditions suitable for IgG cysteine protease activity. After a suitable interval, the assay plate will be washed and a detector antibody which specifically binds to the target-specific antibody will be added under conditions suitable for binding to the target-specific antibody. The detector antibody will bind to any intact target-specific antibody that has bound to the target in each well. After washing, the amount of detector antibody present in a well will be proportional to the amount of target-specific antibody bound to that well. The detector antibody may be conjugated directly or indirectly to a label or another reporter system (such as an enzyme), such that the amount of detector antibody remaining in each well can be determined. The higher the potency of the tested polypeptide that was in a well, the less intact target-specific antibody will remain and thus there will be less detector antibody. Typically, at least one well on a given assay plate will include IdeS instead of a polypeptide to be tested, so that the potency of the tested polypeptides may be directly compared to the potency of IdeS. IdeZ and IdeS / Z may also be included for comparison.

[0135] Other assays may determine the potency of a tested polypeptide by directly visualizing and / or quantifying the fragments of IgG which result from cleavage of IgG by a tested polypeptide. An assay of this type is also described in WO2016 / 128559. Such an assay will typically incubate a sample of IgG with a test polypeptide (or with one or more of IdeS, IdeZ and IdeS / Z as a control) at differing concentrations in a titration series. The products which result from incubation at each concentration are then separated using gel electrophoresis, for example by SDS-PAGE. Whole IgG and the fragments which result from cleavage of IgG can then be identified by size and quantified by the intensity of staining with a suitable dye. The greater the quantity of cleavage fragments, the greater the potency of a tested polypeptide at a given concentration. A polypeptide of the invention will typically produce detectable quantities of cleavage fragments at a lower concentration (a lower point in the titration series) than IdeZ and / or IdeS. This type of assay may also enable the identification of test polypeptides that are more effective at cleaving the first or the second heavy chain of an IgG molecule, as the quantities of the different fragments resulting from each cleavage event may also be determined. A polypeptide of the invention may be more effective at cleaving the first chain of an IgG molecule than the second, particularly when the IgG is an IgG2 isotype. A polypeptide of the invention may be more effective at cleaving IgGl than IgG2.

[0136] In particularly preferred embodiments, the IgG cysteine protease for use in the invention is an IgG cysteine protease having the sequence of SEQ ID NO: 90, or a functional variant thereof. SEQ ID NO: 90 can be considered to be a variant IdeZ sequence which has been shown to be particularly potent. Moreover, because it is derived from IdeZ, there is a benefit that most human subjects will not have any pre-existing antibodies directed against this enzyme because Streptococcus equi ssp. zooepidemicus is not typically a human pathogen.

[0137] Conditioning regime -timing of enzyme administration

[0138] The present invention requires the administration of an IgG cysteine protease before the administration of a vector-based therapy, and typically requires two separate administrations of an IgG cysteine protease before the administration of a vector-based therapy (i.e. steps (a) and (b) of the regimen and methods described herein). Optionally, there is a further administration of an IgG cysteine protease after the vector-based therapy. The use of two separate administrations which are separated by a time interval is intended to increase the time period in which neutralizing factors against the vectorbased therapy is kept at a minimum, such that there is a greater benefit to the subject of the vector-based therapy.

[0139] The invention arises from the discovery that in certain patients who have pre-existing neutralizing antibodies against a vector-based therapy (such as a viral vector), these neutralizing antibodies may remain neutralizing when cleaved to a F(ab’)2 fragment. Therefore, in these cases the initial complete cleavage of serum IgG may not immediately reduce neutralizing titers against the vector-based therapy in the subject. A time interval is first required to allow for F(ab’)2 fragments to disappear from circulation (from its known short half life), but then the inventors find that a second dose is typically required to continue to reduce and maintain a low level of neutralizing factors because the time interval will also allow serum IgG levels to rebound and increase again from normal synthesis and production. Surprisingly, it has been found that such a subsequent dose can lead to a longer lasting reduction in neutralizing activity which would not otherwise be possible from simply increasing a single dose of an IgG cysteine protease. On the other hand, the timing of any subsequent dose will also need to take into account the generation of AD As in the subject, which may reduce the efficacy of any subsequent dosing.

[0140] Accordingly, the methods and regimen of the present invention includes a first dose of IgG cysteine protease in step (a) and typically a second dose of enzyme in step (b), with the vector-based therapy being administered in step (c). Step (a) and (b) may take place with an interval of about 2 to 9 days in between, so that the interval between the first dose of an IgG cysteine protease and the second dose of an IgG cysteine protease is separated by about 2 to 9 days. This will give the body sufficient time to begin clearing the F(ab’)2 fragments generated from the first cleavage and the second dose of IgG cysteine protease will provide activity against the rebounding IgG that is being synthesized. In some embodiments, the interval between the first dose of an IgG cysteine protease and the second dose of an IgG cysteine protease is about 3, 4, 5, 6, 7, 8 or 9 days.

[0141] Preferably, the second dose of an IgG cysteine protease takes place about 3, 4, 5, 6 or 7 days after the first dose. In other words, preferably, the second dose of an IgG cysteine protease takes place about 72 hours to about 192 hours after the first dose. Preferably, the second dose of an IgG cysteine protease takes place about 4 or 5 days after the first dose. In other words, preferably, the second dose of an IgG cysteine protease takes place about 96 hours to about 144 hours after the first dose. More preferably, the second dose of an IgG cysteine protease takes place about 96 hours to about 120 hours after the first dose. In other embodiments, step (b) can take place at any point between 84 hours and 108 hours after step (a). If the second dose of the IgG cysteine protease is given too soon after the first dose, it is believed that it will reduce the effects of said second dose - the total production of new IgG (rebounding IgG) is still relatively low in the first days after the first dose.

[0142] The vector-based therapy (step (c)) may take place up to 4 days after the last dose of IgG cysteine protease that is administered prior to step (c) - this is typically the second dose of IgG cysteine protease. The vector-based therapy may take place, for example, 1 day, 2 days, 3 days, or 4 days after the last dose of IgG cysteine protease that is administered prior to step (c). The vector-based therapy may take place within the first 24 hours after the last dose of IgG cysteine protease that is administered prior to step (c). Preferably, the vector based therapy takes place about 24 to 48 hours after the administration of the final dose of IgG cysteine protease that is administered prior to step (c) (which is typically the second dose of an IgG cysteine protease).

[0143] Any of the methods may comprise a further step (d) wherein after administration of the vector-based therapy there is a further administration of an IgG cysteine protease dose. In these embodiments, the interval between step (c) and step (d) may be no more than 3 days, such as no more than 2 days or 1 day. It is thought that this will help maintain a low neutralizing titer for the duration of the vector-based therapy. In particular embodiments, the regimen or methods disclosed herein comprise the steps of:

[0144] (a) administering to the subject a first dose of an IgG cysteine protease at 0.5 mg / kg BW, 1.0 mg / kg BW, 1.5 mg / kg BW or 2 mg / kg BW;

[0145] (b) administering to the subject at least one further dose of the IgG cysteine protease at 0.5 mg / kg BW, 1.0 mg / kg BW, 1.5 mg / kg BW or 2 mg / kg BW about 4 days after step (a);

[0146] (c) administering the vector-based therapy about 1 day after step (b); and

[0147] (d) optionally administering to the subject one further dose of the IgG cysteine protease at 0.5 mg / kg BW, 1.0 mg / kg BW, 1.5 mg / kg BW or 2 mg / kg BW about 2 days after step (c).

[0148] In particular embodiments, the regimen or methods disclosed herein comprise the steps of:

[0149] (a) administering to the subject a first dose of an IgG cysteine protease at 0.5 mg / kg BW;

[0150] (b) administering to the subject at least one further dose of the IgG cysteine protease at 0.5 mg / kg BW about 4 days after step (a);

[0151] (c) administering the vector-based therapy about 1 day after step (b); and

[0152] (d) optionally administering to the subject one further dose of the IgG cysteine protease at 0.5 mg / kg BW about 2 days after step (c).

[0153] In particular embodiments, the regimen or methods disclosed herein comprise the steps of:

[0154] (a) administering to the subject a first dose of an IgG cysteine protease at 1.0 mg / kg BW;

[0155] (b) administering to the subject at least one further dose of the IgG cysteine protease at 1.0 mg / kg BW about 4 days after step (a);

[0156] (c) administering the vector-based therapy about 1 day after step (b); and

[0157] (d) optionally administering to the subject one further dose of the IgG cysteine protease at 1.0 mg / kg BW about 2 days after step (c). Reference to a day means a period of 24 hours. Thus about 1 day is equivalent to about 24 hours and the terms may be used interchangeably.

[0158] Thus, for the avoidance of doubt, if step (b) takes place about 4 days after step (a), that means step (b) can take place at any point between 96 hours and 120 hours after step (a)

[0159] - i.e. during the 5thday. In other embodiments, step (b) can take place at any point between 84 hours and 108 hours after step (a)

[0160] The time interval between the first and last dose of the IgG cysteine protease should be no more than 14 days, as any further doses of the same IgG cysteine protease would be rendered ineffective through the generation of AD As. Ideally, the time interval between the first and last dose is no more than 9 days.

[0161] Conditioning regime — selection of subjects

[0162] As discussed above, the methods and regimens described herein are particularly relevant for subjects who are in need of a vector-based therapy who have pre-existing neutralizing antibodies against said therapy. Such neutralizing antibodies (Nabs) may be routinely determined in patients who are in need of a vector-based therapy via an in vitro Nab assay. If the result of such an assay means that the neutralizing titers in a subject are above a certain threshold, then that subject - without any further intervention

[0163] - may be denied the vector-based therapy, (see Mendell, Jerry R., et al. "Testing preexisting antibodies prior to AAV gene transfer therapy: rationale, lessons and future considerations." Molecular Therapy-Methods & Clinical Development 25 (2022): 74- 83.)

[0164] The methods and regimens described herein are designed to be particularly effective when (a) the subject in need of a vector-based therapy has pre-existing neutralizing antibodies against said vector-based therapy and (b) the pre-existing neutralizing antibodies remain neutralizing when cleaved into F(ab’)2 fragments. This is because when the pre-existing neutralizing antibodies are dependent on the Fc portion to exert their neutralizing activity against the vector, then it would be expected that a single dose of an IgG cysteine protease would be sufficient to rapidly reduce the neutralizing titer in the subject. Such neutralizing antibodies are known (see - as an example - Mallery, Donna L., et al. "Antibodies mediate intracellular immunity through tripartite motifcontaining 21 (TRIM21)." Proceedings of the National Academy of Sciences 107.46 (2010): 19985-19990). However, and as discussed above, such a single dose would not likely be sufficient if the F(ab’)2 fragments remain neutralizing.

[0165] In order to select the subjects who have neutralizing antibodies that remain neutralizing when cleaved into F(ab’)2 fragments, the inventors have devised a modified in vitro Nab assay which could be used. Such Nab assays simply modify existing Nab assays such that as well as testing the sera from the subject, sera that has been pre-treated with an IgG cysteine protease is also tested. The IgG cysteine protease sera will then provide information on the neutralizing titer in the sera after complete IgG cleavage into F(ab’)2 (and Fc) fragments. If significant neutralizing titer remains, then that may mean that the subject should be selected to receive the regimen and method of the invention.

[0166] In one embodiment, the subject that is selected has been determined to have said preexisting neutralizing IgG antibodies if sera from said subject following in vitro cleavage with an IgG cysteine protease retains a neutralizing titer of at least 1 :5, 1 : 10, 1 :20, 1 :40, 1 :80, 1 : 160, 1 :320, or 1 :640, optionally wherein the in vitro cleavage is performed with the same IgG cysteine protease as the IgG cysteine protease of step (a), step (b) and / or step (d). Typically, the neutralizing titer represents the serum dilution required to inhibit vector transduction by 50%.

[0167] In another embodiment, the subject that is selected has been determined to have said pre-existing neutralizing IgG antibodies if sera from said subject following in vitro cleavage with an IgG cysteine protease retains a neutralizing capacity of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to non-treated sample, optionally wherein the in vitro cleavage is performed with the same IgG cysteine protease as the IgG cysteine protease of step (a), step (b) and / or step (d).

[0168] Vector-based therapy Vector-based therapies are well known in the art, and in the context of the present invention encompasses at least cell-based therapies, viral vector-based therapies and non-viral vector-based therapies.

[0169] Cell therapies

[0170] Cell therapies aim to treat or manage a disease by introducing living cells that will integrate within the host to restore or eliminate dysfunctional tissues. Cell therapies include stem cell (SC) therapies or non-SC therapies. Non-SC therapies utilise derived from autologous, allo- or xenogeneic sources, either unaltered or genetically engineered. Particular examples of cell therapies include hematopoietic SC therapies, adoptive cell transfer therapies, and chimeric antigen receptor T (CAR-T) cell therapies (e.g. for hematologic disorders, autoimmune disorders and cancers). It is well known that cellbased therapies may face challenges from the host immune system following administration, including the presence of neutralizing antibodies against the vector.

[0171] Viral vector therapies - gene therapy

[0172] Gene therapy is typically defined as a therapy which involves the introduction of a (exogenous) nucleic acid into target cells of a subject for therapeutic purposes, such as treating a disease. Various methods which allow for the transfer of nucleic acids to effect a gene therapy are now known, though viral vectors remain the most widely investigated technique.

[0173] A viral gene therapy in accordance with the present invention may utilise any suitable viral vector. Typically, a viral gene therapy vector may be selected from an AAV vector, an adenoviral vector, a chimpanzee adenoviral (ChAd) vector, a lentiviral vector, a Modified vaccinia virus Ankara (MV A), Herpes simplex virus (HSV), or Vesicular stomatitis virus (VSV). Viral gene therapy vectors can additionally be modifications of the vectors such as chimeric vectors, replication defective vectors, helper-dependent vectors, conditionally replicating vectors, capsid-engineered and retargeted vectors as well as combination of such vector approaches. AAV vectors have been particularly studied for their potential role in gene therapy. Accordingly, in some embodiments, the method or regimen of the present invention may be used with a gene therapy which utilizes an AAV vector, optionally wherein the AAV vector is AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVcylO and AAVrhlO, AAVrh74, AAVDJ, AAV-Anc80, AAV- LK03, AAV2i8, AAVpo4 or AAVpo6.

[0174] In certain embodiments, a lentivirus used in the instant invention may be a human immunodeficiency- 1 (HIV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), or caprine arthritis encephalitis virus (CAEV). Lentiviral vectors are capable of providing efficient delivery, integration and long-term expression of heterologous polynucleotide sequences into non-dividing cells both in vitro and in vivo.

[0175] As discussed above, it is well known that viral vectors which are commonly used in gene therapies, particularly AAV, derive from viruses which humans frequently encounter and where there is a significant portion of the population with preexisting immunity because of pre-existing neutralizing antibodies in sera.

[0176] A “transgene” is used herein to conveniently refer to a nucleic acid that is intended or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a heterologous polynucleotide sequence or a heterologous nucleic acid encoding a protein or peptide. The term transgene and heterologous nucleic acid / polynucleotide sequences are used interchangeably herein.

[0177] Non-viral vector gene therapy

[0178] Recently, non-viral vectors, particularly lipid nanoparticles (LNPs) and cationic polymers, have demonstrated robust gene loading capacity, high safety and practicability combined with ease of manufacture. Consequently, non-viral vectors have been considered as gene therapy vectors for further clinical development. Non- viral vectors for use with the present invention include liposomes, lipid nanoparticles (LNPs), highly branched poly(P-amino ester) (HPAE), single-chain cyclic polymer (SCKP), poly(amidoamine) (PAMAM) dendrimers, and polyethyleneimine (PEI). Host immune responses against non-viral vectors can equally dampen therapies using such vectors, and given the vectors are often repurposed for different therapeutic uses, this may lead to a greater proportion of the population being exposed to particular non-viral vectors in the past.

[0179] Viral vector therapy - oncolytic virus Immunotherapy of cancer with oncolytic viruses is an emerging and maturing treatment modality which uses replication-competent viruses that selectively infect and damage tumor cells and may also, preferably, induce an immunological response which can control both the target tumor and distal tumors. Each species of oncolytic virus has a different cellular tropism, which helps determine which tissues are preferentially infected. Engineering of the virus can expand, restrict, or modulate this host range. For example, the oncotoxic effect of oncolytic viruses can be enhanced by inserting various classes of functional genes into the virus genome (as a payload), in order to boost its oncolytic function. This includes the insertion of sequences encoding for e.g. antibodies, bi-specific T cell engagers (BiTEs), bispecific adapter proteins or various forms of fusion proteins, possibly also in combination with chimeric antigen receptor (CAR)-T cell therapies. By these strategies, it is possible to deliver immune checkpoint inhibitors, anti -angiogenic antibodies, or other functional entities such as e.g. cytotoxic fusion proteins to the tumour or to hematologic neoplasms. A variety of species of virus have been investigated for use in oncolytic therapies.

[0180] An oncolytic viral vector in accordance with the present disclosure and for use with the methods of the present invention may be selected from adenovirus (such as Chimeric adenovirus vectors Ad5 / 3, Ad5 / 32, Adl lp / Ad3), Herpesvirus Parvovirus, Poxvirus, Semiliki Forest virus, Sindbis virus, Zika virus, Measles virus, Newcastle disease virus, Coxsackievirus, Polio virus, Seneca valley virus, Reovirus, Vesicular stomatitis virus (VSV), Maraba virus and Vaccinia virus. It is well known that the presence of neutralizing antibodies (nAbs) against an oncolytic viral vector may also lead to the inhibition and (premature) clearance of the vector. Various strategies have previously been proposed to remove or avoid the effect of such nAbs, including PEGylation, encapsulation or polymers for blocking and shielding the viral vector. It is believed that the methods and regimen of the present invention will also allow for improved therapeutic benefit from oncolytic viral vector therapy.

[0181] Methods of treating or preventing a disease or condition

[0182] The present invention also provides a method for the prevention or treatment of a disease or condition in a subject. The method and regimens described herein comprises a step of administering a vector-based therapy to the subject, wherein the vector-based therapy is administered to treat said disease or condition. Expressed another way, the invention also provides a method for treating a disease or condition in a subject by administering a vector-based therapy which to treat said disease or condition, the method comprising (carrying out) the regimen or method of the invention.

[0183] As used herein, the term "treatment" or "treat" refers to both prophylactic or preventive treatment like gene therapy as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0184] As such, the present disclosure also provides a method for the prevention or treatment of a disease or condition in a subject, wherein the disease is:

[0185] (i) selected from a lung disease (e.g . cystic fibrosis), a bleeding disorder {e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, a blood disorder {e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, a lysosomal storage disease {e.g., aspartylglucosaminuria, Batten disease, late infantile neuronal ceroid lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease II (Pompe disease), GM2-gangliosidosis type I (Tay Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis types I (sialidosis type I and II), II (I-cell disease), III (pseudo- Hurler disease) and IV, mucopolysaccharide storage diseases (Hurler disease and variants, Hunter, Sanfilippo Types A,B,C,D, Morquio Types A and B, Maroteaux-Lamy and Sly diseases), Niemann-Pick disease types A / B, Cl and C2, and Schindler disease types I and II), hereditary angi oedema (HAE), a copper or iron accumulation disorder (e.g., Wilson’s or Menkes disease), lysosomal acid lipase deficiency, a neurological or neurodegenerative disorder, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, a metabolic defect (e.g., glycogen storage diseases), a disease of solid organs (e.g., brain, liver, kidney, heart), or an infectious viral (e.g., hepatitis B and C, HIV, etc.), bacterial or fungal disease. In certain embodiments, a subject has a blood clotting disorder. In certain embodiments, a subject has hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, a deficiency in any coagulation Factor: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or a combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase Cl deficiency or gamma-carboxylase deficiency;

[0186] (ii) selected from anemia, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anti coagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e. FXa inhibitors), or a platelet disorder such as, Bernard Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency;

[0187] (iii) selected from a disease that affects or originates in the central nervous system (CNS). In certain embodiments, the disease is a neurodegenerative disease. In certain embodiments, the CNS or neurodegenerative disease is Alzheimer’s disease, Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy’s disease, a polyglutamine repeat disease, or Parkinson's disease. In certain embodiments, the CNS or neurodegenerative disease is a polyglutamine repeat disease. In certain embodiments, the polyglutamine repeat disease is a spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17); or

[0188] (iv) cancer, optionally a cancer selected from brain cancer, breast cancer, colon cancer, esophageal cancer, prostate cancer, bladder cancer, kidney cancer, endometrial cancer, thyroid cancer, gastric cancer, liver cancer, lung cancer, pancreatic cancer, uterine cancer, tracheal cancer, testicular cancer, cervical cancer, head and neck cancer, skin cancer, soft tissue sarcoma, bone cancer, pancreatic cancer and ovarian cancer, or a hematological cancer such as leukemia, lymphoma and multiple myeloma.

[0189] The invention also provides an IgG cysteine protease in a subject for use in a method for the prevention or treatment of a disease or condition, wherein the method is as described herein.

[0190] The invention also provides the use of an IgG cysteine protease in a subject in the manufacture of a medicament, wherein the medicament is for the prevention or treatment of a disease or condition in a method as described herein.

[0191] Methods of improving the benefit to a subject of a vector-based therapy

[0192] The present invention also provides a method of improving the benefit to a subject of a vector-based therapy.

[0193] The benefits of using the regimen and methods of the present invention are clear for any vector-based therapy, particularly for subjects who have pre-existing neutralizing antibodies against said vector. The consequences of reducing the neutralizing titer in serum means that there is a reduced chance that the vector-based therapy will fail.

[0194] In this regard, the benefit of using the regimen would result in a reduction in neutralizing titer in sera from the subject against the vector-based therapy following step (b), and preferably immediately before step (c). For example, the reduction in neutralizing titer may be at least 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, 80-fold, 100- fold, 200-fold, 400-fold, 1000-fold, lOOOO-fold or more.

[0195] It is also envisaged that such a benefit to the vector-based therapy in some cases may be quantifiable as well.

[0196] For example, in the case of gene therapy or oncolytic viral therapy, the therapeutic benefit may be quantified as the expression level of a transgene that was delivered by the gene therapy vector (whether this vector was viral or non-viral). In this case, the improvement of benefit may be expressed as an increase in the expression level of the transgene, such as an increase of at least 10%, 20%, 40%, 80%, 160%, 320% or more.

[0197] In the case of oncolytic viral therapy, the therapeutic benefit may be quantified as the expression level of a transgene that was delivered by the gene therapy vector (whether this vector was viral or non-viral). In this case, the improvement of benefit may be expressed as an increase in the expression level of the transgene, such as an increase of at least 10%, 20%, 40%, 80%, 160%, 320% or more.

[0198] In the case of oncolytic viral therapy, the therapeutic benefit may also be an improvement in one or more endpoint measurements, such as a decrease in tumour mass, an increase in the time to disease progression, increase in the duration of progression free survival, or an increase in overall survival. In one embodiment, the decrease in tumour mass may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In one embodiment, the increase in the time to disease progression, increase in the duration of progression free survival may be an increase of such as an increase of at least 10%, 20%, 40%, 80%, 160%, 320% or more.

[0199] Production of polypeptides

[0200] The IgG cysteine proteases used in the methods of the invention are polypeptides and may be produced by any suitable means. For example, a polypeptide may be synthesised directly using standard techniques known in the art, such as Fmoc solid phase chemistry, Boc solid phase chemistry or by solution phase peptide synthesis. Alternatively, a polypeptide may be produced by transforming a cell, typically a bacterial cell, with a nucleic acid molecule or vector which encodes said polypeptide. Production of enzyme polypeptides by expression in bacterial host cells is described and exemplified in WO2016 / 128558 and WO2016 / 128559.

[0201] Compositions and formulations comprising polypeptides

[0202] The present invention also provides compositions comprising an IgG cysteine protease for use in the methods of the invention. For example, the invention provides a composition comprising one or more polypeptides, and at least one pharmaceutically acceptable carrier or diluent. The carrier(s) must be 'acceptable' in the sense of being compatible with the other ingredients of the composition and not deleterious to a subject to which the composition is administered. Typically, carriers and the final composition are sterile and pyrogen free.

[0203] Formulation of a suitable composition can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the reasonably skilled artisan. For example, the enzyme can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, reducing agents and the like, may be present in the excipient or vehicle. Suitable reducing agents include cysteine, thioglycerol, thioredoxin, glutathione and the like. Excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, thioglycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, mal onates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in dry (for e.g., a powder or granules) form for reconstitution with a suitable vehicle (e. g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides.

[0204] Other parenterally-administrable compositions which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. The compositions may be suitable for administration by any suitable route including, for example, intradermal, subcutaneous, percutaneous, intramuscular, intra-arterial, intraperitoneal, intraarticular, intraosseous or other appropriate administration routes. Preferred compositions are suitable for administration by intravenous infusion. Kits

[0205] The invention also provides a kit for carrying out the methods described herein. The kit of the invention may include an enzyme or a composition comprising an enzyme, as described above. The kit may include means for administering the IgG cysteine protease or composition to a subject in accordance with the methods and / or regimens described herein. The kit may include instructions for use of the various components in any method as described herein.

[0206] EXAMPLES

[0207] Unless indicated otherwise, the methods used are standard biochemistry and molecular biology techniques. Examples of suitable methodology textbooks include Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley and Sons, Inc.

[0208] Serum samples

[0209] Serum samples were collected in clinical studies, predose and at different timepoints post dosing with the IgG cysteine proteases (EudraCT Number 2022-033409-31 and EudraCT number 2019-002770-31).

[0210] Example 1 — Pharmacokinetics and pharmacodynamics of IgG cysteine proteases

[0211] Materials and methods

[0212] Pharmacokinetics

[0213] In brief, a hen-anti -enzyme antibody (raised against the IgG cysteine protease in question) is coated on a MULTI-ARRAY 96-well plate (MSD). To enable detection of total enzyme concentration the potential binding between enzyme and blocking antienzyme antibodies present in serum, a dissociation step is introduced by incubation of the serum samples with Gentle Elution Buffer (GEB, Thermo Scientific #21027) at 37°C. The plate is blocked and washed, and calibrators, QCs and study samples incubated with GEB are added to the plate followed by incubation at room temperature. The plate is washed, and a detection mix containing biotinylated goat-anti-enzyme and SULFO-TAG labelled streptavidin is added to the plate. After a final incubation and wash, Read buffer is added, and the SULFO-TAG emits light when a voltage in the MSD instrument is applied to the plate electrodes. The instrument measures the intensity of emitted light to provide a quantitative measure of the concentration of enzyme in the samples.

[0214] Pharmacodynamics

[0215] In brief, a goat-anti-human F(ab’)2-specific IgG is coated on a MULTI-ARRAY 96-well plate (MSD). After blocking, the plate is washed and calibrators, QC and study samples are added to the plate followed by incubation at room temperature. The plate is washed and a detection mix containing biotinylated goat-anti-human Fc-specific F(ab')2- fragment and SULFO-TAG conjugated streptavidin is added to the plate. After a final incubation and wash, Read buffer is added, and the SULFO-TAG emits light when a voltage in the MSD instrument is applied to the plate electrodes. The instrument measures the intensity of emitted light to provide a quantitative measure of intact and single cleaved IgG in the samples.

[0216] Results and discussion

[0217] To develop an effective treatment regimen which would allow a greater number of people to receive vector-based therapies such as gene therapy or oncolytic viral therapy, we first studied the pharmacokinetics and pharmacodynamics of N240 treatment in subjects to identify potential routes to improving the clearance of neutralizing factors which would inhibit said vector-based therapies.

[0218] The serum concentration of N240 in subjects dosed with various doses of N240 is shown in Figure 1, where the concentration is shown for 3 days after dosing. Figure 1 also show the comparison against previous known pharmacokinetic data for the IgG cysteine protease imlifidase (IdeS) in healthy subjects. The study found that high doses (including as high as 1 mg / kg) were well tolerated and safe in healthy subjects, and also that a higher dose does provide a higher Cmax and higher AUC (Table 1). On the other hand, a higher dose does not provide any impact on distribution or elimination half life.

[0219] Consequently, in all doses tested, there is a rapid reduction in enzyme concentration in all cases, and this suggests that increasing a single dose of an IgG cysteine protease such as N240 would not substantially prolong IgG cleaving activity in serum.

[0220] To further explore these data, with pharmacodynamics, the concentration of intact IgG and single cleaved IgG (scIgG) in serum was followed in subjects treated with various doses of N240 and is shown in Figure 2. Figure 2A shows the concentration of intact IgG and single cleaved IgG (scIgG) in serum up to 2 months after dosing, while Figure 2B shows in more detail the effects in the first ~15 days. Like in Figure 1, these results show a comparison against previous known pharmacodynamic data for the IgG cysteine protease imlifidase (IdeS, dosed at 0.25 mg / kg).

[0221] In all cases apart from the lowest tested dosages, we see the expected rapid complete cleavage of serum IgG. On the other hand, it is surprising that once a certain dose threshold is reached, there appears to be no further significant impact on the duration of the response with higher doses despite the increase in Cmax and AUC seen with higher doses. In particular, a higher dose does not appear to significantly impact the speed at which the serum IgG levels recover.

[0222] In conclusion, despite N240 being well tolerated at high doses which also leads to an increased Cmax and AUC, the higher doses do not correlate with a longer duration of response, i.e. to maintain low IgG levels, which may be required to enable therapies such as vector-based therapies. In cases where a prolonged response is required, a redosing strategy may be required.

[0223] Example 2 — Immunogenicity of N240

[0224] Materials and methods

[0225] In brief, the immunogenicity profile of N240 was assessed by a multi-tiered approach consisting of a screening step, a confirmatory step and a titration step. The basis for the assay is a bridging step where biotin-conjugated N240 and SULFO-TAG conjugated N240 is allowed to form a complex with potential ADA present in a serum sample prior to applying the whole complexed sample to a blocked and washed MSD streptavidin plate. After a final incubation and wash, Read Buffer was added, and the SULFO-TAG emitted light when a voltage in the MSD instrument was applied to the plate electrodes. The instrument measured the intensity of emitted light to provide a quasi-quantitative measure of ADA in the samples. The assay detected intact Ig, single cleaved IgG and (Fab’)2 fragments, but not Fab fragments. Samples reported as positive both in the screening and confirmatory assays were analysed with the titration assay, where samples were diluted until the mean ECL signal of the sample was below the titration cut point (TCP).

[0226] Results and discussion

[0227] It is known that other IgG cysteine proteases such as imlifidase can induce the production of anti -drug antibodies (ADA) in subjects. The immunogenicity data for N240 (Figure 3) similarly shows that following administration of N240 to a subject, the development of ADA begins shortly after 7 days and peaks around 14 to 21 days. If a redosing strategy is to be considered, it would be important for the second (and any further) dose of N240 to be given prior to the peak of ADA.

[0228] Example 3 — In vitro determination of N240 redosing efficacy

[0229] Materials and methods

[0230] Given the potential challenges of developing a regimen where e.g. at least two separate doses of N240 are administered to a subject, an in vitro redosing efficacy assay was developed that provides an excellent representation of in vivo efficacy.

[0231] In this assay, serum samples from subjects dosed with N240 are treated in vitro with doses within a range (0-80 pg / mL) of N240. The samples are incubated at 37°C for approx. 20h to allow time for cleavage. The IgG content (intact IgG and scIgG) in the sample is detected and quantified in a validated ECL assay (this is essentially equivalent to the pharmacodynamics assay described in Example 1). The pre-treatment samples are used to assign the baseline for the individual subjects IgG level and to assess the treatment effect of the sample at start.

[0232] A high likelihood to a good treatment effect (in this assay) is determined by being able to reach a low level (10% of baseline levels) after in vitro treatment of N240.

[0233] Results and discussion

[0234] Figure 4 shows the results of the in vitro redosing assay using serum samples taken from subjects who were dosed with N240 on study Day 1. As shown on the Figure, redosing was assessed at the specified dose on samples taken from the subjects on study Days 7, 9, 11, 14, 21 and 28 following the initial N240 dosage. The Figure shows the ratio of subjects where one would expect to have a further effective treatment of N240 at the specified concentration. In this regard, 75% would mean that 75% of subjects would be expected to respond to a further dose (corresponding to that specific Cmax) of N240. In such a scenario, it would be envisaged that approximately 25% of subjects would be expected to have already developed significant neutralizing AD As which would render further doses ineffective.

[0235] In agreement with the results from the immunogenicity / ADA assay, the results demonstrate that after Day 7, the effects of increasing ADA can be seen. From Day 11, there is already a significant decrease in the expected effectiveness of any subsequent N240 dose. Increasing the dose amount (i.e. increasing the Cmax) overcomes some of the ADA related lack of efficacy. However, even doubling the dose to 2 mg / kg (corresponding to approximate Cmax of 40 pg / mL) would only marginally increases the ability to redose in some individuals and during peak ADA duration it would still not be possible to dose in at least half of the individuals; increasing the dosage does not appear to have the ability to completely restore the ability to redose an individual who have developed significant AD As against N240.

[0236] Example 4 — Pharmacodynamics of neutralizing F(ab’)2 fragments

[0237] A further complication when considering some vector-based therapies is that in certain circumstances, the neutralizing factors present in the serum target epitopes for which even the F(ab’)2 fragments may inhibit the vector therapy. In other words, these epitopes present on the vectors can be targeted and bound by F(ab’)2 fragments leading to neutralization even in the absence of any Fc-mediated response. This would mean that in these situations, the initial complete cleavage of serum IgG by e.g. an IgG cysteine protease would not be sufficient to eliminate the neutralizing factors completely because the F(ab’)2 fragments generated as a cleavage product remains neutralizing. For situations where the F(ab’)2 fragments are neutralizing, a regimen needs to be developed which allow for their clearance prior to administering a specific therapy such as a vector-based therapy. F(ab’)2 fragments are known to have a significantly shorter half life compared to intact IgG (and single cleaved IgG) but this will still require sufficient time to allow for clearance. At the same time, it will also be equally important to consider and potentially remove newly produced IgG or scIgG prior to administering the specific therapy such as a vector-based therapy. Materials and methods

[0238] In order to investigate the potential effects of F(ab’)2 fragments further, the following methods were employed to further consider the pharmacodynamics of N240 after administration. This included:

[0239] SDS-PAGE evaluation of F(ab’)2 presence and re-appearance of scIgG / intact IgG

[0240] - Reevaluating the PD (ECL) data from Example 1 using additional pharmacodynamic data collected using the IgG concentration method available at hospitals to detect F(ab’)2 fragments as well as scIgG and intact IgG

[0241] The IgG concentration method at hospitals is a turbidimetry or nephelometry method used to measure total IgG concentration in a serum or plasma sample. Those methods build on the addition of a specific antibody against IgG to the sample and this antibody forms complexes with IgG molecules present in the sample. The sample is then passed through a cuvette or a flow cell in the nephelometer instrument where a light source is directed through the sample. As the light passes through the sample the IgG-antibody complexes cause the light to scatter in multiple directions. The nephelometer detects and measures the intensity of the scattered light and the intensity of the scattered light is directly proportional to the concentration of IgG in the sample.

[0242] Commercially available polyclonal anti Ig-specific antibodies are added to generate the antigen-antibody complexes is not specific for intact IgG but also form complexes with the cleavage-products post enzymatic IgG-cleavage e.g. scIgG and F(ab’)2 fragments. Therefore when all IgG is cleaved (within hours after enzyme treatment) the decline of the IgG concentration reported with this method follows the clearance of the F(ab’)2 fragments and continues reporting the sum of all IgG-related molecules that includes F(ab’)2 fragments (i.e. the sum of IgG, scIgG and F(ab’)2 fragments in the sample).

[0243] Results and discussion On the SDS-PAGE gel (Figure 5), it is clear that the generation of F(ab’)2 fragments is fast and no signs of scIgG or intact IgG can be seen in the first few days. Using this method, it can be seen that new intact IgG cannot be seen before day 7. Additionally, F(ab’)2 fragments do clear quickly and are significantly decreased in number by day 3 / 5.

[0244] In Figure 6, the pharmacodynamics data from Example 1 is compared with the nephelometry data which includes F(ab’)2 fragments. As discussed above, the MSDZECL method detects only molecules with an Fc-portion i.e. scIgG and intact IgG.

[0245] The data is clear in showing that higher doses - to a limit - enable reaching a deeper nadir on both measurements.

[0246] From studying the SDS-PAGE gels we can determine that the early increase in signal with the PD ECL / MSD method is a consequence of measuring scIgG - we know that this is not from intact IgG because they are not detected on the gels. This is likely a combination of residual enzyme activity remaining and capable of cleaving newly produced IgG into scIgG, as well as potentially remaining scIgG in extravascular space that flows back to circulation. On the other hand, when considering the nephelometry data which concerns all F(ab’)2 fragments as well, it can be seen that the total amount continues to fall due to F(ab’)2 fragments gradually leaving circulation. Therefore, in scenarios where F(ab’)2 fragments are neutralising, this time period still leads to an overall decrease in neutralizing titer.

[0247] Regardless, the consequence is that around 4-6 days after the first dose the sum of all available molecules with a F(ab’)2 portion starts to increase and hence this is the timepoint where the second dose would be desirable when F(ab’)2 fragments remain neutralising. The (vector-based) therapy could then be applied within about 48 hours from the second enzyme dose to allow for initial clearance of newly generated F(ab’)2 fragments and allow for transduction without impact from returning scIgG and IgG.

[0248] Example 5 — exemplary conditioning regimen As alluded to above, one problem is that current methods to reduce serum IgG are not sufficiently effective and / or sufficiently long-lasting in the contexts where clearing all F(ab’)2 containing products is desirable, and the F(ab’)2 containing products from IgG- cleaving enzymes are not sufficiently cleared before new IgGs are produced.

[0249] This clearance period of F(ab’)2 containing products such as F(ab’)2 fragments during an IgG-free window might be especially important in situations where the presence of F(ab’)2 containing products is not permissive for an effective treatment with a subsequent vector-based therapy such as a gene therapy, oncolytic virus or cell therapies. This is because in some cases, the F(ab’)2 fragments derived from neutralising antibodies against the vector are directed to specific epitopes on the vector which mean that the F(ab’)2 fragments remain neutralising (i.e. the neutralising activity is Fc-independent).

[0250] For some vector-based therapies neutralising antibody (Nab) assays are used to exclude patients from treatment with non-permissive antibody titers. By using an in vitro Nab assays it might on the one hand be observed that the activity of a single dose of an IgG- degrading enzyme is sufficient to convert patient NAb serum titers below the required threshold for their treatment despite the presence of F(ab’)2 fragments. For this group a single preconditioning dose of IgG-cleaving enzymes would be sufficient. Neutralizing antibodies that prohibit transduction as intact IgG but not as their F(ab’)2 fraction have been described previously (Mallery et al. (2010)).

[0251] On the other hand and as discussed above, it can also be observed from such an assay that following complete IgG cleavage, the cleavage by-products such as F(ab’)2 fragments can neutralize GT vectors in vitro Nab assays to levels which do not permit the administration of the subsequent therapy. For patients with this category of Nabs it will be imperative to adapt the treatment regime with IgG-cleaving enzymes in a way that allows for the wash-out of neutralizing F(ab’)2 fragments while at the same time to keep cleaving de novo intact IgG. As such, the present inventors have devised a regimen which will firstly allow for the determination and stratification of subjects who would require additional dosing with an IgG cysteine protease in order to receive a vector-based therapy. The regimen then provides a redosing schedule which seeks to reduce all F(ab’)2 containing products in the subject over a longer duration so that the benefit of the vector-based therapy is improved, particularly when compared to previous strategies which have used just a single conditioning dose of an IgG cysteine protease. This strategy is illustrated in Figure 7.

[0252] In the first step, the regimen would further stratify subjects who have already been determined through a NAb assay to have pre-existing neutralising antibodies against the vector-based therapy.

[0253] This first step could involve a further NAb assay to determine whether the neutralising antibodies against the vector-based therapy remain neutralising following cleavage by an IgG cysteine protease.

[0254] Principle of NAb assay in the context of a gene therapy

[0255] Briefly, target cells are seeded into culture plates. Human test serum or anti-vector-mAb (positive control) are serially diluted with matrix (e.g. sero negative human serum) and incubated with and without IgG-cleaving enzymes (15 pg / mL) for 2 hr before being added to the gene therapy vectors at 37°C for Ihr and subsequently to the target cells. After approx. 48 hrs, vector transduction can be examined based on the expression of reporter genes (e.g. luciferase). Inhibition of vector transduction by intact Nabs can be expressed as the percentage of transduction when compared to the intact IgG sample. Alternatively, a neutralising titer can be calculated from the cleaved sample, whereby serial dilutions are used to determine the titer at which 50% inhibition of transduction is achieved.

[0256] Principle of viral neutralization assay to measure NAb levels in the context of an oncolytic virus (OV) therapy Target cells are seeded in flat-bottom 96-well plates and allowed to adhere overnight in the incubator (37°C, 5% CO2, 90% humidity). The next day, human serum samples are incubated with or without IgG-cleaving enzymes (15 pg / mL) for 2hr at 37°C. For serum samples, a 2-fold dilution series (starting with 1 :5) can be prepared in duplicate in matrix (e.g. DMEM with 2% FCS, or sero-negative serum). Pool of OV seropositive IgG donors can be used as a positive control. Serum samples are mixed with OV for 30 minutes at 37°C to allow the binding of NAbs and F(ab’)2 fragments to viral particles. Next, the serum / OV complexes were transferred in duplicate onto target cells. Cell growth can be determined 3 days postinfection by viability staining (e.g. crystal violet staining).

[0257] Whether the sample following IgG cysteine protease cleavage is determined to be sufficiency neutralising may be determined using known thresholds for receiving a vector-based therapy such as a gene therapy. For example, previous studies into AAV- based gene therapies have considered a cut-off threshold of neutralizing titers of at least 1 :5, 1 :10, 1 :40 and 1 :50 (Mendell el al. (2022)).

[0258] If the in vitro assay determines that the subject has neutralising antibodies against the vector-based therapy which remain sufficiency neutralising following cleavage by an IgG cysteine protease, then a multi-dose regimen should be employed (e.g. using imlifidase or N240) in order to provide long-lasting enzymatic activity to reduce neutralising titers from all F(ab’)2 containing species. This protocol could be in accordance with the right hand side of Figure 7, which involves:

[0259] - Preconditioning with an IgG-degrading enzyme (such as an IgG cysteine protease such as imlifidase or N240, typically at a dose of 0.25 mg / kg BW, 0.5 mg / kg BW, 1 mg / kg BW, 1.5 mg / kg BW or 2.0 mg / kg BW) After an interval of time (e.g. 4-7 days, typically 4 or 5 days) which allows for the reduction in the initial levels of F(ab’)2 fragments, administering a further dose of an IgG-degrading enzyme (such as an IgG cysteine protease such as imlifidase or N240, typically at a dose of 0.25 mg / kg BW, 0.5 mg / kg BW, 1 mg / kg BW. 1.5 mg / kg BW, or 2.0 mg / kg BW) Subsequently carrying out the vector-based therapy which could be e.g. an oncolytic viral therapy or a gene therapy.

[0260] A further dose of an IgG-degrading enzyme such as an IgG cysteine protease (such as imlifidase or N240, typically at a dose of 0.25 mg / kg BW, 0.5 mg / kg BW, 1 mg / kg BW. 1.5 mg / kg BW, or 2.0 mg / kg BW) may be administered 1, 2 or 3 days after the vectorbased therapy. This may help maintain a low neutralizing antibody titer in the serum to improve the benefit of the vector-based therapy further. Without wishing to be bound by theory, it is expected that vector-based therapies may remain in circulation for several days (such as 2, 3 or 4 days) following administration and maintaining a low neutralizing titer in the serum of a subject will continue to provide benefit for the vector-based therapy.

[0261] The time interval between the first and last dose of the IgG cysteine protease should be no more than 14 days, as any further doses of the same IgG cysteine protease would be rendered ineffective through the generation of AD As. Ideally, the time interval between the first and last dose is no more than 9 days.

[0262] Example 6 — Pre-existing Anti-AAV8 neutralising antibodies in a patient

[0263] Material and methods - Anti-AAV8 cell-based NAb titer assay

[0264] Human serum samples, both non-cleaved and cleaved with IgG cleaving enzyme (either in vivo or in vitro), were analysed in a direct cell-based NAb assay. AAV8-neutralizing antibodies in serum were detected by measuring the reduction compared to negative control in relative light units (RLU) in the presence of antibodies that block AAV8-Luc gene transduction and consequently decreased gene expression.

[0265] Briefly, HEK293 (ACC 305, DSMZ) were cultured in complete cell culture media at 37 °C and in 5% CO2 (10% heat inactivated (HI) FBS + 1% penicillin-streptomycin in Gibco DMEM, high glucose, GlutaMAX). 20 000 cells / well were seeded on tissue culture treated 96-well plates and incubated overnight at 37 °C and in 5% CO2. On the next day, sera were diluted 1 :5 (MRD) followed by serial two-fold dilutions in dilution buffer (DMEM + 1 % HI FBS). The AAV8-CMV-LUC vector (CV10026-AAV8-100, Amsbio) was diluted to a calculated concentration to reach the desired multiplicity of infection (MOI) in the next step. The serial diluted serum samples and controls were mixed with virus, in equal volumes, and pre-incubated for 1 h at 37 °C. Dilutions of anti-AAV8 mouse monoclonal, ADK8 were used as high (200 ng / mL), medium (80 ng / mL) and low (40 ng / mL) positive controls and an anti-AAV8 negative serum pool (30 individuals) served as negative control. In all steps virus was handled in low binding plastics.

[0266] Thereafter, the serum / virus mixtures were transferred to the HEK293 cells at a MOI of 20 000. The cell plates were further incubation at 37 °C for gene transduction to take place. After 48 h, the cells were lysed by addition of LAR substrate (One-Gio Luciferase Assay System, #E6110 / E6120, Promega) and the luciferase signal was measured on a luminometer (SpectraMax i3, Molecular Devices). A high signal indicates that there was no or low level of anti-AAV8 NAb present in the serum sample and AAV8 was able to transduce the cells. On the contrary, a low signal indicates high anti-AAV8 NAb level in the sample. A high NAb level results in no or low reporter gene translation, since the NAbs bind to the AAV8 capsid preventing it from transducing the HEK-293 cells. The luminescence readings are expressed as relative light units (RLU) per second. Assay cut point (ACP) is 50%I (percent inhibition). Meaning that a serum dilution with above 50%I compared to plateau value for the analysed serum was reported as positive. If the inhibition was below ACP, the sample was reported as negative.

[0267] Material and methods - In vitro cleavage

[0268] Serum samples (TRINA Bioreactives, Switzerland) known to be positive for total IgG anti-AAV8 antibodies were in vitro cleaved with the IgG cleaving enzyme N240 at a final cleavage concentration of 21 pg / mL. Samples were either incubated with enzyme or with equal volume of PBS to get comparable samples. Cleavage was done in 80% serum and 20% enzyme / PBS. Incubation was performed at 37 °C on gentle agitation overnight. The next day all samples were heat inactivated at 56 °C for 30 min. The samples were run on SDS- PAGE, 10 well 4-20% Mini-PROTEAN® TGXTM precast gels, 200V for 40 min. ChemiDocTM MP Imaging System (ID-33) and software Image LabTM were used for analysing the gels.

[0269] Subject A - Pharmacodynamics

[0270] Subject A was a subject who was seropositive for anti-AAV8 antibodies. Subject A was administered with 0.25 mg / kg BW of imlifidase, and serum was analysed in accordance with the ECL method of Example 1 and nephelometry method of Example 4. Figure 8 shows the results in graphical form.

[0271] This further confirms the fact that in the context of a subject known to be seropositive for anti-AAV8 neutralising antibodies, the nephlometry method shows that the total amount of F(ab’)2 containing species decreases over time in the first ~5-6 days before reaching a nadir - this is much later than the initial decrease of the intact IgG species as shown in the ECL curve. The consequence is that around 5-6 days after the first dose the sum of all available molecules with a F(ab’)2 portion starts to increase. It can also be seen from the ECL curve that the intact IgG and scIgG species gradually increases after the initial sharp drop in concentration.

[0272] Subject A - cell-based NAb assay

[0273] Pre-serum and serum 48 hrs post imlifidase treatment were analysed in the anti-AAV8 cell -based NAb assay described above for evaluation of neutralizing F(ab’)2. Figure 9 shows the raw data from the luciferase assay. Anti-AAV8 NAb titers for Subject A, before and 48 hrs after treatment with 0.25 mg / kg imlifidase were calculated to be 1 :20 and 1 :5, respectively. The neutralizing capacity of serum from this individual has clearly decreased due to cleavage of intact IgG and clearance of F(ab’)2. Residual F(ab’)2 fragments still provide some degree of inhibition on gene transduction but to a lower extent than pre-treatment. This shows the potential utility of the dosing strategy of the present invention to further reduce the neutralising titer in such individuals.

[0274] In vitro cleaved sera tested in cell-based NAb assay

[0275] Serum was extracted from several subjects known to be seropositive for anti-AAV8 antibodies. The serum was subjected to in vitro cleavage in accordance with the methods described above. Anti-AAV8 NAb titers were calculated for the sera both before and after cleavage. In these experiments, the cleaved products including the F(ab’)2 fragments remain in the sample during the titer determination. The results are shown in the table below.

[0276] In almost every case, just cleaving the intact IgG into Fc and F(ab’)2 fragments reduces the NAb titer. This shows that while the initial cleavage is effective in reducing neutralising titer as expected (particularly in combination with rapid clearance of F(ab’)2 fragments from circulation), some subjects - particularly those with high preexisting neutralising titers - would benefit from one or more additional doses of the IgG cysteine protease to allow for further clearance of any neutralising F(ab’)2 fragments while keeping rebound IgG levels down, so that the neutralising titer can be kept as low as possible.

Claims

CLAIMS1. A method of treating a subject in need of a vector-based therapy, the method comprising:(a) administering to the subject a first dose of an IgG cysteine protease;(b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and(c) subsequent to step (b), administering the vector-based therapy; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a).

2. A method of improving the benefit to a subject of a vector-based therapy, the method comprising:(a) administering to the subject a first dose of an IgG cysteine protease;(b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and(c) subsequent to step (b), administering to the subject the vector-based therapy; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a).

3. A method of treating a disease in a subject, the method comprising:(a) administering to the subject a first dose of an IgG cysteine protease;(b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and(c) subsequent to step (b), administering a vector-based therapy which treats the disease; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a).

4. A method for reducing or inhibiting an immune response against a vectorbased therapy, the method comprising:(a) administering to the subject a first dose of an IgG cysteine protease;(b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and(c) subsequent to step (b), administering the vector-based therapy; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a).

5. The method according to any one of claims 1-4, wherein the vector-based therapy comprises a cell therapy, a non-viral vector, or a viral vector; optionally wherein the vector-based therapy comprises a viral vector, more optionally wherein the viral vector comprises a transgene.

6. A method for increasing or improving transduction of a transgene into a cell in a subject, the transgene delivered by way of a viral or non-viral vector, the method comprising:(a) administering to the subject a first dose of an IgG cysteine protease;(b) subsequent to step (a), administering to the subject at least one further dose of an IgG cysteine protease; and(c) subsequent to step (b), administering the viral or non-viral vector comprising the transgene; wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of step (b) is the same as the IgG cysteine protease of step (a).

7. The method according to claim 5 or 6, wherein the viral or non-viral vector is a gene therapy vector; or wherein the viral vector is an oncolytic virus.

8. A method according to any one of claims 5-7, wherein the:(i) Non-viral gene therapy vector is selected from liposomes, lipid nanoparticles (LNPs), highly branched poly(P-amino ester) (HPAE), single-chain cyclic polymer (SCKP), poly(amidoamine) (PAMAM) dendrimers, and polyethyleneimine (PEI);(ii) Viral gene therapy vector is selected from an AAV vector, an adenoviral vector, a chimpanzee adenoviral (ChAd) vector, a lentiviral vector, a Modified vaccinia virus Ankara (MV A); Herpes simplex virus (HSV); Vesicular stomatitis virus (VSV); optionally wherein the AAV vector is AAV1 , AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAVcylO and AAVrhlO, AAVrh74, AAVDJ, AAV-Anc80, AAV-LK03, AAV2i8, AAVpo4 or AAVpo6; or(iii) oncolytic virus is selected from adenovirus (such as Chimeric adenovirus vectors Ad5 / 3, Ad5 / 32, Adi Ip / Ad3), Herpesvirus Parvovirus, Poxvirus, Semiliki Forest virus, Sindbis virus, Zika virus, Measles virus, Newcastle disease virus, Coxsackievirus, Polio virus, Seneca valley virus, Reovirus, Vesicular stomatitis virus (VSV), Maraba virus, Vaccinia virus.

9. The method according to any one of the preceding claims, wherein the interval between doses of an IgG cysteine protease is 2, 3, 4, 5, 6, 7, 8 or 9 days.

10. The method according to any one of the preceding claims, wherein the interval between doses of an IgG cysteine protease is 4, 5, or 6 days.

11. The method according to any one of the preceding claims, wherein the interval between doses of an IgG cysteine protease is 4 or 5 days.

12. The method according to any one of the preceding claims, wherein the interval between doses of an IgG cysteine protease is at from about 96 hours to about 120 hours.

13. The method according to any one of claims 1 to 12, wherein the interval between doses of an IgG cysteine protease is at from about 84 hours to about 108 hours.

14. The method according to any one of the preceding claims, comprising a further step (d) comprising: subsequent to step (c) administering to the subject one further dose of an IgG cysteine protease, optionally wherein the interval between step (c) and step (d) is no more than 3 days, more optionally no more than 2 days or 1 day.

15. The method of claim 14, wherein the IgG cysteine protease of step (d) is the same as the IgG cysteine protease in step (a) and / or (b).

16. The method according to any one of the preceding claims, wherein the method consists of two doses of an IgG cysteine protease prior to step (c).

17. The method according to any one of claims 1-15, wherein the method consists of three doses of an IgG cysteine protease prior to step (c) and the interval between doses of an IgG cysteine protease is independently selected from 2, 3, 4, 5, or 6 days.

18. The method according to any one of the preceding claims, wherein the interval between the first and final doses of an IgG cysteine protease is no more than 9 days.

19. The method according to any one of the preceding claims, wherein the first dose of an IgG cysteine protease is administered at a dosage of 0.1 to 2 mg / kg BW.

20. The method according to any one of the preceding claims, wherein the first dose of an IgG cysteine protease is administered at a dosage of 0.2 to 2 mg / kg BW, optionally at a dosage of 0.2 to 1 mg / kg BW, 0.2 to 0.5 mg / kg BW, 0.5 to 1 mg / kg BW, 0.5 to 2 mg / kg BW, or 1 to 2 mg / kg BW.

21. The method according to any one of the preceding claims, wherein the first dose of an IgG cysteine protease is administered at a dosage of about 0.25 mg / kg BW, 0.5 mg / kg BW, 1 mg / kg BW, 1.5 mg / kg BW or 2 mg / kg BW.

22. The method according to any one of the preceding claims, wherein the at least one further dose of an IgG cysteine protease is administered at a dosage of 0.1 to 2 mg / kg BW.

23. The method according to any one of the preceding claims, wherein the at least one further dose of an IgG cysteine protease is administered at a dosage of 0.2 to 2 mg / kg BW, optionally at a dosage of 0.2 to 1 mg / kg BW, 0.2 to 0.5 mg / kg BW, 0.5 to 1 mg / kg BW, 0.5 to 2 mg / kg BW, or 1 to 2 mg / kg BW.

24. The method according to any one of the preceding claims, wherein the at least one further dose of an IgG cysteine protease is administered at a dosage of about, 0.25 mg / kg BW 0.5 mg / kg BW, 1 mg / kg BW, 1.5 mg / kg BW or 2 mg / kg BW.

25. The method according to any one of the preceding claims, wherein the interval between steps (b) and (c) is no more than 4 days.

26. The method according to any one of the preceding claims, wherein the interval between steps (b) and (c) is no more than 3 days, optionally no more than 2 days or 1 day.

27. The method according to any one of the preceding claims, wherein the subject has pre-existing neutralising IgG antibodies against the vector-based therapy, wherein said pre-existing neutralising IgG antibodies remain neutralising when digested to F(ab’)2 fragments.

28. The method according to claim 27, wherein the subject has been determined to have said pre-existing neutralising IgG antibodies if sera from said subject following in vitro cleavage with an IgG cysteine protease retains a neutralising titre of at least 1 :5, 1 :10, 1 :20, 1 :40, 1 :80, 1 : 160, 1 :320, or 1 :640, optionally wherein the in vitro cleavage is performed with the same IgG cysteine protease as the IgG cysteine protease of step (a), step (b) and / or step (d).

29. The method according to claim 27, wherein the subject has been determined to have said pre-existing neutralising IgG antibodies if sera from said subject following in vitro cleavage with an IgG cysteine protease retains a neutralising capacity of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to non-treated sample, optionally wherein the in vitro cleavage is performed with the same IgG cysteine protease as the IgG cysteine protease of step (a), step (b) and / or step (d).

30. The method according to any one of the preceding claims, wherein the IgG cysteine protease of step (a), step (b) and / or step (d) is an IgG cysteine protease from a Streptococcus bacterium, such as Streptococcus pyogenes, Streptococcus equi or Streptococcus zooepidemicus, optionally wherein said IgG cysteine protease is a IdeS, MAC2, SpeB, IdeZ, IdeS / Z or IgdE polypeptide.

31. The method according to any one of the preceding claims, wherein the IgG cysteine protease of step (a), step (b) and / or step (d) comprises or consists of a sequence that is at least 80% identical to SEQ ID NO: 2, 4, 5 or 90 such as at least 85%, 90%, 95%, 99% or 100% identical, or wherein said IgG cysteine protease comprises or consists of the sequence of any one of SEQ ID NOs: 6 to 25, 56 to 69 optionally wherein said sequence includes an additional methionine at the N terminus and / or a histidine tag at the C terminus.

32. The method according to any one of the preceding claims, wherein said IgG cysteine protease is a polypeptide comprising or consisting of a sequence that is at least 80% identical to SEQ ID NO: 90 or 91, such as such as at least 85%, 90%, 95%, 99% or 100% identical, optionally wherein said sequence includes an additional methionine at the N terminus and / or a histidine tag at the C terminus.

33. The method according to any one of claims 3, 5 and 7-32, wherein the disease is:(i) selected from a lung disease ( e.g ., cystic fibrosis), a bleeding disorder {e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, a blood disorder {e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis (ALS), epilepsy, a lysosomal storage disease {e.g., aspartylglucosaminuria, Batten disease, late infantile neuronal ceroid lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease II (Pompe disease), GM2-gangliosidosis type I (Tay Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis types I (sialidosis type I and II), II (I-cell disease), III (pseudo- Hurler disease) and IV, mucopolysaccharide storage diseases (Hurler disease and variants, Hunter, Sanfilippo Types A,B,C,D, Morquio Types A and B, Maroteaux-Lamy and Sly diseases), Niemann-Pick disease types A / B, Cl and C2, and Schindler disease types I and II), hereditary angi oedema (HAE), a copper or iron accumulation disorder {e.g., Wilson’s or Menkes disease), lysosomal acid lipase deficiency, a neurological or neurodegenerative disorder, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, a metabolic defect {e.g., glycogen storage diseases), a disease of solid organs {e.g., brain, liver, kidney, heart), or an infectious viral {e.g., hepatitis B and C, HIV, etc.), bacterial or fungal disease. In certain embodiments, a subject has a blood clotting disorder. In certain embodiments, a subject has hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, a deficiency in any coagulation Factor: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or a combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase Cl deficiency or gamma-carboxylase deficiency;(ii) selected from anemia, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anti coagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e., FXa inhibitors), or a platelet disorder such as, Bernard Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency;(iii) selected from a disease that affects or originates in the central nervous system (CNS). In certain embodiments, the disease is a neurodegenerative disease. In certain embodiments, the CNS or neurodegenerative disease is Alzheimer’s disease, Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy’s disease, a polyglutamine repeat disease, or Parkinson's disease. In certain embodiments, the CNS or neurodegenerative disease is apolyglutamine repeat disease. In certain embodiments, the polyglutamine repeat disease is a spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17); or (iv) cancer, optionally a cancer selected from brain cancer, breast cancer, colon cancer, esophageal cancer, prostate cancer, bladder cancer, kidney cancer, endometrial cancer, thyroid cancer, gastric cancer, liver cancer, lung cancer, pancreatic cancer, uterine cancer, tracheal cancer, testicular cancer, cervical cancer, head and neck cancer, skin cancer, soft tissue sarcoma, bone cancer, pancreatic cancer and ovarian cancer, or a hematological cancer such as leukemia, lymphoma and multiple myeloma.

34. A conditioning regimen for administering a vector-based therapy to a subject, comprising:(a) administering to the subject a first dose of an IgG cysteine protease;(b) subsequent to step (a), optionally administering to the subject at least one further dose of an IgG cysteine protease; and(c) subsequent to step (b), administering the vector-based therapy;(d) subsequent to step (c) administering to the subject one further dose of an IgG cysteine protease, optionally wherein the interval between step (c) and step (d) is no more than 3 days, more optionally no more than 2 days or 1 day wherein the interval between doses of an IgG cysteine protease is at least 2 days and no more than 9 days, optionally wherein the IgG cysteine protease of steps (a), (b) and (d) are the same.

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