Synthetic polymeric conjugates to inhibit Anti-adeno-associated virus neutralizing antibodies and medical use thereof

Abstract

The present invention provides a group of compounds for the sequestration of Adeno-associated virus (AAV) and adenovirus (AdV) neutralizing antibodies (NAbs) in a patient. The compounds comprise an inert backbone conjugated with at least one recombinant peptide comprising an epitope. The epitope is a conserved epitope that is targeted by human anti-AAV or anti-AdV NAbs. The backbone where the conserved epitope is conjugated to is a poly-lysine (PLys) or poly-acrylamide (PAA) backbone, among others. Also provided is a pharmaceutical composition comprising this type of compounds, as well as a method for use to treat an individual to inhibit the immune reaction against an AAV or AdV-based therapy. It is also possible to use synthetic compounds for an in vitro detection of anti-AAV or anti-AdV NAbs in a sample.

Description

[0001] P7120PC00 1

[0002] TITLE: Synthetic polymeric conjugates to inhibit anti-adeno-associated virus neutralizing antibodies and medical use thereof

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the field of therapeutic treatment, particularly to gene therapy, vaccine technology, and in vitro methods. Specifically, it relates to a group of polymeric conjugated compounds comprising conserved epitopes to inhibit anti-Adeno-associated virus (AAV) and antiAdenovirus (AdV) neutralizing antibodies (NAbs) for increasing efficiency of non-pathogenic viral vectors and reducing immune reactions to these viral vectors, such as used in vaccines or in gene therapy. It further relates to new polymeric conjugate compounds, compositions comprising thereof, a method for obtaining thereof, their therapeutic or prophylactic uses, and a kit for the detection and / or quantification of anti-AAV and anti-AdV NAbs, comprising said group of compounds.

[0005] BACKGROUND OF THE INVENTION

[0006] Vaccines and human gene therapies using recombinant adeno-associated virus (rAAV) vectors have demonstrated enormous promise. In fact, the rAAV vector-based gene therapy is currently the only in vivo gene therapy approved by regulatory agencies for clinical use. The success of AAV as the leading gene delivery modality is based on a multitude of factors: (i) even wild-type AAV has not been shown to cause any disease, (ii) in contrast to other currently available gene delivery methods, in non-dividing or very slowly dividing cells AAV results in the long-term expression of the therapeutic payload, even though the AAV vector DNA persists mostly as extrachromosomal episomes, (iii) rAAV vectors are not strongly immunogenic. While rAAVs lack many characteristics of the wild-type viruses from which they are derived, they nevertheless retain legacy immunogenic features that affect their safety and efficacy (Hamilton BA, Wright JF, 2021).

[0007] AAVs infect humans and other mammalian species starting from the first years of life, but the exposure to these viruses has not been clearly associated with any clinical pathology or disease. The natural AAV Infection results in the formation of a humoral response against the virus followed by a large proportion (ranging from 30% to 60%) of the infected population developing cross-reactive anti- AAV NAbs possibly due to successive infections and / or broad cross-reactivity between AAV serotypes. Although the infection with wild-type AAV results in antibodies from all IgG subclasses, lgG1 appears to be the predominant immunoglobulin subclass in wild-type AAV seropositive individuals. The levels of IgG, in general, correlate with the neutralizing antibody titers, although it has been reported the presence of IgG that while binding to AAV vectors did not neutralize their capacity to enter the cells in vitro and in vivo. More specifically, titers of lgG1 , lgG2, and IgM are well correlated with the level of NAbs, which is not the case for lgG3 and lgG4. Similarly, in subjects undergoing AAV vector gene transfer, it has been documented a significant development of high-titer IgG 1 antibodies together with NAbs (Gross DA et al., 2022; Weber T, 2021 ; Hamilton BA, Wright JF, 2021 ; P7120PC00 2

[0008] Costa Verdera H et al., 2020; Mingozzi F, High KA, 2013).

[0009] There are several different natural AAV serotypes (AAV1-AAV13) that have been isolated in nature, which differ in the sequence of their capsid. The capsid serotype and the presence of a specific receptor on the host cells determine the tropism of each AAV serotype for a tissue. This is a property that makes AAVs versatile vectors adaptable to a broad range of therapeutic applications (Hadi M et al., 2024; Costa Verdera H et al., 2020).

[0010] AAV seroprevalence varies geographically, with anti-AAV2 NAbs displaying the highest prevalence, ranging from 30% to 60% of the population. The prevalence of total anti-AAV antibodies is close to 70% of the population for AAV1 and AAV2, 45% for AAV6 and AAV9, and 38% for AAV8. The systemic delivery of rAAV vectors in the presence of NAbs has been shown to result in the accumulation of vector components in lymphoid organs. Additionally, due to the broad cross-reactivity between AAV serotypes, NAbs recognizing virtually all serotypes can be found in almost all subjects. This cross-reactivity reflects the amino acid sequence and structural homology across capsids of different AAV serotypes. Cross-reactivity also represents a big throwback for the use of AAV gene therapy since the anti-AAV NAbs can have a profound impact on the efficacy of gene transfer and it is also a concern in terms of safety (Weber T, 2021 ; Costa Verdera H et al., 2020; Mingozzi F, High KA, 2013).

[0011] Pre-existing anti-AAV neutralizing antibodies are a vexing problem for AAV gene therapy because they can severely limit the patient population that could benefit from it. In addition, the induction of anti-AAV NAbs with long persistence and wide specificity after injection of rAAV constitutes a significant barrier to vector re-administration in patients who have received rAAV and subsequently experienced loss of therapeutic transgene expression. Thus, overcoming of humoral immune response to AAV is key to extend therapy to patients with NAbs, either as a result of natural infection by wild type AAV (pre-existing antibodies) or prior treatment with an AAV vector (induced antibodies). So far, the approach to the problem of preexisting humoral immunity to AAV in clinical trials has been the exclusion of seropositive subjects from enrollment; however, this solution is far from being optimal, as it results in the exclusion of a significant proportion of otherwise potentially eligible patients (Weber T, 2021 ; Bertin B et al., 2020; Gross DA et al., 2022).

[0012] Different methods have been developed to reduce the impact of anti-AAV NAbs in a patient. Among them, immunosuppression strategies have always seemed to be an alternative with a high potential in the clinical setting. Unfortunately, clinical data so far demonstrate that the efficacy of this approach is not consistently reliable, and appears to be largely confined to individuals with low baseline anti- AAV NAbs titers or those who are likely naive to AAVs (Gross DA et al., 2022).

[0013] In theory, the most appealing approach to overcome the challenge of pre-existing NAbs is to introduce mutations into the AAV capsid that prevent the binding of NAbs. However, the significant cross- P7120PC00 3 reactivity among AAV serotypes shows that this is not an easy feat to achieve. Nonetheless, it has been demonstrated that the introduction of mutations into the AAV2 capsid can attenuate the sensitivity of these mutant viruses to neutralization. However, for most viruses including AAV, a large fraction of the neutralizing antibodies is directed against the receptor binding domain(s). Consequently, one would think that mutating residues in the receptor binding region(s) could constitute the most straightforward way to ameliorate neutralization. However, mutations in the receptor binding region(s) are also very likely to affect viral tropism and / or transduction efficiencies (Weber T, 2021).

[0014] Among the rest of strategies to address the issue of AAV NAbs, plasmapheresis is an attractive approach as it represents a safe and clinically established technology broadly available. Plasmapheresis is a process used to selectively remove blood components (e.g. plasma replacement) used to treat a variety of conditions including those caused by the acute overproduction of antibodies (e.g. autoimmunity, transplant rejection), in which removal of pathogenic immunoglobulins results in clinical benefit. In gene therapy with AAV vectors, plasmapheresis offers some potential advantages, including (i) it is a relatively simple and low-risk procedure, and (ii) the antibody depletion is only transient thus unlikely to result in prolonged immunosuppression (Gross DA et al., 2022). This strategy had been only tested in a non-human primate model of AAV gene transfer, and required the development of an AAV-specific immune absorption column able to highly-efficiently deplete anti- AAV antibodies from a variety of plasma samples. Depletion of antibodies directed against the capsid did not affect total IgG levels and resulted in a dramatic decrease in neutralizing activity of plasma. Matrix optimization and the use of empty AAV particles to capture antibodies further improved the efficacy of the approach as confirmed in vivo in a passive immunization model of gene transfer. Despite its advantages, plasmapheresis is also characterized by potential drawbacks associated with the need for repeated cycles over time to completely eliminate detrimental antibodies and the lack of specificity of antibody removal, which inevitably can result in hypogammaglobulinemia, potentially exposing patients to an enhanced risk of infection (Berlin B et al., 2020).

[0015] An alternative to the use of the prior strategies could be the reduction of circulating IgG with the use of IgG-cleaving proteases. This method is particularly attractive because of its ease of use. It has been demonstrated recently the efficacy of the IgG degrading enzyme of Streptococcus pyogenes (IdeS) for the degradation of anti-AAV antibodies and the successful administration of AAV vectors in seropositive non-human primates (NHPs). In particular, IdeS pre-treatment improved liver transduction efficacy in seropositive NHPs both in the context of first administration in monkeys naturally exposed to AAVs and in a re-administration setting. IdeS has the potential to enable AAV vectors administration in seropositive patients even in the context of repeated administration of the same vector. One limitation toward the clinical use of IdeS in this setting could be that the repeated administration of IdeS may trigger a hypersensitivity reaction. The use of IdeS is also a non-specific approach since it depletes or reduces the total levels of circulating immunoglobulins, which may be associated with an increasing risk of developing infections (Gross DA et al., 2022). P7120PC00 4

[0016] Different strategies trying to accomplish the inhibition / adsorption / removal of natural antibodies have been disclosed in the state of the art, involving in vitro, extracorporeal, or in vivo treatments. Some of these examples are listed next:

[0017] EP 4217006 A1 discloses a compound for the sequestration of undesirable NAbs against viral vectors, such as adeno-associated virus vectors used in vaccines and gene therapy. The compound comprises a biopolymer scaffold with at least two peptides derived from a capsid of a viral vector. The biopolymer scaffold is selected from the group consisting of albumins, alpha-globulins, alpha2- globulins, beta-globulins and immunoglobulins. The viral vector is an adenovirus (AdV) vector or an adeno-associated virus (AAV) vector. The compound is used to overcome the preexisting humoral immunity against the viral vector by removing the immunoglobulins.

[0018] US 6022544 A1 discloses a method for reducing an undesired antibody response in a mammal using a non-immunogenic construct comprising a biopolymer scaffold to which epitopes are conjugated via linker. The biopolymer scaffold is described as dextran, albumin, and immunoglobulin. However, when peptides are conjugated to a scaffold, only dextran is used.

[0019] EP 1832600 A1 discloses a peptide bound to a solid phase scaffold, which is useful to be used in detecting, binding, complexing, or neutralizing antibodies directed against beta-2-adrenergic receptor. The invention is also potentially used for diagnosing, treating, preventing, and to follow-up glaucoma disease. The peptides are bound to the scaffold surface by a linker or spacer. The scaffold is listed as agarose, cellulose, silica gel, polyamide and polyvinyl alcohols.

[0020] WO 2020 / 193486 A1 discloses a compound for the sequestration of undesirable NAbs against viral vectors used in vaccines and gene therapy. The compound comprises a biopolymer scaffold with at least two peptides that are each bound to the biopolymer scaffold via a linker. The biopolymer scaffold is selected from the group consisting of alpha-globulins, alpha2-globulins and beta-globulins.

[0021] WO 00 / 20041 A2 discloses the extracorporeal removal of anti-adenovirus antibodies from plasma using affinity columns comprising hexon, penton and fiber as immunosorbents.

[0022] Mingozzi F, High KA, 2013 discloses empty AAV8 or AAV2 capsids acting as decoys used for removing intravenous immunoglobulins (Mg) which are administered in excess together with an AAV8 vector expressing human factor IX.

[0023] Bertin B et al., 2020 discloses the use of empty AAV capsids bound to a resin in a column for extracorporeal depleting anti-AAV antibodies from plasma.

[0024] Assessment of anti-adeno-associated virus (AAV) antibodies in patients prior to systemic gene therapy administration is an important consideration regarding efficacy and safety of the therapy. There P7120PC00 5 is a need for a reliable approach to measuring anti-AAV neutralizing antibodies in patient blood, plasma or serum, to understand the potential safety and efficacy consequences associated with the presence of these antibodies, and to support the development of strategies to mitigate the effects of both pre-existing and treatment-induced anti-AAV neutralizing antibodies in patients. (Schulz M et al., 2023).

[0025] For AAV-based gene therapies to be applicable to a broader range of indications and reach more patients, immune responses to rAAV must be reduced and better controlled. To date, the reality is that the formation of anti-capsid NAbs restricts rAAV gene therapy to a single administration, at least for the commonly used systemic route of administration. Furthermore, while capsid-specific cytotoxic T-lymphocyte responses can sometimes be controlled by immune suppression, many diseases necessitate systemic administration of rAAV vectors at high doses where immunomodulation is less effective, and inflammatory toxicities are likely to be worsened by strong innate immunogenic features of the vector.

[0026] However, although many approaches to remove AAV neutralizing antibodies have been disclosed in the prior art, they come with many shortcomings. Neither of them has been approved for regular clinical use. With the aim of overcoming the current drawbacks in the technical field, it is thus a goal of the present invention to provide improved compounds and methods forthe removal, sequestration, or reduction of AAV neutralizing antibodies or AdV neutralizing antibodies in an individual. In particular a compound for use prior to or concurrently with an AAV vector-based therapy or an AdV vectorbased therapy for lowering, reducing, sequestering, or depleting the corresponding neutralizing antibodies, with the object of improving the efficacy of such therapy.

[0027] SUMMARY OF THE INVENTION

[0028] The invention provides a product, a method for obtaining said product, a pharmaceutical composition comprising the claimed product, a method for use of the claimed product, and a kit for performing said claimed method involving the claimed product.

[0029] Based on the experimental evidence provided herein, the present invention discloses a group of synthetic conjugates having a polymeric backbone which is conjugated efficiently and in a stable manner with at least one recombinant peptide comprising an AAV epitope. Each of the recombinant peptides are bound to the polymeric backbone by means of a linker each. The invention also comprises a non-immunogenic polymeric backbone or scaffold (e.g., see EXAMPLE 1 , Figure 3 for toxicity assay) with the capacity to functionally display a plurality of AAV or AdV epitopes increasing the avidity of the polymeric conjugate by anti-AAV neutralizing antibodies. Epitopes are spaced from the structure through a linker that allows the effective exposition of epitopes and avoids steric hindrance and the collapse of the conjugates. P7120PC00 6

[0030] EXAMPLE 1 provides experimental data on a polymeric conjugate employed by the applicant prior to the present invention, demonstrating its ability to remove harmful antibodies while showing no evidence of toxicity.

[0031] EXAMPLE 2 shows the multi-step process for the identification of neutralizing AAV epitopes. Table 1 represents the specific matrix of human anti-AAV neutralizing antibodies obtained by peptide arrays. Table 2 shows a list of consensus sequences and singletons conserved across serotypes obtained by a further bioinformatic analysis of the identified sequences. Table 3 shows a selection of 12 consensus and singletons sequences that were used as free peptides as well as recombinant peptides conjugated to PLys or PAA for the in vitro proof of concept.

[0032] EXAMPLE 3 demonstrates the synthesis of free peptides and different synthetic polymeric conjugates.

[0033] EXAMPLE 4 demonstrates the AAV-NAbs inhibitory activity of free peptides (i.e., neutralization capacities exerted by the free peptides) by suspension treatment. Peptide 1.1 (SEQ ID NO: 1), peptide 2.1 (SEQ ID NO: 4), peptide 3.1 (SEQ ID NO: 7), peptide 4.1 (SEQ ID NO: 9), peptide 5.1 (SEQ ID NO: 10), peptide 8.1 (SEQ ID NO: 13), peptide 13.1 (SEQ ID NO: 18), peptide 14.1 (SEQ ID NO: 19), peptide 17.1 (SEQ ID NO: 22), peptide 19.1 (SEQ ID NO: 24), peptide 19.1 M (SEQ ID NO: 303), and peptide 23.1 (SEQ ID NO: 28) were tested.

[0034] EXAMPLE 5 demonstrates the neutralization capacities exerted by PLys conjugates by suspension treatment. Peptide 1.1 C (SEQ ID NO: 191), peptide 2.1 C (SEQ ID NO: 192), peptide 3.1 C (SEQ ID NO: 193), peptide 5.1 C (SEQ ID NO: 195) and peptide 17.1 C (SEQ ID NO: 199) conjugates were tested.

[0035] EXAMPLE 6 demonstrates the neutralization capacities exerted by PLys conjugates by solid-phase treatment to mimic in vivo conditions. Peptide 1.1 C (SEQ ID NO: 191), peptide 2.1 C (SEQ ID NO: 192), peptide 3.1 C (SEQ ID NO: 193), peptide 5.1 C (SEQ ID NO: 195) and peptide 17.1 C (SEQ ID NO: 199) conjugates were tested. As shown in EXAMPLES 4-6, it is emphasized that the infectivity of the AAV serotypes increases as a result of the inhibition of anti-AAV neutralizing antibodies.

[0036] EXAMPLE 7 demonstrates that peptide 1 .10, peptide 2.1 C, peptide 3.1 C, peptide 5.1 C and peptide 17.1 C conjugates effectively inhibit AAV-neutralizing antibodies from normal human serum samples. As a result of this inhibition, the infectivity of the chimeric AAVs is substantially increased in cellbased neutralization assays.

[0037] EXAMPLE 8 shows the identification of commercial mouse-derived anti-AAV monoclonal antibodies. The selected antibodies were tested against their corresponding AAV serotypes, as well as the chimeric vectors used in this study, AAV-DJ and AAV9P31 . P7120PC00 7

[0038] EXAMPLE 9 shows the identification of peptide epitopes for antibodies A1 , B1 , A20, ADK1 a, ADK6, ADK8 and ADK9 by peptide arrays. Table 10 represents the list of the top-ranked antibody-inhibitory sequences selected for peptide synthesis.

[0039] EXAMPLE 10 demonstrates the linear epitope validation for antibodies A1 , B1 , A20, ADK1 a, ADK6, ADK8 and ADK9. Free peptides A20.2.2 (SEQ ID NO: 283) and AD6.1 .3 (SEQ ID NO: 299) demonstrated excellent performance in neutralizing their respective antibodies, A20 and ADK6 (Figure 25).

[0040] EXAMPLE 11 represents a comparative analysis between the previously identified peptide sequences in human serum using peptide arrays (Tables 1-3) and the newly identified peptide sequences using anti-AAV monoclonal antibodies and the same peptide arrays.

[0041] EXAMPLE 12 represents the in vitro characterization of A20-specific binding to PLys conjugates. EXAMPLE 12.1 shows the synthesis of newly developed PLys conjugates, and EXAMPLES 12.2 and 12.3 demonstrate the specific binding of the anti-AAV antibodies to these PLys conjugates. Figure 26 shows the results of the specific binding for conjugates 13.1 M (SEQ ID NO: 305), 2.1 (SEQ ID NO: 4), 2.1 C (SEQ ID NO: 192), 3.1 (SEQ ID NO: 7), 3.1 C (SEQ ID NO: 193), and 17.1 (SEQ ID NO: 22).

[0042] EXAMPLE 13 describes the in vitro evaluation of A20 monoclonal antibody removal following treatment with PLys conjugates. These assays were performed using both conjugate 13.1 M and A20.2.2M. EXAMPLE 13.1 represents the synthesis of PLys conjugates and, EXAMPLES 13.2 and 13.3 show the in vitro treatment to validate the antibody-blocking effect mediated by these PLys conjugates.

[0043] EXAMPLE 14 shows the synthesis of newly synthetic polymeric conjugates with cellobiose.

[0044] The present invention concerns a laborious and non-obvious process for the identification of specific linear sequences capable of inhibiting anti-AAV antibodies. Unlike conventional approaches that merely identify sequences present in AAVs, the present identification method enables the detection of linear, non-conformational epitopes that effectively bind to and neutralize antibodies responsible for AAV inactivation. Many of these epitopes correspond to short linear peptide sequences, and it is not obvious that such short linear motifs would be sufficient to mediate specific recognition or inhibition of neutralizing antibodies involving conformational paratopes. The inventors have successfully identified binding sequences with inhibitory capacity against neutralizing antibodies, thereby providing a novel means to overcome immune interference in AAV-based applications. All identified sequences were derived from the human anti-AAV neutralizing pool and mouse monoclonal anti-AAV antibodies, and were obtained solely through experimental procedures, without the use of theoretical or computational predictions. The sequences disclosed in Table 1 and Table 10 represent the result P7120PC00 8 of an empirical screening process that allowed the selection and validation of linear motifs exhibiting specific anti-AAV neutralizing activity.

[0045] The success of the present invention relies on:

[0046] (i) the identification of a conserved pattern of AAV neutralizing epitopes from polyclonal human NAbs, which were exclusively isolated from normal human serum (NHS) samples (Figure 13) and also mouse monoclonal anti-AAV antibodies. The inventors identified high-affinity peptides through peptide array screening, enabling the selection of sequences that specifically bind anti-AAV antibodies, including those targeting conformational epitopes. Notably, A20.2.2 and AD6.1.3 emerged as top candidates with exceptional neutralization performance (EXAMPLE 13). Furthermore, many sequences identified in EXAMPLES 7-13 were consistent with previously reported neutralizing sequences of EXAMPLE 2, reinforcing the reliability of the peptide array screening approach;

[0047] (ii) the use of a non-immunogenic polymeric backbone, linkers, and capping agents allowing the clinical use of the polymeric conjugates for the intracorporeal removal of circulating NAbs without side effects (Figures 1 to 3). The compounds in this invention could diminish the degree of the immune response against rAAV -based therapy, which in turn would affect directly to the efficacy of the therapy at stake. For instance, the treatment with PLys-conjugates 13.1 M and A20.2.2M efficiently blocked A20 antibody (EXAMPLE 13), achieving up to ~80% removal with 13.1 M and nearly 100% with A20.2.2M, even at low concentrations. This validates the potential of these conjugates to neutralize circulating anti-AAV antibodies and improve vector availability;

[0048] (iii) the backbone's polymeric nature allows the stable conjugation of a high number of recombinant peptides in the same polymeric conjugate, this number of recombinant peptides bound to the polymeric backbone being higher than any other compound described in the prior art, maximizing the avidity of compounds by NAbs. This property is critical to allow high rates of NAbs inhibition and increase the safety and efficacy of rAAV-based therapies. The inventors also demonstrated the specific antibody-conjugate binding, e.g., in EXAMPLES 12 and 13, conjugation of the selected peptides to PLys maintained their binding specificity. A20 antibody exhibited strong and specific binding to PLys-conjugate 13.1 M, whereas interactions with non-target conjugates (17.1) or the PLys scaffold alone were minimal, confirming low nonspecific binding;

[0049] (iv) the use of the identified epitopes and polymeric conjugates for in vitro methods to identify and measure the levels of NAbs in human samples.

[0050] These results collectively indicate that these conjugates, particularly peptide-PLys conjugates, can specifically and efficiently bind and inhibit anti-AAV neutralizing antibodies, including those recognizing conformational epitopes, providing a promising strategy to enhance the efficacy of chimeric AAV- mediated gene therapy. P7120PC00 9

[0051] The first object of protection of this application describes a group of novel synthetic polymeric conjugates for the removal or the detection of anti-AAV or anti-AdV neutralizing antibodies.

[0052] The present invention, according to the first aspect, is firstly directed to a synthetic polymeric conjugate comprising:

[0053] (i) a polymeric backbone;

[0054] (ii) one or more recombinant peptides; and

[0055] (iii) a linker; wherein the polymeric backbone is made of a plurality of monomeric units, each unit being bound to the following consecutive unit forming a chain, and to a single recombinant peptide; the polymeric backbone being selected from the group consisting of: homologous a-amino acid polymers (homopolypeptides), heterologous a-amino acid polymers (heteropolypeptides), protein polymers, lipid polymers, nucleic acid polymers, acrylic acid polymers, methacrylic acid polymers, polysaccharides, polyethylene polymers, polypropylene polymers, polystyrene polymers, polyvinyl butyrate polymers, poly (vinyl chloride) polymers and dendrimer polymers; wherein the percentage of the monomeric units that are bound to the recombinant peptide is between 1 % and 100% of the total number of the monomeric units of the polymeric backbone; wherein the recombinant peptide comprises an AAV epitope or an AdV epitope, each peptide being bound to a single monomeric unit through the linker; wherein the linker is a linear alkylene having between 2 and 30 carbon atoms that spatially separates the recombinant peptide from the monomeric unit of the polymeric backbone to which is bound to, and the carbon atoms are substituted or unsubstituted.

[0056] Alternatively, the synthetic polymeric conjugate comprises:

[0057] (i) a polymeric backbone made of a plurality of monomeric units, each monomeric unit being bound to the following consecutive unit forming a chain, and wherein the polymeric backbone is selected from the group consisting of: homologous a-amino acid polymers (homopolypeptides), heterologous a-amino acid polymers (heteropolypeptides), protein polymers, lipid polymers, nucleic acid polymers, acrylic acid polymers, methacrylic acid polymers, polysaccharides, polyethylene polymers, polypropylene polymers, polystyrene polymers, polyvinyl butyrate polymers, poly (vinyl chloride) polymers and dendrimer polymers;

[0058] (ii) one or more recombinant peptides, wherein the recombinant peptides are bound to from 1 % to 100% of the total number of monomeric units;

[0059] (iii) one or more capping agents, wherein the capping agents are bound to a plurality of monomeric units that are not bound to the recombinant peptide; and

[0060] (iv) a linker which is positioned between each recombinant peptide and a single monomeric unit.

[0061] A second aspect relates to a pharmaceutical composition comprising a synthetic polymeric conjugate P7120PC00 10 according to the first aspect, and at least one pharmaceutically acceptable excipient, carrier or vehicle.

[0062] A third aspect relates to a synthetic polymeric conjugate according to the first aspect or a pharmaceutical composition according to the second aspect, for use as a medicament.

[0063] Another aspect relates to a synthetic polymeric conjugate as defined in the first aspect or a pharmaceutical composition as defined in the second aspect, for the treatment or prevention of one disease selected from the group consisting of: cancer, ocular disease, neurological disease, metabolic disease, hematological disease, neuromuscular disease, cardiovascular disease, autoimmune disease, and infectious disease.

[0064] Another aspect relates to a synthetic polymeric conjugate as defined in the first aspect or a pharmaceutical composition as defined in the second aspect, for use in the treatment of a subject for the removal of one or more anti-AAV or anti-AdV neutralizing antibodies, wherein the treatment is intra- corporeal or extracorporeal.

[0065] Another aspect relates to a use of a synthetic polymeric conjugate of the first aspect or the pharmaceutical composition of the second aspect for the in vitro detection and / or quantification of an anti- AAV neutralizing antibody in a sample from a subject.

[0066] Another aspect relates to a recombinant peptide with a sequence selected from the group consisting of SEQ ID NO: 1 to 202 and SEQ ID NO: 283 to 305.

[0067] Another aspect relates to a method for obtaining a synthetic polymeric conjugate of the first aspect, comprising the following steps: (a) isolating a human anti-AAV neutralizing antibody or a human anti- AdV neutralizing antibody from a human sample using cell-based assays and isolation procedures; (b) evaluating the neutralization capacity of the isolated human anti-AAV neutralizing antibody or the human anti-AdV neutralizing antibody by cell-based assays; (c) determining a recombinant peptide comprising an epitope targeted by the human anti-AAV neutralizing antibody or the human anti-AdV neutralizing antibody by printed peptide-arrays; (d) synthesizing the recombinant peptide comprising the epitope; (e) evaluating the capacity of the peptide comprising the epitope to bind and block / inhibit the target human anti-AAV neutralizing antibody or the human anti-AdV neutralizing antibody; and (f) synthesizing the synthetic polymeric conjugate by covalently attaching the recombinant peptide comprising the epitope to the polymeric backbone.

[0068] Another aspect relates to a method for obtaining a synthetic polymeric conjugate, comprising attaching a recombinant peptide selected from the group consisting of SEQ ID NO: 1 to 202 and SEQ ID NO: 283 to 305, to a polymeric backbone as described herein. P7120PC00 11

[0069] Another aspect relates to an in vitro method for detecting and / or quantifying an anti-AAV neutralizing antibody or an anti-AdV neutralizing antibody in a sample from a subject by means of a synthetic polymeric conjugate of the first aspect, wherein the method comprises: (a) contacting the sample with the synthetic polymeric conjugate and a detection agent, (b) incubating the sample with the synthetic polymeric conjugate and a detection agent to form a detection-polymeric conjugate-neutralizing antibody complex; (c) separating the detection-polymeric conjugate-neutralizing antibody complex from the remaining sample containing the unbound detection agent; and (d) detecting the anti-AdV or anti-AAV neutralizing antibody.

[0070] Another aspect relates to a kit for detecting and / or quantifying an anti-AAV neutralizing antibody or an anti-AdV neutralizing antibody in a sample from a subject, comprising: (a) a capture agent comprising a solid support or carrier coated with a synthetic polymeric conjugate of the present invention or the recombinant peptide comprising an AAV epitope selected from the group consisting of Table 1 and / or 10 (SEQ ID NO: 1 to 202 and SEQ ID NO: 283 to 305); (b) a labeled detection agent comprising an antibody that binds the anti-AAV neutralizing antibody or an anti-AdV neutralizing antibody; (c) an incubator configured to incubate the sample with the capture agent and detection agent to form a polymeric conjugate-neutralizing antibody-detection agent complex or a peptide-neutraliz- ing antibody-detection agent complex; and (d) means for detecting and / or quantifying the presence of the polymeric conjugate-neutralizing antibody-detection agent complex or the peptide-neutralizing antibody-detection agent complex; wherein the sample is cell supernatant, blood, serum, plasma or any other organic fluid.

[0071] DESCRIPTION OF DRAWINGS

[0072] Figure 1 represents the Harmful Antibody Removal Technology (HART). The polymeric conjugate molecule utilized for this Figure as an example was RA0127 (see Scheme 11). The RA0127 molecule has a poly-L-lysine backbone derivatized with the oligosaccharide Galal ,3Galp1 ,4GlcNAc- (aGal) to remove anti-aGal antibodies and was developed to protect ICU patients from Gram-negative bacterial infections. A). Scheme of RA0127 intravenous administration in Cynomolgus monkeys and the different time points of blood collection. Animals received 50 mg / kg (or only the vehicle) through a 30-minute intravenous infusion three times a week for four weeks. The blood samples were taken before administration (marked as “B. Inf’ in Figure 1 (B)), at 24 hours (day 1) after the first administration, 72 hours after the last administration (day 29), 7 days after the last administration (day 33) and 18 days after the last administration (day 44). Control animal only received the vehicle. B). Baseline antibody levels varied among individuals and by isotype (IgG or IgM). 50 mg / kg administration resulted in a removal of 90% (IgG) and 97% (IgM) within 24 hours. After 18 days following the final administration (day 44), antibody levels showed minimal recovery, demonstrating a long- lasting anti-aGal antibodies intracorporeal inhibition. Constant levels of anti-aGal antibodies were observed in the control animal. P7120PC00 12

[0073] Figure 2 represents the administration regimen and pharmacokinetics of RA0127 molecule at different concentrations. A). Scheme of the RA0127 intravenous administration in Cynomolgus monkeys and the different time points of blood collection. Animals received 5, 15 or 50 mg / kg through a 30- minute intravenous infusion three times a week for four weeks. The blood samples were taken on days 1 (first dose) and 26 (last dose) before infusion (marked as “B. inf’ in Figure 2(B)), just after finishing the infusion (TO) and at 2, 4, 8, 24, 36, 48 hours after the administration (n=1). B). The time at which maximum RA0127 serum concentration was observed (tmax) was 0.5 hours after infusion start (TO, just after infusion end) in all experimental groups. RA0127 showed a fast kinetic; it was cleared from circulation rapidly in a dose-dependent manner with an elimination half-life (tic) ranging from 4 to 10 h. No serum RA0127 levels were observed in any of the samples obtained from control animals. No accumulation effect was observed in animals treated with the different doses of RA0127 when administered by 30-minute intravenous infusion three times a week for four weeks.

[0074] Figure 3 represents the non-immunogenic profile of RA0127 molecule. A). Scheme of the RA0127 intravenous administration in Cynomolgus monkeys and the different time points of blood collection. Animals received 50 mg / kg (or only the vehicle) through a 30-minute intravenous infusion three times a week for four weeks. The blood samples were taken on days 1 (first dose) and 26 (last dose) before infusion (marked as “B. inf’ in Figure 3(B)), just after finishing the infusion (TO), and at 2, 4, 8, 24, 36, and 48 hours after the administration (n=10). B). No complement activation was detected after administration of RA0127 (50 mg / Kg). Additionally, there were no differences in C3a levels between monkeys that received 50 mg / kg and those given the vehicle.

[0075] Figure 4 represents the synthesis of peptides and shows SEQ ID NO: 22 peptide: Asp Vai Phe Met He Pro Gin Tyr Gly Tyr Leu Thr Leu Asn Asp (DVFMIPQYGYLTLND). The peptide corresponding to EXAMPLE 3 was purified by reversed-phase HPLC on column C8, 20x250mm using a gradient of acetonitrile in water (from 10% to 70% in 60 min). A) Absorbance spectrum at 226 nm of peptide 17.1 (SEQ ID NO: 22) by HPLC. The fraction corresponding to the main absorbance peak was collected and freeze-dried. B) ESI mass-spectrometry of SEQ ID NO: 22 (200 u / ml scan 100-1500 acetonitrile / water gradient KROM ASIL100-1.8-C18. The molecular mass of the peptide determined by ESI mass-spectrometry was 1788.8 Da, calculated molecular mass - 1787.8 Da.

[0076] Figure 5 represents the Poly-L-lysine polymer of 400 degree of polymerization (DP400).1H NMR spectra of PLys_DP400*HBr (section 3, top) in D2O; PLys400-N-(CO)CH2CI (section 2, middle) in D7-DMF and PLys_DP400-TGA (thioglycolate control) (section 1 , bottom) in D2O (303K, 800 MHz). To a stirred solution of PLys-N-(CO)CH2CI (4.37 mg, 21.35 micromole of chloride) in DMF (0.874 mL) thioglycolic acid (85.4 micromole, 9.17 pL of 70% aq. thioglycolic acid) and DBU (120 micromole, 18 pL, pH was 9.0) were added. After 5 minutes, the reaction mixture became cloudy, and precipitate was formed. The mixture was stirred for 40 min and after addition of AcOH (6.8 pL) was diluted to volume 13 mL with ethyl acetate, thoroughly mixed and centrifuged (5000 g for 8 min). The precipitate was dried in vacuum (waterjet pump), dissolved in 4 mL of water + 64 pL 1 M NaHCOs (pH of solution P7120PC00 13

[0077] 6.9). Ultrafiltration on Amicon® Ultra-15 Centrifugal Filter Units (50 kDa), with subsequent washing with PBS (5 mL) and water (2 x 5 mL) gave small residue, that was dissolved in water (4 mL), filtered (filter # 2) and freeze-dried. Yield of PLys_DP400-N-(CO)CH2SCH2COONa 5.52 mg (92%). PLys400-N-(CO)CH2CI. (section 2). To a stirred solution of PLys_DP400xHBr (50.9 mg, 243 micromoles of NH2groups) in DMF (1 .7 mL) chloroacetic anhydride (125 mg, 731 micromole) in DMF (0.624 mL) and then DBU (55 pL, 368 micromole) were added. After 30 min the solution was diluted with ethyl acetate + 1 % AcOH to the volume 45 mL, thoroughly mixed and centrifuged (5000 g for 8 min). The precipitate was suspended in methanol + 5% AcOH (3 mL), diluted with ethyl acetate to the volume 45 mL, thoroughly mixed and centrifuged (5000 g for 8 min). The last procedure was repeated. The precipitate was dried in vacuum (water jet pump), suspended in water and freeze- dried. Yield 43.2 mg (87%) as white solid. The poly-L-lysine hydrobromide DP 400 (section 1) was synthesized, via ring-opening polymerization of N-carboxy-anhydrides using N6-trifluoroacetyl-L-ly- sine as precursor, as disclosed in Hadjichristidis N et al., 2009. PLys: Poly-L-lysine polymer; DMF: dimethylformamide; DBU: 1 ,8-Diazabicyclo[5.4.0]undec-7-ene.

[0078] Figure 6 represents PLys-oligoethyleneglycol (OEG) derivatives.1H NMR spectra of Plys_DP400*HBr (section 2, top) in D2O and PLys_DP400-OEGi2-NH2*HCI (section 1 , bottom) in D2O + 0.5% d-AcOH (303K, 800 MHz). PLys_DP400-GEGi2-NH2. To a stirred solution of PLys_DP400xHBr (18 mg, 86.1 micromole of NH2groups) in dimethyl sulfoxide (DMSO) (0.9 mL) a solution of Fmoc-OEGi2-ONSu (121 mg, 129 micromole) in DMSO (0.9 mL) and i-Pr2NEt (22.5 pL, 129 micromole) were added. The solution was stirred for 40 min, then ethanolamine (10.4 pL, 172 micromole) was added to deactivate excessive Fmoc-OEGi2-ONSu active ester. 20 minutes later pyperidine (100 pL) was added and solution was stirred for 2 h. After addition of AcOH (300 pL) the solution with precipitate of Fmoc-pyperidine was diluted with water to 10 mL volume and filtered. PLys_DP400-OEGi2-NH2was isolated by ultrafiltration on Amicon® Ultra-15 Centrifugal Filter Unit (50 kDa) in 0.2% AcOH. The residue on the filter was dissolved in water and after addition of 2M HCI (43 pL) freeze-dried. Yield of PLys_DP400-OEGi2-NH2*HCI was 58.6 mg (89%). PLys: Poly-L-lysine polymer; DP: degree of polymerization.

[0079] Figure 7 represents 3.1 C (10%)-Peptide-PLys400-TGA conjugate.1H NMR spectra of free peptide 3.1 C (SEQ ID NO: 193) and 3.1 C (10%)-Plys_DP400 conjugate in D2O (303K, 800 MHz), top (section 2) and bottom (section 1) respectively. For peptide 3.1 C sequence see SEQ ID NO: 193 on Table 1. Integration of the signals in the regions 0.65-2.38 ppm and 2.56-4.69 ppm with subtracting of the peptide signals in these regions gives mole content of the peptide in the conjugate ~ 9%. To a stirred solution of PLys- / V-(CO)CH2CI in DMF (2.95 mg in 590 mL; 0.036 micromole, 14.4 micromole of chloride) a solution of 3.1 C peptide (5.65 mg, 2.158 micromole mono-CFsCOOH salt) in DMF (283 pL) and DBU (10% solution by volume in DMF, 26.4 pL ~ 8 mole / mole of peptide) were added (2 pL of reaction mixture diluted with 6 pL of water has pH 8.5-9.0) and the mixture was stirred for 1 h. The binding of peptide to polylysine was controlled by TLC. Then thioglycolic acid (57.6 micromole, 4 mole / mole of total chloride, 6.2 pL of 10% solution in DMF of 70% aq. TGA) and DBU (12.4 pL) were P7120PC00 14 added. After 5 minutes of stirring the reaction mixture became cloudy and precipitate was formed. The mixture was stirred for 30 min and after addition of AcOH (to pH 7) was diluted with water or methanol\water 3:1 to ~ 10 mL. The conjugate was isolated by ultrafiltration of the solution on Amicon® Ultra-15 Centrifugal Filter Units (100 kDa) with subsequent washing with PBS and water (twice). The remainder on the filter was dissolved / suspended in water and evaporated. The pH of aqueous solution / suspension of conjugate (2-5 mL) was adjusted to 6.6-6.8 with an aqueous Na- HCO3 and ultrasound treatment was done. The solution was filtered (glass filter #2), evaporated and freeze-dried from water. PLys: Poly-L-lysine polymer; TGA: thioglycolate; DP: degree of polymerization; DMF: dimethylformamide; DBU: 1 ,8-Diazabicyclo[5.4.0]undec-7-ene.

[0080] Figure 8 represents 17.1 C (15%)-Peptide-PLys400-TGA conjugate.1H NMR spectra of free peptide 17.1 C (SEQ ID NO: 199) and 17.1 C (15%)-Plys_DP400 conjugate in D2O (303K, 800 MHz), top (section 2) and bottom (section 1) respectively. For peptide 17.1 C sequence see SEQ ID NO: 199 on Table 1. Integration of the signals in the regions 0.40-2.33 ppm and 2.33-4.67 ppm with subtracting of the peptide signals in these regions gives mole content of the peptide in the conjugate ~ 15%. To a stirred solution of PLys- / V-(CO)CH2CI in DMF (2.94 mg in 588 mL; 0.0359 micromole, 14.35 micromole of chloride) a solution of 17.1 C peptide (4.32 mg, 2.153 micromole mono-CFsCOOH salt) in DMF (360 pL) and DBU (10% solution by volume in DMF, 35.7 pL ~ 11 mole / mole of peptide) were added (2 pL of reaction mixture diluted with 6 pL of water has pH 8.5-9.0) and the mixture was stirred for 1 h. The binding of peptides to poly-lysine was controlled by TLC. Then thioglycolic acid (57.4 micromole, 4 mole / mole of total chloride, 6.2 pL of 10% solution in DMF of 70% aq. TGA) and DBU (12.4 pL) were added. After 5 minutes of stirring the reaction mixture became cloudy and precipitate was formed. The mixture was stirred for 30 min and after addition of AcOH (to pH 7) was diluted with water or methanol\water 3:1 to ~ 10 mL. The conjugate was isolated by ultrafiltration of the solution on Amicon® Ultra-15 Centrifugal Filter Units (100 kDa) with subsequent washing with PBS and water (twice). The remainder on the filter was dissolved / suspended in water and evaporated. The pH of aqueous solution / suspension of conjugate (2-5 mL) was adjusted to 6.6-6.8 with an aqueous Na- HCO3 and ultrasound treatment was done. The solution was filtered (glass filter #2), evaporated and freeze-dried from water. PLys: Poly-L-lysine polymer; TGA: thioglycolate; DP: degree of polymerization; DMF: dimethylformamide; DBU: 1 ,8-Diazabicyclo[5.4.0]undec-7-ene.

[0081] Figure 9 represents 1 ,1 C (15%)-Peptide-PLys400-TGA conjugate.1H NMR spectra of free peptide 1.1 C (SEQ ID NO: 191) and 1.1 C (15%)-Plys_DP400 conjugate in D6-DMSO / D2O 1 :1 (303K, 800 MHz), top (section 2) and bottom (section 1) respectively. For peptide 1.1 C sequence see SEQ ID NO: 191 on Table 1. Integration of the signals in the regions 0.41-2.40 ppm and 2.81-4.45 ppm with subtracting of the peptide signals in these regions gives mole content of the peptide in the conjugate ~ 14%. PLys: Poly-L-lysine polymer; TGA: thioglycolate; DP: degree of polymerization.

[0082] Figure 10 represents 2.1 C (10%)-Peptide-PLys400-TGA conjugate.1H NMR spectra of free peptide 2.1 C (SEQ ID NO: 192) and 2.1 C (10%)-Plys_DP400 conjugate in D2O (303K, 800 MHz), top (section P7120PC00 15

[0083] 2) and bottom (section 1) respectively. For peptide 2.1 C sequence see SEQ ID NO: 192 on Table 1. Integration of the signals in the regions 0.51-2.40 ppm and 2.40-4.67 ppm with subtracting of the peptide signals in these regions gives mole content of the peptide in the conjugate ~ 9%. PLys: Poly- L-lysine polymer; TGA: thioglycolate; DP: degree of polymerization.

[0084] Figure 11 represents 5.1 C (15%)-Peptide-PLys400-TGA conjugate.1H NMR spectra of free peptide 5.1 C (SEQ ID NO: 195) and 5.1 C (15%)-Plys_DP400 conjugate in D2O (303K, 800 MHz), top (section 2) and bottom (section 1) respectively. For peptide 5.1 C sequence see SEQ ID NO: 195 on Table 1. Integration of the signals in the regions 0.50-2.33 ppm and 2.33-4.67 ppm with subtracting of the peptide signals in these regions gives mole content of the peptide in the conjugate ~ 15%. PLys: Poly-L-lysine polymer; TGA: thioglycolate; DP: degree of polymerization.

[0085] Figure 12 represents the Synthetic pathways to obtain Polylysine (PLys)-PEG-peptide conjugates.

[0086] A). Synthesis pathway PLys derivatives from Cys-PEG-peptide. PLys400 is derivatized with a mix of 3-Maleimidopropionic acid N-hydroxysuccinimide ester and succinic, and then functionalized with Cys-peptide (15%). The rest of monomeric units in the polymer are capped with succinic acid (85%).

[0087] B). Synthesis pathway PLys derivatives from Cys-PEG-peptide. PLys400 is derivatized with (i) MalpAla-NH-PEG4-CH2COONSu and (ii) succinic, and then functionalized with the Cys-peptide (15%). The rest of monomeric units in the polymer are capped with succinic acid (85%). PEG4: 15- amino-4,7,10,13-tetraoxapentadecanoic acid, acting as spacer; Mai: maleimide; PEG: polyethylene glycol); Cys: Cysteine; Ala: Alanine; Sue: succinic acid.

[0088] Figure 13 represents isolation and characterization of anti-AAV neutralizing antibodies (NAb) from NHS (normal human serum). Scheme of NAbs purification and sequence identification. For the identification of neutralizing AAV epitopes a multi-step process of antibody selection and isolation was used in order to obtain a precise and specific matrix of neutralizing anti-AAV antibodies. First, NHSwas exposed to each AAVX to isolate serotype-specific AAV-binding antibodies (BAbs) and then used in an in vitro AAV neutralization assay. BAbs were incubated with their corresponding AAV serotype to allow the formation of the BAb-AAVX complexes. The mixture Bab-AAVX was used to infect HEK 293T cells. After 24h of infection, cell supernatant which contained AAV particles bound to BAbs that were unable to enter the cells, indicating neutralization by neutralizing antibodies. The NAbs were then purified using magnetic beads, isotyped, and utilized as a refined and specific matrix of AAV-neutralizing antibodies forthe identification of neutralizing AAV epitopes Peptide arrays. AAV: Adeno-associated virus.

[0089] Figure 14 represents the neutralization assays. Neutralization assays were performed using pooled- NHS (122 donors, Spain) or human binding anti-AAV antibodies for each serotype, and the previously selected multiplicity of infection (MOIs) for each serotype. After GFP quantification by ELISA, results were analyzed using MyAssays (https: / / myassays.com / ) to calculate the % of inhibition / infec- tion for each of the assessed AAV-serotype (Bass-Stringer S et al., 2021 ; Guo P et al., 2019; Meliani P7120PC00 16

[0090] A et al., 2015). The percentage of inhibition was calculated using the following formulae 00 - [(Test sample signal readout (virus + serum or sample of interest) - baseline signal readout (media only control)) I (maximum signal readout (media and virus only) - baseline signal readout) x 100] = % Transduction inhibition. The results of the neutralizing assay plotted in GraphPad Prism 9 software are shown in Figure 14 (results from three independent experiments). NHS: normal human serum; AAV: Adeno-associated virus; Bab: binding antibodies.

[0091] Figure 15 represents isotyping of neutralizing anti-AAV antibodies. The isolated binding and neutralizing antibodies samples were isotyped by ELISA. The most predominant subclasses of IgG antibodies were lgG1 and lgG2 with almost inexistent levels of IgM. This result suggested that IgG is the main isotype involved in the neutralization of the AAV. For that reason, we focused our attention on studying IgG neutralizing activity. A). Quantification by ELISA of different antibodies isotypes (lgG1 , lgG2, lgG3, lgG4 and IgM) from eluted binding anti-AAV antibodies. B). Neutralizing anti-AAV antibodies samples. AAV: Adeno-associated virus.

[0092] Figure 16 represents the alignment of all consensus sequences identified in each serotype. The phylogenetic relation between consensus sequences was identified by obtaining a guide tree using the CLUSTAL O (1 .2.4) multiple sequence alignment. The alignment shows a similar sequence clustering compared with submitting all sequences. The number on the left represents the serotype of origin of the consensus sequence; the number on the right represents the number of amino acids.

[0093] Figure 17 represents the spatial localization of conserved epitope sequences identified in this work, in AAV8 structure (sequences from four independent experiments). The molecular graphics images were produced using the UCSF Chimera package.

[0094] Figure 18 represents the antibody neutralizing assays utilizing free peptides at different serum dilutions. The percentage of AAV infection was calculated by measuring the concentration of GFP by ELISA. The results show the mean of three independent experiments, except for 23.1 (SEQ ID NO: 28) in AAV8 where n=1 . #, statistical significance found. AAV: Adeno-associated virus.

[0095] Figure 19 represents the antibody neutralizing assays utilizing conjugated peptides at different serum dilutions. The percentage of AAV infection was calculated by measuring the concentration of GFP by ELISA. The results show the mean of three independent experiments. #, statistical significance found. Peptide 1 ,1 C, peptide 2.1 C, peptide 3.1 C, peptide 5.1 C, and peptide 17.1 C correspond to SEQ ID NO: 191 , SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 195, and SEQ ID NO: 199, respectively. AAV: Adeno-associated virus.

[0096] Figure 20 represents the solid phase treatment. The solid-phase treatment was exclusively used for conjugated neutralizing epitopes, designed to mimic in vivo gene therapy conditions. This protocol P7120PC00 17 involved two main steps: immunoadsorption to remove anti-AAV antibodies and an in vitro neutralization assay with the antibody-depleted NHS. (1) Representation of an example of an immunoadsorption assay on a plate coated with peptide conjugate. (2) The plate used for the immunoadsorption was processed for ELISA assay. (3) The antibody-depleted NHS obtained in step 1 was diluted and mixed with AAV, (4) and used to infect the cell plate. AAV: Adeno-associated virus; NHS: normal human serum.

[0097] Figure 21 represents the solid-phase-antibody neutralizing assays utilizing PLys Conjugates at different serum dilutions. The percentage of AAV infection was calculated by measuring the concentration of GFP by ELISA. The results show the mean of two or three independent experiments. PLys: Poly-L-lysine polymer; AAV: Adeno-associated virus.

[0098] Figure 22 represents anti-AAV antibodies removal assays utilizing PLys Conjugates. NHS was subjected to a solid-phase treatment with PLys Conjugates for specific anti-AAV antibody capture and elimination. Followed by the immunoadsorption of the depleted-NHS in AAV-coated plates to analyze the AAV Ab binding. The percentage of anti-AAV IgG removal was calculated from the basal antibody measurements from non-depleted NHS. PLys: Poly-L-lysine polymer; AAV: Adeno-associated virus; NHS: normal human serum. Peptide 1.1 C and peptide 17.1 C correspond to SEQ ID NO: 191 , and SEQ ID NO: 199, respectively.

[0099] Figure 23 represents the NHS Neutralization Assays with the conjugated Peptides at different serum dilutions. NHS dilutions were treated with conjugated peptides for specific antibody inhibition, after which, AAV DJ or AAV9 P31 vector was used to infect HEK 293T cells. The percentage of AAV infection was calculated by measuring the concentration of GFP by ELISA. NHS: normal human serum; AAV: Adeno-associated virus.

[0100] Figure 24 shows the AAV Neutralization Assays of the different assessed mouse anti-AAV monoclonal antibodies. A specific AAV was diluted in DMEM and treated with varying concentrations of the specified antibodies for 1 h at 37°C. Then the mixture was used to infect HEK 293T cells, and the AAV-infectivity was calculated by measuring the concentration of GFP by ELISA. AAV: Adeno-associated virus; DMEM: Dulbecco's Modified Eagle Medium.

[0101] Figure 25 shows the antibody Neutralization Assays for the Free Peptides. A specific monoclonal anti-AAV antibody was diluted in DMEM and treated with varying concentrations of the specified free peptide for 1 h at 37°C. Then the mixture was incubated with a specific AAV for 1 h at 37°C. The infectivity was calculated by measuring the concentration of GFP by ELISA. DMEM: Dulbecco's Modified Eagle Medium.

[0102] Figure 26 shows the Antibody-Conjugate Binding Assays. (A) Varying concentrations of A20 antibody were incubated on a plate coated with 10 pg / ml of 13.1-, 17.1-PLys conjugates and control of P7120PC00 18

[0103] PLys for 1 h at RT and revealed with an anti-mouse IgG-HRP antibody. The antibody binding OD is represented. (B) anti-AAV antibody capture assay - mouse monoclonal antibodies: A specific mouse monoclonal anti-AAV antibody was diluted in PBS 1X at a concentration of 1 pg / ml and incubated on a plate coated with 10 pg / ml of the indicated PLys conjugates for 1 h at RT, followed by an incubation with an anti-mouse IgG-HRP (for A20, ADK6 and ADK8) or anti-mouse IgA-HRP (ADK9) antibody. The antibody binding OD is represented.

[0104] Figure 27 shows the antibody-Conjugate in vitro Assays. Two concentrations of A20 antibody were treated with varying concentrations of 13.1 peptide conjugate, ranging from 7.81-250 pg / ml, for 45- 60 min at RT. Then, the mixture was incubated on a plate coated with 10 pg / ml of 13.1 conjugate for 1 h at RT and revealed with an anti-mouse IgG-HRP antibody. The percentage of antibody removal compared to the untreated A20 is represented.

[0105] Figure 28 represents the antibody-conjugate in vitro Assays. A20 antibody was treated with varying concentrations of A20.2.2 peptide conjugate, ranging from 1-200 pg / ml, for 45-60 min at RT. Then, the mixture was incubated on a plate coated with 10 pg / ml of A20.2.2 conjugate for 1 h at RT and revealed with an anti-mouse IgG-HRP antibody. The percentage of antibody removal compared to the untreated A20 is represented.

[0106] Figure 29 represents the1H NMR spectra of Mal(15%)-PLys400-Suc(85%) (section 1 , top) and Mal- PEG4(15%)-PLys400-Suc(85%) (section 2, bottom) in D6-DMSO / D2O 4:1 + 1 % CD3COOD (800 MHz, 303 K). PLys: Poly-L-lysine polymer; Sue: Succinic acid; Mai: maleimide; PEG4: 15-amino- 4,7,10,13-tetraoxapentadecanoic acid, acting as spacer.

[0107] Figure 30 represents the1H NMR spectra of PLys400 x HBr (section 1 , top), PLys400-Suc (section 2, middle-top), ME-PLys400-Suc (section 3, middle-bottom) and ME-PEG4-PLys400-Suc (section 4, bottom) in D2O (800 MHz, 303 K). PLys: Poly-L-lysine polymer; Sue: Succinic acid; ME: mercaptoethanol; PEG4: 15-amino-4,7,10,13-tetraoxapentadecanoic acid, acting as spacer.

[0108] Figure 31 represents the1H NMR spectra of: (A) peptide_13.1-Cys (without PEG4, pH = 8.5) (section 1 , top) and of conjugate 13.1 M-PEG4-Cys-PLys400-Suc (section 2, bottom) in D2O (800 MHz, 303 K). (B) Peptide_13.1-Cys (without PEG4, pH = 8.5) (section 1 , top) and conjugate 13.1-Cys- PEG4-PLys400-Suc in D2O (800 MHz, 303 K) (section 2, bottom). (C) Peptide_2.1-Cys (without PEG4) (section 1 , top) and conjugate 2.1-PEG4-Cys-PLys400-Suc in D2O (800 MHz, 303 K) (section 2, bottom). (D) Peptide_2.1-Cys (without PEG4) (section 1 , top) and conjugate 2.1-Cys-PEG4- PLys400-Suc in D2O (800 MHz, 303 K) (section 2, bottom). (E) Peptide_3.1-Cys (without PEG4) (section 1 , top) and conjugate 3.1-PEG4-Cys-PLys400-Suc in D2O (section 2, bottom) (800 MHz, 303 K). (F) Peptide_3.1-Cys (without PEG4) (section 1 , top) and (2) conjugate 3.1-Cys-PLys400-Suc in D2O (section 2, bottom) (800 MHz, 303 K). (G) Peptide_17.1-Cys (without PEG4) (section 1 , top) and conjugate 17.1-PEG4-Cys-PLys400-Suc in D2O (section 2, bottom) (800 MHz, 303 K). Cys: P7120PC00 19

[0109] Cysteine; PEG4: 15-amino-4,7,10,13-tetraoxapentadecanoic acid, acting as spacer; PLys: Poly-L- lysine polymer.

[0110] Figure 32 represents the1H NMR spectra of peptide A20.2.2-Cys in D2O (800 MHz, 30 °C) (section 1 , top), conjugate A20.2.2-Cys-PLys-Suc in D2O, pH = 9 (700 MHz, 30 °C) (section 2, middle), and conjugate A20.2.2-PEG4-Cys-PLys-Suc in D2O, pH = 7.2 (700 MHz, 30 °C) (section 3, bottom). Cys: Cysteine; PLys: Poly-L-lysine polymer; Sue: succinic acid; PEG4: 15-amino-4,7,10,13-tetraoxapen- tadecanoic acid, acting as spacer.

[0111] Figure 33 represents the1H NMR spectra (30 °C, 700 MHz) of: (A) of PLys400 x HBr in D2O (section 1 , top); of Cellobiose-NH-glutarate in D2O (section 2, middle); of Mal(15%)-PLys400-Cellobi- ose(85%) in D2O + 1 % CD3COOD (section 3, bottom); (B) Mal(15%)-PLys400-Cellobiose(85%) in D2O + 1 % CD3COOD. PLys: Poly-L-lysine polymer; HBr: hydrogen bromide; Mai: maleimide.

[0112] Figure 34 represents the1H NMR spectra of peptide AD6.1 ,3-Cys in D2O (800 MHz, 30 °C) (section 1 , top); conjugate AD6.1 ,3-Cys-PLys-Suc in D2O + 0.5% CD3COOD (700 MHz, 30 °C) (secton 2, middle); conjugate AD6.1 ,3-PEG4-Cys-PLys-Suc in D2O + 0.5% CD3COOD (700 MHz, 30 °C) (section 3, bottom). Cys: Cysteine; PLys: Poly-L-lysine polymer; Sue: succinic acid; PEG4: 15-amino- 4,7,10,13-tetraoxapentadecanoic acid, acting as spacer.

[0113] DETAILED DESCRIPTION OF THE INVENTION

[0114] Synthetic polymeric conjugate

[0115] Polymeric backbone

[0116] In the context of the present invention, the term “polymer” is a generic term used to describe a substance made of molecules that have one type of repeating monomeric units or monomers that are connected by chemical bonds. The term “monomer” refers to a small molecule of low molecular weight that can become chemically bonded to other monomers to form a polymer. The term “polymer backbone” refers to the main chain of the polymer that is composed of one type of repeating monomeric units or monomers. The term “chain” refers to various lengths of repetitive monomeric units or monomers which make up the polymer backbone and the polymer. Based on monomer unit organization, the polymer structure could be linear or branched. The term "linear polymer" refers to a polymer chain that has the monomeric units linked in a single and continuous chain, where each monomer unit is connected to the following monomer unit by a covalent bond. The term “branched polymer” refers to a polymer chain of monomer units having at least 1 branching side chain that is attached to a monomer by a covalent bond. One or more side chains can be attached to the polymer backbone and can have a multitude of structures. Branching side chains can vary in length and frequency, and their presence has an impact on the melt rheology, mechanical behavior, solution P7120PC00 20 properties, and density. In the particular case when the polymeric backbone is formed by amino acids, there are some amino acids that due to a unique asymmetrical AB2 structure, display special abilities to form branching side chains and dendrimers, such as L-lysine, L-glutamic acid, and L- aspartic. In the case of poly(L-lysine), dendrimers, N-Boc- or N-Fmoc-protected L-Lysine is coupled via an amide bond to an initiator core carrying a certain number of amino groups. After deprotection, the first generation of the dendrimer is completed and now displays two amino-groups for each one present in the initiator. These amino groups are now available for the coupling of two more protected L-lysine molecules per initiating amino function. Thus, the number of reactive sites doubles in each generation. These coupling reactions are typically base-catalyzed and often use activating reagents. (Thompson M, Scholz C, 2021).

[0117] In the context of the present invention, the term “polymeric backbone” is also called “polymer chain” or simply “backbone” of a polymer, and as it has been defined herein it refers to the main chain of a polymer. Polymers are often classified according to the elements in the main chains. The character of the backbone, i.e. its flexibility, determines the properties of the polymer. For instance, polymers with rigid backbones are prone to crystallization (e.g. polythiophenes) in films and in solution. Common organic synthetic polymers have main chains composed of carbon, i.e. C-C-C-C. Examples include polyolefins such as polyethylene, polystyrene, polypropylene, and acrylates. Other major classes of organic polymers are polyesters and polyamides. They have respectively -C(O)-O- and - C(O)-NH- groups in their backbones in addition to chains of carbon. The term “biopolymer” is generally understood as an organic polymer that is produced naturally by living organisms. Based on structure, major subsets of biopolymers are polysaccharides (carbohydrates), peptides, and polynucleotides. Peptide polymers or protein polymers are characterized by amide linkages (-N(H)-C(O)-) formed by the binding of amino acids. The sequence of the amino acids in the polypeptide backbone is known as the primary structure of the protein. Like almost all polymers, protein fold and twist, forming into the secondary structure, which is rigidified by hydrogen bonding between the carbonyl oxygens and amide hydrogens in the backbone, i.e. C=O-HN. Further interactions between residues of the individual amino acids form the protein's tertiary structure. Carbohydrates arise by binding of monosaccharides such as glucose. The polymers can be classified into oligosaccharides (up to 10 residues) and polysaccharides (up to about 50,000 residues). The backbone chain is characterized by an ether bond between individual monosaccharides, which is called the glycosidic linkage.

[0118] In the context of the present invention, the polymeric backbone load with a recombinant peptide is represented by the percentage of the total number of monomeric units that are bound to a recombinant peptide. Particularly, a load between 1 % and 100% of the total number of the monomeric units of the polymeric backbone are bound to a recombinant peptide. In a more particular embodiment, a load between 1 % and 50% of the total number of the monomeric units of the polymeric backbone are bound to a recombinant peptide.

[0119] In the context of the present invention, the polymer backbone is a non-immunogenic and / or non-toxic P7120PC00 21 polymer (see EXAMPLE 1 , Figures 1 to 3 for toxicity assay).

[0120] In a particular embodiment, the polymeric backbone is selected from homologous and heterologous a-amino acid polymers (homo- and hetero-polypeptides), proteins, lipids, nucleic acids, acrylic acid polymers, methacrylic acid polymers, acrylic and methacrylic acid copolymers, polysaccharides such as chitosan or agarose, polyethylene, polypropylene, polystyrene, polyvinyl butyrate, poly (vinyl chloride) and dendrimers.

[0121] The polymeric backbone is particularly selected from the group consisting of homologous a-amino acid polymers such as poly-L-lysine, or hydrogel polymers such as polyacrylamide, or agarose polymer. In a particular embodiment, the polymeric backbone is a homologous a-amino acid polymer or an acrylic acid polymer. The selection of the particular polymer backbone for preparing the polymeric conjugate of the invention will be based on the desired end use of the polymeric conjugate.

[0122] In a particular embodiment, the polymeric backbone is selected from the group consisting of polyacrylic acid polymer (PAA), agarose polymer, and poly-lysine polymer (pLys). Particularly, the polymeric backbone is a polyacrylic acid polymer (PAA) or a poly-lysine polymer (pLys).

[0123] In the context of the present invention, the average sum of monomeric units of the polymeric backbone, also referred herein as degree of polymerization, can be in the range of from 2 to 3,000 total number of monomeric units, particularly from 2 to 1 ,200 total number of monomeric units. In some embodiments, the average sum of monomeric units of the polymeric backbone is in the range of from 2 to 1 ,000 total number of monomeric units, particularly from 2 to 600 total number of monomeric units, more particularly from 2 to 400 total number of monomeric units, even more particularly from 2 to 200 total number of monomeric units, and even more particularly from 2 to 100 total number of monomeric units. For instance, the polymeric backbone can be about: 50, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100, or 2500 monomeric units. In a particular embodiment, the polymeric backbone has a total number of monomeric units of 400 units. In another particular embodiment, the polymeric backbone has a total number of monomeric units of 150 units.

[0124] In a particular embodiment, the polymeric backbone has a total number of monomeric units comprised between 2 units and an upper limit selected from the group consisting of 1200 units, 1000 units, 600 units, 400 units, 200 units and 100 units.

[0125] In the embodiment wherein the polymeric backbone is comprised by poly-lysine (pLys) units, the poly-lysine backbone can be either linear poly-L-lysine or branched poly-L-lysine. The average sum of lysine units that are comprised within the poly-lysine backbone is referred as degree of lysine polymerization herein. In the context of the present invention, suitable degrees of polymerization of poly-lysine are comprised between about 50 and 3000 Lys units. In a particular embodiment, the poly-lysine degree of polymerization is between about 100 Lys units and about 1 ,000 Lys units, or P7120PC00 22 between about 200 Lys units and about 600 Lys units, or between about 300 Lys units and about 500 Lys units, these poly-Lys backbone are commercially available. In a particular embodiment, the poly-lysine degree of polymerization is about 400 Lys units. See e.g., the polymeric conjugate synthesized in EXAMPLE 3.2.2 (Scheme 2), wherein the degree of polymerization is 400 Lys units. In this particular example, 60 Lys units are conjugated to the recombinant peptide, while 340 Lys units are capped.

[0126] In a particular embodiment, the average sum of lysine units within the polymeric backbone, also referred herein as degree of polymerization, can be in the range of from 2 to 3,000 total number of lysine units, particularly from 2 to 1 ,200 total number of lysine units. In some embodiments, the average sum of lysine units of the polymeric backbone is in the range of from 2 to 1 ,000 total number of lysine units, particularly from 2 to 600 total number of lysine units, more particularly from 2 to 400 total number of lysine units, even more particularly from 2 to 200 total number of lysine units, and even more particularly from 2 to 100 total number of lysine units. For instance, the polymeric backbone can be about 50, 150, 300, 400, 500, 900, 1 100, or 2500 lysine units.

[0127] Agarose is a linear polysaccharide made up of repeating units of agarobiose. In the embodiment wherein the polymeric backbone is comprised by agarose, the polymeric backbone is a linear polymer comprised of agarobiose units. For preparing the polymeric conjugates with agarose, typically, activated forms of agarose are used, which are commercially available as resin beads of different sizes. In the context of the present invention, suitable activated agarose forms are, but not limited to: N-hydroxysuccinimide-activated agarose, aldehyde-activated agarose, azlactone-activated, and 1 ,1 '-carbonyl diimidazole (CDI) activated agarose. Particular forms of agarose are those having molecular weight comprised between 25-1000 kDa.

[0128] In a particular embodiment, the polymeric backbone is an agarose polymer. Agarose is a linear polysaccharide of repeating units of agarobiose, which is a dimer of d-galactose and 3-6- anhydro-l- galactopyranose. The average sum of agarobiose units within the polymeric backbone, also referred herein as degree of polymerization, can be in the range of from 1 to 1 ,500 total number of agarobiose units, particularly from 1 to 600 total number of agarobiose units. In some embodiments, the average sum of agarobiose units of the polymeric backbone is in the range of from 1 to 500 total number of agarobiose units, particularly from 1 to 300 total number of agarobiose units, more particularly from 1 to 200 total number of agarobiose units, even more particularly from 1 to 100 total number of agarobiose units, and even more particularly from 2 to 50 total number of agarobiose units. For instance, the polymeric backbone can be about 25, 75, 150, 250, 450, 550, or 1250 agarobiose units.

[0129] In a particular embodiment, the polymeric backbone is comprised by poly-acrylic acid (PAA) units. For preparing the polymeric conjugate with a polyacrylic acid backbone, the acidic -COOH groups on the polyacrylic acid can be activated, for example, with N-hydroxysuccinimide, forming N-succin- imide ester groups, or can be activated as 4-nitrophenyl ester groups (Tuzikov A et al., 2021). P7120PC00 23

[0130] In a particular embodiment, the polymeric backbone is a poly-acrylamide polymer. The average sum of acrylic acid units within the polymeric backbone, also referred herein as degree of polymerization, can be in the range of from 2 to 3,000 total number of acrylic acid units, particularly from 2 to 1 ,200 total number of acrylic acid units. In some embodiments, the average sum of acrylic acid units of the polymeric backbone is in the range of from 2 to 1 ,000 total number of acrylic acid units, particularly from 2 to 600 total number of acrylic acid units, more particularly from 2 to 400 total number of acrylic acid units, even more particularly from 2 to 200 total number of acrylic acid units, and even more particularly from 2 to 100 total number of acrylic acid units. In a particular embodiment, the average sum of acrylic acid units of the polymeric backbone is in the range of from 100 to 200 total number of acrylic acid units. For instance, the polymeric backbone can be about 50, 100, 150, 200, 300, 400, 500, 900, 1100, or 2500 acrylic acid units, and more particularly 150 acrylic acid units. See e.g., the polymeric conjugate synthesized in EXAMPLE 3.2.5 (Scheme 5), wherein the degree of polymerization is 150 acrylic acid units. In this particular example, 7-8 acrylic acid units are conjugated to the recombinant peptide, 22-23 acrylic acid units are capped, and 120 acrylic acid units are not bound to either a recombinant peptide or a capping agent (not functionalized).

[0131] Capping agent

[0132] In the context of the present invention, the synthetic polymeric conjugate comprises a plurality of monomeric units that are not bound to a recombinant peptide and that are present in capped form, i.e., they are reacted with a suitable capping substance, with the purpose of suppressing the reactivity of the free functional groups on the polymer. That is, the synthetic polymeric conjugate comprises some monomeric units that are bound to the recombinant peptide, and some that are bound to a capping agent.

[0133] In some embodiments, the capping agents are bound to the remaining monomeric units that are not bound to a recombinant peptide, i.e., the synthetic polymeric conjugate comprises some monomeric units that are bound to the recombinant peptide, and all the remaining units are capped. This is the case of e.g., pLys conjugates such as that of EXAMPLE 3.2.2 (Scheme 2), wherein 15% of the monomeric units are bound to the recombinant peptide, while the remaining units (i.e., 85%) are capped, in this particular embodiment with thioglycolate (TGA).

[0134] In an embodiment wherein the polymeric backbone is comprised by poly-lysine (pLys) units, the capping agents are bound to the remaining monomeric units that are not bound to a recombinant peptide. Particularly, the capping agents are bound to about 75%, 80%, 85%, 90%, or 95% of the total number of monomeric units, more particularly to about 85%, 90%, or 95%, and even more particularly to about 85%.

[0135] Alternatively, in other embodiments, the synthetic polymeric conjugate comprises some monomeric P7120PC00 24 units that are bound to the recombinant peptide, some that are bound to a capping agent, and the remaining monomeric units are not bound to either a recombinant peptide or a capping agent. This is the case of e.g., PAA conjugates such as that of EXAMPLE 3.2.5 (Scheme 5), wherein 5% of monomeric units are conjugated to the recombinant peptide, 15% of monomeric units are capped, and the remaining monomeric units (i.e., 80%) are not bound to either a recombinant peptide or a capping agent (i.e., some monomeric units are not functionalized).

[0136] In a particular embodiment, the capping agents are bound to from 1 % to 99% of the total number of monomeric units, and wherein the remaining monomeric units are not bound to a capping agent (i.e., some monomeric units are not functionalized). In a more particular embodiment, the capping agents are bound to from: 1 % to 99%, or 1 % to 80%, or 1 % to 70%, or 1 % to 60%, or 1 % to 50%, or 1 % to 40%, or 5% to 30%, or 5% to 20%, or 10% to 20%, of the total number of monomeric units. Particularly, the capping agents are bound to about 1 %, 5%, 10%, 15%, or 20% of the total number of monomeric units, more particularly to about 15%.

[0137] In an embodiment wherein the polymeric backbone is comprised by poly-acrylic acid (PAA) units, the capping agents are bound to about 1 %, 5%, 10%, 15%, or 20% of the total number of monomeric units, more particularly to about 15%.

[0138] In a particular embodiment, the recombinant peptides are bound to from 1 % to 25% of the total number of monomeric units; the capping agents are bound to from 1 % to 50% of the total number of monomeric units, and the remaining monomeric units are not bound to either a recombinant peptide or a capping agent.

[0139] In a more particular embodiment, the recombinant peptides are bound to from 1 % to 10% of the total number of monomeric units; the capping agents are bound to from 5% to 20% of the total number of monomeric units, and the remaining monomeric units are not bound to either a recombinant peptide or a capping agent. Particularly, the polymeric backbone is a polyacrylic acid polymer.

[0140] The capping agent to be used depends on the functional group of the polymer. The capping agent is particularly hydrophilic to avoid loss of solubility in water, not antigenic, and not highly charged. Commonly used capping agents are but not limited to: glycolic acid, gluconic acid, succinic acid, ethanol amine or other amine derivatives, thio-compounds such as thioglycerol, and any combinations thereof.

[0141] In a particular embodiment, the capping agent is selected from the group consisting of glycolic acid, gluconic acid, succinic acid, ethanol amine or other amine derivatives, a thio-compound, a disaccharide, a polyethylene glycol (PEG), and any combination thereof. P7120PC00 25

[0142] In a particular embodiment, the capping agent is glycolic acid. In a particular embodiment, the capping agent is succinic acid (e.g., Schemes 12 to 15). In another embodiment, the capping agent is a thio-compound, particularly thioglycerol or thioglycolic acid (TGA, e.g., Scheme 2). In another embodiment, the capping agent is a disaccharide, particularly cellobiose (e.g., Schemes 19 and 20) or lactose. In another embodiment, the capping agent is polyethylene glycol), particularly a tri(ethylene glycol) (OEG3) or a tri(ethylene glycol) functionalized with a maleimide (OEGs-maleimide, e.g., Scheme 5).

[0143] In a particular embodiment, the capping agent is selected from the group consisting of succinic acid, thioglycolic acid, cellobiose, lactose, and tri(ethylene glycol) functionalized with a maleimide.

[0144] Recombinant peptide

[0145] In the context of the present invention, the term “recombinant peptide", also called “peptide” herein, refers to an amino acid sequence comprising a serotype-specific neutralizing epitope or a conserved neutralizing epitope that is a fragment from a capsid protein sequence of a non-pathogenic viral vector, such as AAV or AdV, determined by peptide arrays from human isolated anti-AAV or Anti- AdV Nabs or mouse anti-AAV monoclonal antibodies. The “neutralizing epitope”, is also called “epitope” or “AAV epitope” or “AdV epitope” herein. The AAV epitope or AdV epitope (or the neutralizing epitope or the epitope) is a conserved epitope that is comprised within the recombinant peptide and that are targeted by anti-AAV or Anti-AdV NAbs from pre-existing human immunity or by mouse anti-AAV monoclonal antibodies.

[0146] According to some particular embodiments, the epitope is indistinctly selected from the group consisting of Table 1 in EXAMPLE 2 and / or of Table 10 in EXAMPLE 9.

[0147] In the context of the present invention, the recombinant peptide has an amino acid sequence comprising a conserved epitope that is a fragment from a capsid protein sequence of a non-pathogenic viral vector, such as AAV or AdV. In a particular embodiment, the recombinant peptides comprise conserved epitopes derivative from anti-AAV NAbs or anti-AdV NAbs that are coming from pre-existing human humoral immunity. Particularly suitable epitopes for depleting neutralizing antibodies against AAV and AdV, were determined and developed by a method to determine the conserved epitopes that are targeted by pre-existing anti-AAV neutralizing antibodies or by pre-existing anti- AdV neutralizing antibodies, both from normal human sera, said method is described herein.

[0148] In EXAMPLE 2 described herein, the inventors employed a multi-step process of antibody selection and isolation (Figure 13) to obtain a precise and specific matrix of neutralizing anti-AAV antibodies, that were utilized to determine the sequence and develop the group of neutralizing epitopes (Table 1) that are ultimately the foundation of this new technology described herein. Additionally, EXAMPLE 9 describes the process for identifying linear epitopes for the mouse monoclonal anti-AAV antibodies P7120PC00 26

[0149] A1 , B1 , A20, ADK1 a, ADK6, ADK8, and ADK9 (Table 10).

[0150] The resulting group of epitopes accounts for only the proteins from the AAV capsid that are targeted and bound by NAbs. Non-toxic, highly conserved, and highly antigenic epitopes were identified to minimize adverse effects and maximize the efficacy of the therapies that could be developed from the conjugated polymers comprising these epitopes. This new approach for obtaining the AAV epitopes constitutes a direct way of obtaining only the functional epitopes that are targeted by neutralizing antibodies avoiding epitopes recognized by binding antibodies that lack neutralizing activity. Therefore, the compounds in this invention could diminish the degree of the immune response against rAAV and rAdV-based therapy, which in turn would affect directly to the efficacy of the therapy at stake. All the epitopes comprised in the unique epitope sequence group Table 1 and Table 10 have in common that are derivative from anti-AAV NAbs.

[0151] In some embodiments, the recombinant peptide is a linear sequence comprising between 5 and 50 amino acids. In a particular embodiment, the recombinant peptide is a linear sequence comprising between 5 and 35 amino acids, or between 8 and 35 amino acids, or between 10 and 40 amino acids, or between 10 and 30 amino acids. Particularly, the recombinant peptide is a linear sequence comprising 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 amino acids, more particularly 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.

[0152] In some embodiments, the recombinant peptide specifically binds to and / or inhibits a target anti-AAV neutralizing antibodies.

[0153] According to the art and as understood by the skilled person, the terms "specificity" or "binding specificity" or "specifically binding" refer to the ability of a binding molecule, e.g., a recombinant peptide of the present invention, to bind preferentially to an epitope versus a different epitope and does not necessarily imply high affinity. The terms "binding specificity" and "specificity" are used interchangeably and can refer both to (i) a specific portion of a binding molecule (e.g., a recombinant peptide), and ii) the ability of the binding molecule to specifically bind to a particular epitope. A binding molecule, e.g., a recombinant peptide, "specifically binds" when there is an specific interaction between the recombinant peptide and its target epitope. The term "specifically binds" means that the recombinant peptide has been generated to bind to its target epitope. The term "non-specific binding" means that a recombinant peptide has not been generated to specifically bind to a target epitope but does somehow bind to the epitope through non-specific means.

[0154] The terms “inhibits a target anti-AAV neutralizing antibody", “inhibition of a target anti-AAV neutralizing antibody", “target anti-AAV neutralizing antibody inhibition”, “blocks a target anti-AAV neutralizing antibody", and grammatical variants thereof, refer to the inhibition and / or reduction of the binding activity of a target antibody, e.g., its ability to recognize and interact with its natural antigen. In the P7120PC00 27 context of the present invention, a target anti-AAV neutralizing antibody is considered to be inhibited by a recombinant peptide of the present invention if the binding activity of said antibody is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the binding activity observed in the absence of the recombinant peptide and in the presence of the antibody’s natural antigen. Neutralizing antibodies typically recognize specific conformational or linear epitopes within viral capsid proteins, and such recognition mediates the neutralization of AAV particles. In some embodiments, the term “inhibit an antibody” refers, for example, to (i) inhibition or complete prevention of antibody binding to its antigen, (ii) reduction or partial inhibition of antibody binding, (iii) inhibition or complete prevention of antibody- mediated neutralizing activity, (iv) reduction or partial inhibition of antibody-mediated neutralizing activity, or (v) any combination thereof, by the recombinant peptides of the present invention. As a consequence of such inhibition of anti-AAV neutralizing antibodies, the infectivity of the AAV vectors (including chimeric AAVs or any serotype) is correspondingly increased. In this description, “inhibition”, "blocking" and “reduction” of antibody activity can be used interchangeably.

[0155] The ability of a recombinant peptide of the present invention to specifically bind to a target anti-AAV neutralizing antibody can be determined by in vitro binding assays, including enzyme-linked immunosorbent assay (ELISA), fluorescence-based binding assays, surface plasmon resonance (SPR), kinetic capillary electrophoresis, fluorescence microscopy or flow cytometry, all of which are routinely used to quantify peptide-antibody interactions. Likewise, the capacity of the recombinant peptide to inhibit the target anti-AAV neutralizing antibody can be evaluated by methods well known to the person skilled in the art, such as competition ELISA, SPR-based inhibition analysis or fluorescencebased competitive binding.

[0156] The term “a target anti-AAV neutralizing antibody” refers to an antibody that specifically recognizes and binds to the capsid of an AAV and / or that inhibits, reduces, or prevents the ability of the virus to enter cells and mediate transduction. Such antibodies can arise naturally through previous exposure to wild-type AAV or can be generated in response to therapeutic AAV vectors. The term encompasses antibodies that neutralize a specific AAV serotype or clade, as well as antibodies displaying cross-reactivity among different AAV variants.

[0157] In some embodiments, the anti-AAV neutralizing antibodies targeted by the recombinant peptides of the present invention include antibodies that specifically recognize and neutralize common AAV serotypes used in gene therapy, such as AAV1 , AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, AAV10 as well as additional clinical or research serotypes such as AAV-DJ, AAV9P31 , and synthetic AAV variants including sAAV1 through sAAV12. In certain embodiments, the targeted anti-AAV neutralizing antibodies also include those directed against engineered or naturally occurring variants such as AAVrhI O, AAVhu37, AAVLK03, and other next-generation or rationally designed capsids. In further embodiments, the term encompasses cross-reactive neutralizing antibodies that recognize conserved epitopes shared among multiple AAV serotypes, thereby reducing or preventing transduction P7120PC00 28 across a broader range of viral vectors.

[0158] In some embodiments, the anti-AAV neutralizing antibodies include mouse monoclonal anti-AAV antibodies such as A1 , A69, B1 , A20, ADK8, ADK1 a, ADK6, and ADK9.

[0159] In particular embodiments, the target anti-AAV neutralizing antibody is a human antibody, for example an antibody naturally present in human serum as a consequence of prior exposure to wild-type AAV or following administration of an AAV-based gene therapy vector. In certain embodiments, the target anti-AAV neutralizing antibody is of murine origin, including antibodies generated in experimental mouse models for the evaluation of AAV vector immunogenicity. In other embodiments, the target antibody is of non-human primate origin, such as cynomolgus or rhesus macaque antibodies commonly used in preclinical studies of AAV-based therapeutics. In further embodiments, the target antibody is of canine, porcine, or ovine origin, depending on the species used as a preclinical model for AAV biodistribution, safety, or immunogenicity assessments. In additional embodiments, the target antibody includes recombinant, monoclonal, or engineered neutralizing antibodies generated in vitro for the purpose of evaluating peptide-antibody binding, cross-reactivity, or blocking capacity.

[0160] In some embodiments, when the recombinant peptide naturally contains one or more internal cysteine residues, such internal cysteines can be substituted with methionine or other non-reactive amino acids to prevent undesired reactions during conjugation. This substitution ensures that only the additional cysteine introduced as a dedicated conjugation handle reacts with the linker, thereby avoiding non-specific attachment, intermolecular disulfide formation, or misorientation, while preserving the overall structure and functionality of the peptide.

[0161] The recombinant peptides described herein, unless otherwise specified, refer to both free and conjugated forms. For free peptides, it should be noted that, upon conjugation, any internal cysteine residues present in the peptide sequence can be substituted with methionine, as described above, to prevent undesired side reactions. In addition, a cysteine residue is introduced as a dedicated conjugation handle, which can be positioned at the N-terminus of the recombinant peptide or at another appropriate location within the synthetic polymeric conjugate, depending on the nature and orientation of the linker, spacer (if any), or type of polymeric backbone to which the peptide is attached.

[0162] The conserved epitopes that have been identified by said method are comprised in the unique epitope sequence group Table 1. Accordingly, in a particular embodiment, the recombinant peptide as used herein comprises a sequence of a unique epitope that is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 202, SEQ ID NO: 303 and SEQ ID NO: 305.

[0163] In another particular embodiment, the recombinant peptide as used herein comprises a sequence of a unique epitope that is selected from the group consisting of: SEQ ID NO: 1 to SEQ ID NO: 30, SEQ P7120PC00 29

[0164] ID NO: 303 and SEQ ID NO: 305 (from 1 .1 peptide to 25.1 peptide, see Table 2). In another particular embodiment, the recombinant peptide is selected from the group consisting of: SEQ ID NO: 1 (1.1 peptide), SEQ ID NO: 4 (2.1 peptide), SEQ ID NO: 7 (3.1 peptide), SEQ ID NO: 9 (4.1 peptide), SEQ ID NO: 10 (5.1 peptide), SEQ ID NO: 13 (8.1 peptide), SEQ ID NO: 18 (13.1 peptide), SEQ ID NO: 19 (14.1 peptide), SEQ ID NO: 22 (17.1 peptide), SEQ ID NO: 24 (19.1 peptide), SEQ ID NO: 303 (19.1 M peptide) and SEQ ID NO: 28 (23.1 peptide). Particularly, the recombinant peptide is selected from the group consisting of: SEQ ID NO: 1 (1.1 peptide), SEQ ID NO: 4 (2.1 peptide), SEQ ID NO: 7 (peptide 3.1), SEQ ID NO: 9 (4.1 peptide), SEQ ID NO: 10 (5.1 peptide), SEQ ID NO: 13 (8.1 peptide), SEQ ID NO: 18 (13.1 peptide), SEQ ID NO: 22 (17.1), SEQ ID NO: 24 (19.1) and SEQ ID NO: 28 (23.1 peptide). More particularly, the recombinant peptide is SEQ ID NO: 18 (13.1 peptide).

[0165] In another particular embodiment, the recombinant peptide as used herein comprises a sequence of a unique epitope that is selected from the group consisting of: SEQ ID NO: 191 to SEQ ID NO: 202 (from 1 ,1 C peptide to 23.1 C peptide, see Table 3), or their corresponding SEQ ID NOs for free peptides (without Cys). Particularly, the recombinant peptide is selected from the group consisting of: SEQ ID NO: 191 (1.1 C peptide), SEQ ID NO: 192 (2.1 C peptide), SEQ ID NO: 193 (3.1 C peptide), SEQ ID NO: 195 (5.1 C peptide), and SEQ ID NO: 199 (17.1 C peptide). Alternatively, the recombinant peptide is selected from the group consisting of: SEQ ID NO: 1 (1.1 peptide), SEQ ID NO: 4 (2.1 peptide), SEQ ID NO: 7 (3.1 peptide), SEQ ID NO: 10 (5.1 peptide), and SEQ ID NO: 22 (17.1 peptide).

[0166] The conserved epitopes that have been identified are also comprised in the unique epitope sequence group Table 10. Accordingly, in a particular embodiment, the recombinant peptide as used herein comprises a sequence of a unique epitope that is selected from the group consisting of: SEQ ID NO: 283 to SEQ ID NO: 302 and SEQ ID NO: 304. Particularly, the recombinant peptide is SEQ ID NO: 283 (A20.2.2 peptide) or SEQ ID NO: 299 (AD6.1 .3 peptide).

[0167] In some embodiments, the recombinant peptide is selected from SEQ ID NO: 1 to SEQ ID NO: 30, SEQ ID NO: 303, SEQ ID NO: 305 (Table 2), SEQ ID NO: 191 to SEQ ID NO: 202 (Table 3), SEQ ID NO: 283 to SEQ ID NO: 302 and SEQ ID NO: 304 (Table 10). In a particular embodiment, the recombinant peptide is selected from SEQ ID NO: 1 (1.1 peptide), SEQ ID NO: 4 (2.1 peptide), SEQ ID NO: 7 (3.1 peptide), SEQ ID NO: 9 (4.1 peptide), SEQ ID NO: 10 (5.1 peptide), SEQ ID: 13 (8.1 peptide), SEQ ID NO: 18 (13.1 peptide), SEQ ID NO: 305 (13.1 M peptide), SEQ ID NO: 19 (14.1 peptide), SEQ ID NO: 22 (17.1 peptide), SEQ ID NO: 24 (19.1 peptide), SEQ ID NO: 303 (19.1 M peptide), SEQ ID NO: 28 (23.1 peptide), SEQ ID NO: 191 (1.1 C peptide), SEQ ID NO: 192 (2.1 C peptide), SEQ ID NO: 193 (3.1 C peptide), SEQ ID NO: 194 (4.1 C peptide), SEQ ID NO: 195 (5.1 C peptide), SEQ ID NO: 196 (8.1 C peptide), SEQ ID NO: 197 (13.1 CM peptide), SEQ ID NO: 198 (14.1 C peptide), SEQ ID NO: 199 (17.1 C peptide), SEQ ID NO: 200 (19.1 C peptide), SEQ ID NO: 201 (19.1 CM peptide), SEQ ID NO: 202 (23.1 C peptide), SEQ ID NO: 283 (peptide A20.2.2), SEQ ID NO: 304 (peptide A20.2.2M), and SEQ ID NO: 299 (AD6.1.3 peptide). P7120PC00 30

[0168] In some embodiments, the recombinant peptide is selected from SEQ ID NO: 1 to SEQ ID NO: 30, SEQ ID NO: 303, SEQ ID NO: 305 (Table 2), SEQ ID NO: 191 to SEQ ID NO: 202 (Table 3), SEQ ID NO: 283 to SEQ ID NO: 302 and SEQ ID NO: 304 (Table 10). In a particular embodiment, the recombinant peptide is selected from SEQ ID NO: 18 (13.1 peptide), SEQ ID NO: 305 (13.1 M peptide), SEQ ID NO: 197 (13.1 CM peptide), SEQ ID NO: 1 (1 .1 peptide), SEQ ID NO: 191 (1.1 C peptide), SEQ ID NO: 4 (2.1 peptide), SEQ ID NO: 192 (2.1 C peptide), SEQ ID NO: 7 (3.1 peptide), SEQ ID NO: 193 (3.1 C peptide), SEQ ID NO: 10 (5.1 peptide), SEQ ID NO: 195 (5.1 C peptide), SEQ ID NO: 22 (17.1 peptide), SEQ ID NO: 199 (17.1 C peptide), SEQ ID NO: 283 (A20.2.2 peptide), SEQ ID NO: 304 (A20.2.2M peptide), and SEQ ID NO: 299 (AD6.1.3 peptide).

[0169] In some embodiments, the polymeric conjugate has a plurality of recombinant peptides, and all the recombinant peptides have the same sequence.

[0170] In the context of the present invention, and for a stronger reduction of the titer of neutralizing antibodies that engage the same epitope, or for enhancing the identification of the presence in a sample of neutralizing antibodies that engage the same epitope, in a particular embodiment, the polymeric conjugate has a plurality of recombinant peptides that are distributed along the polymeric backbone, all the recombinant peptides share the same sequence comprising the same epitope.

[0171] The conserved epitope comprised within the recombinant peptides that are bound to the polymeric backbone has been identified by the method described herein and it is comprised in the unique epitope sequence group Table 1 and / or Table 10. Accordingly, in the context of this particular embodiment, the recombinant peptides share the sequence of a unique epitope that is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 202 and SEQ ID NO: 283 to SEQ ID NO: 305 (comprised in Table 1 and / or Table 10).

[0172] Particularly, the conjugate has a plurality of recombinant peptides, and the corresponding epitopes have different sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 202 and SEQ ID NO: 283 to SEQ ID NO: 305 (Table 1 in EXAMPLE 2 and / or Table 10 in EXAMPLE 9). Particularly, the plurality of recombinant peptides comprise epitopes with five different sequences; more particularly the plurality of recombinant peptides comprise epitopes with four different sequences; more particularly the plurality of recombinant peptides comprise epitopes with three different sequences; more particularly the plurality of recombinant peptides comprise epitopes with two different sequences.

[0173] In a particular embodiment, the polymeric conjugate comprises a plurality of recombinant peptides that are distributed along the polymeric backbone, the recombinant peptides sharing a combination of five different sequences. Each recombinant peptide different sequence comprises a different conserved epitope. Accordingly, in the context of this particular embodiment, the polymeric conjugate P7120PC00 31 can present any combination of five different conserved epitopes that are selected from the epitope group of Table 1 and / or Table 10.

[0174] In a more particular embodiment, the polymeric conjugate comprises a plurality of recombinant peptides that share a combination of four different sequences. Each recombinant peptide different sequence comprises a different conserved epitope. Accordingly, in the context of this more particular embodiment, the polymeric conjugate can present any combination of four different conserved epitopes that are selected from the epitope group of Table 1 and / or Table 10.

[0175] In an even more particular embodiment, the polymeric conjugate comprises a plurality of recombinant peptides that share a combination of three different sequences. Each recombinant peptide different sequence comprises a different conserved epitope. Accordingly, in the context of this even more particular embodiment, the polymeric conjugate can present any combination of three different conserved epitopes that are selected from the epitope group of Table 1 and / or Table 10.

[0176] In an even more particular embodiment, the polymeric conjugate comprises a plurality of recombinant peptides that share a combination of two different sequences. Each recombinant peptide different sequence comprises a different conserved epitope. Accordingly, in the context of this even more particular embodiment, the polymeric conjugate can present any combination of two different conserved epitopes that are selected from the epitope group of Table 1 and / or Table 10.

[0177] This particular embodiment aims for a stronger reduction of the titer of neutralizing antibodies that engage the same epitope, and for enhancing the identification of the presence in a sample of neutralizing antibodies that engage the same epitope.

[0178] In some embodiments, the percentage of the monomeric units bound to the recombinant peptide sequences is selected from the group of ranges consisting of: between 1 % and 80%, between 1 % and 60%, between 1 % and 50%, between 1 % and 40%, and between 1 % and 30%, and between 1 % and 25%. In a particular embodiment, the percentage of the monomeric units bound to the recombinant peptide sequences is between 1 % and 50%.

[0179] In the context of the present invention, the polymeric backbone load with a recombinant peptide is represented by the percentage of the total number of monomeric units that are bound to a recombinant peptide. In a particular embodiment, a load between 1 % and 80% of the total number of the monomeric units of the polymeric backbone are bound to a recombinant peptide. In another particular embodiment, the percentage of the monomeric units bound to the recombinant peptide sequences is selected from the group of ranges consisting of: between 1 % and 45%, between 1 % and 40%, and between 1 % and 30%, and between 1 % and 25%. Particularly, a load between 1 % and 45% or between 1 % and 50% of the total number of the monomeric units of the polymeric backbone are bound to a recombinant peptide. More particularly, a load between 1 % and 30% of the total number P7120PC00 32 of the monomeric units of the polymeric backbone are bound to a recombinant peptide. Even more particularly, a load between 1 % and 25% or between 5% and 15% of the total number of the monomeric units of the polymeric backbone are bound to a recombinant peptide. Particularly, the percentage of the total number of monomeric units that are bound to a recombinant peptide is of about: 5%, 10%, or 15%.

[0180] In some embodiments, the polymeric backbone is poly-L-lysine and the percentage of the total number of monomeric units that are bound to a recombinant peptide is of about 10% or 15%.

[0181] In some embodiments, the polymeric backbone is polyacrylic acid and the percentage of the total number of monomeric units that are bound to a recombinant peptide is of about 5%.

[0182] The monomeric units that are not bound to a recombinant peptide are usually capped, as described in Sections “Polymeric Backbone” and "Capping agent" above.

[0183] Another aspect relates to a recombinant peptide with a sequence selected from the group consisting of SEQ ID NO: 1 to 202 and SEQ ID NO: 283 to 305.

[0184] Linker and spacer

[0185] The synthetic polymeric conjugate of the present invention comprises a linker. In the context of the present invention, the term "linker" refers to a moiety positioned between each recombinant peptide and a single monomeric unit and acts as a binding molecule that provides the operative attachment between them. This definition does not require the recombinant peptide and the monomeric unit to be directly bonded to one another; intermediate groups (e.g., a spacer) may be present between the monomeric unit and the recombinant peptide, provided that the linker constitutes the connecting moiety that establishes the linkage between these components.

[0186] In some embodiments, the linker binds the recombinant peptide to the monomeric unit (e.g., Scheme 12), or the monomeric unit to a spacer (if present) (e.g., Scheme 13), or a spacer (if present) to the recombinant peptide (e.g., Scheme 14), or a spacer to another spacer (e.g., Scheme 15).

[0187] In some embodiments, the linker comprises a linear alkylene chain having between 2 and 30 carbon atoms, particularly between 2 and 20 carbon atoms, more particularly between 2 and 10 carbon atoms. The linear alkylene can be unsubstituted or substituted. In the case of being substituted, the linear alkylene can be mono-, di- or trisubstituted by groups such as, but not limited to: alkyl, alkylamino, hydroxyl, alkoxy, hydroxyalkyl, halogen, thiol, alkylthio, or cyano groups. Besides, up to 5 non- consecutive -CH2- groups within the linear alkylene can be replaced by other groups such as, but not limited to: ether, thioether, ketone, amine, and / or an amide group. In certain embodiments, one or more -CH2CH2O- units are present within the linker backbone, thereby enabling PEG-like or mixed P7120PC00 33 alkylene-PEG linkers. In a particular embodiment, the linear alkylene is substituted with a thiol group, an halogen group, or a amide group.

[0188] In an embodiment, the linker is selected from the group consisting of thiol-reactive linkers, carbonylreactive linkers, disulfide-based linkers, PEG-based linkers, click-chemistry linkers, Michael-acceptor linkers, and diacid-based linkers.

[0189] In an embodiment, the thiol-reactive linker is selected from the group consisting of maleimide-based linkers, S-acyl thioester, thioester linkers, haloacetamide linkers (e.g., bromoacetamide, iodoacetyl, chloroacetyl), or other thiol-reactive electrophiles. In another embodiment, the thiol-reactive linker is a maleimide-based linker or a thioester linker.

[0190] In a particular embodiment, the thiol-reactive linker is a S-acyl thioester linker. In a particular embodiment, the thiol-reactive linker is a thioester. In a particular embodiment, the thiol-reactive linker is a thio-methyl-carbonyl linker (R-S-CH2-C(=O)-R). In a particular embodiment, the thiol-reactive is a thioester (S-acyl) linker followed by an amide bond (-C(=O)-NH-) and an aminopropyl linker.

[0191] In a particular embodiment, the linker is a maleimide-based linker. Maleimide linkers are widely used for thiol-specific conjugation and include maleimide itself and substituted derivatives such as N- methylmaleimide, N-ethylmaleimide, N-phenylmaleimide, and bifunctional NHS(N-hydroxysuccin- imide)-maleimide reagents including SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1- carboxylate), sulfo-SMCC, EMCS (N-(s-maleimidocaproic acid) N-hydroxysuccinimide ester), GMBS (N-y-maleimidobutyryl-oxysuccinimide ester), and BMPS (N-p-maleimidopropionic acid N-hydroxy- succinimide ester), each comprising a maleimide moiety for thiol coupling and an NHS ester for amine reactivity. Cleavable maleimide-containing linkers include sulfo-LC(long chain)-SPDP(Sul- fosuccinimidyl 3-(2-pyridyldithio)propionate) and related reagents that incorporate disulfide-cleava- ble motifs.

[0192] In a particular embodiment, the linker is a maleimide group. In another particular embodiment, the linker is a PEG-maleimide linker, a maleimide-PEG linker, or a PEG-maleimide-PEG linker. In a particular embodiment, the linker is tri(ethylene glycol) functionalized with a maleimide (OEG3-malei- mide).

[0193] In an embodiment, the carbonyl-reactive linker is a hydrazone-forming or an oxime-forming linker suitable for conjugation to aldehyde- or ketone-containing substrates, including hydrazide, aminooxy, or alkoxyamine derivatives.

[0194] In an embodiment, the disulfide-based linker is selected from the group consisting of N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), sulfo-SPDP, SATP, SPDP-PEG derivatives, and other pyridyl- disulfide-containing linkers. P7120PC00 34

[0195] In an embodiment, the PEG-based linker is selected from the group consisting of PEG-NHS, PEG- maleimide, PEG-azide, PEG-thiol, PEG-vinyl sulfone, PEG-acrylate, and heterobifunctional PEG spacers. In a particular embodiment, the PEG-based linker is a tri(ethylene glycol) (OEG3, or PEG3).

[0196] In an embodiment, the click-chemistry linker is an azide-alkyne linker (e.g., CuAAC reagents or SPAAC reagents such as DBCO or BCN), a tetrazine-TCO linker, or a cyclopropene-based linker.

[0197] In an embodiment, the Michael-acceptor linker is selected from vinyl sulfone linkers, acrylate linkers, methacrylate linkers, and fumarate-derived linkers.

[0198] In an embodiment, the diacid-based linker is selected from the group consisting of a linear aliphatic diacid (e.g., oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pamelic acid, suberic acid, azelaic acid, sebacic acid), a branched aliphatic diacid (e.g., 2-methylsuccinic acid, 2,2-dime- thylglutaric acid), an aromatic diacid (e.g., terephthalic acid, isophthalic acid, phthalic acid), an unsaturated diacid (e.g., maleic acid, fumaric acid), and a functionalized diacid (e.g., diglycolic acid, dioxo-diacids, PEG-diacids). In a particular embodiment, the diacid-based linker is a linear aliphatic diacid selected from the group consisting of oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pamelic acid, suberic acid, azelaic acid, and sebacic acid. In a more particular embodiment, the linear aliphatic diacid is adipic acid.

[0199] In some embodiments, conjugation of a recombinant peptide to the polymeric backbone requires the incorporation of an additional cysteine (Cys) residue within the synthetic polymeric conjugate. This cysteine functions as a dedicated conjugation handle, providing a reactive thiol group that ensures site-specific, controlled, and oriented attachment to thiol-reactive linkers (e.g., maleimides, haloace- tyl groups, vinyl sulfones). The introduction of an extra cysteine prevents competition with endogenous cysteines present in the peptide sequence, minimizes structural perturbation of the functional domain, and enables a reproducible 1 :1 conjugation stoichiometry. The conjugation cysteine can be placed at the N-terminus or C-terminus of the recombinant peptide, or within the structure of the synthetic polymeric conjugate depending on the desired spatial orientation.

[0200] Accordingly, in a particular embodiment, the synthetic polymeric conjugate further comprises an additional cysteine residue within the synthetic polymeric conjugate.

[0201] In a particular embodiment, the cysteine residue is placed at the N-terminus of the recombinant peptide. In another particular embodiment, the cysteine residue is placed between the spacer and the linker. In another embodiment, the cysteine residue is placed between the recombinant peptide and the spacer.

[0202] Instead of a cysteine, other residues or functional groups can serve as conjugation handles, including P7120PC00 35 lysine or an engineered N-terminus for amine-based conjugation, selenocysteine as a thiol analogue, or non-natural amino acids bearing bioorthogonal groups such as azides or alkynes, which enable selective click chemistry reactions. These alternative handles allow site-specific attachment, controlled stoichiometry, and preservation of the recombinant peptide functional structure.

[0203] In some embodiments, the polymeric conjugate of the present invention further comprises at least one spacer which binds each recombinant peptide to a single monomeric unit and spatially separates each recombinant peptide from the polymeric backbone. In the context of the present invention, the term “spacer” refers to a moiety that spatially separates the recombinant peptide at a suitable distance from the polymeric backbone in the polymeric conjugate compound. This spacer is additional to the linker, i.e., the spacer can optionally be present. In some embodiments, the spacer binds the cysteine residue to the recombinant peptide (e.g., Scheme 13), or the linker to the cysteine residue, or the monomeric unit to the linker (e.g., Scheme 14).

[0204] In the context of the present invention, any compound defined herein as a “linker” is also suitable for use as a “spacer”. Accordingly, all linkers described throughout this specification, including but not limited to thiol-reactive linkers, carbonyl-reactive linkers, disulfide-based linkers, PEG-based linkers, click-chemistry linkers, Michael-acceptor linkers, and diacid-based linkers, can function as spacers providing a defined distance between two molecular entities.

[0205] In an embodiment, the spacer is selected from the group consisting of alkylene spacers, heteroatomcontaining spacers (optionally comprising one or more ether, thioether, ketone, amine or amide units), PEG or mixed alkylene-PEG spacers, diacid-derived spacers, disulfide-containing spacers, click-compatible spacers, and Michael-acceptor spacers.

[0206] In a particular embodiment, the spacer is a PEG-based spacer. As used herein, “PEG” refers to polyethylene glycol, a polymer formed by repeating ethylene oxide units of the formula -CH2-CH2- O— . PEG spacers can be linear, branched, or mixed alkylene-PEG structures. PEG spacers are preferred due to their flexibility, hydrophilicity, and tunable length, which is determined by the number of ethylene oxide repeat units.

[0207] In a more particular embodiment, the PEG spacer comprises between 1 and 10 ethylene oxide units (PEG1-PEG10), or between 1 and 8 units (PEG1-PEG8), particularly between 4 and 6 units (PEG4- PEG6), and more particularly between 4 and 5 units (PEG4-PEG5). In a particular embodiment, the PEG-based linker is a tri(ethylene glycol) (OEG3, or PEG3). In another particular embodiment, the PEG-based linker is a tetra(ethylene glycol) linker (OEG4, or PEG4).

[0208] In another particular embodiment, the spacer is derived from a diacid, such as succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, or longer-chain fatty diacids including palmitic acid. P7120PC00 36

[0209] Longer-chain diacids confer extended spacer length, increased hydrophobicity, and enhanced spatial separation between conjugated entities. Particularly, the spacer is succinic acid or palmitic acid.

[0210] Any of the foregoing spacers can be used alone or in combination, and can optionally include further substituents or heteroatom insertions as defined for the general linker structure.

[0211] In an embodiment, the spacer is placed between the recombinant peptide and the cysteine residue. In another embodiment, the spacer is placed between the linker and the monomeric unit. In another embodiment, the synthetic polymeric conjugate comprises two spacers, wherein the first spacer is placed between the recombinant peptide and the cysteine residue, wherein the second spacer is placed between the linker and the monomeric unit.

[0212] In some embodiments, the monomeric units bound to a recombinant peptide have one of the following structures:

[0213] (i) monomeric unit - linker - cysteine - recombinant peptide (e.g., Scheme 12);

[0214] (ii) monomeric unit - linker - cysteine - spacer - recombinant peptide (e.g., Scheme 13);

[0215] (iii) monomeric unit - linker - spacer - cysteine - recombinant peptide;

[0216] (iv) monomeric unit - spacer - linker - cysteine - recombinant peptide (e.g., Scheme 14); or

[0217] (v) monomeric unit - spacer - linker - cysteine - spacer - recombinant peptide (e.g., Scheme 15).

[0218] Particular combinations

[0219] In an embodiment, the synthetic polymeric conjugate of the invention comprises:

[0220] (i) a polymeric backbone made of a plurality of monomeric units, each monomeric unit being bound to the following consecutive unit forming a chain, wherein the polymeric backbone has a total number of monomeric units between 2 units and 1200 units, and wherein the polymeric backbone is an homologous a-amino acid polymer or an acrylic acid polymer;

[0221] (ii) one or more recombinant peptides, wherein the recombinant peptides are bound to from 1 % to 50% of the total number of monomeric units, wherein each recombinant peptide is a linear sequence comprising between 8 and 35 amino acids which binds to and / or inhibits a target anti-AAV neutralizing antibody;

[0222] (iii) one or more capping agents, wherein the capping agents are bound to a plurality of monomeric units that are not bound to the recombinant peptide, and wherein the capping agent is selected from the group consisting of glycolic acid, gluconic acid, succinic acid, ethanol amine or other amine derivatives, a thio-compound, a disaccharide, a polyethylene glycol, and any combination thereof; and

[0223] (iv) a linker which binds each recombinant peptide to a single monomeric unit, wherein the linker is selected from the group consisting of thiol-reactive linkers, carbonyl-reactive linkers, disulfide-based linkers, PEG-based linkers, click-chemistry linkers, Michael-acceptor linkers, and diacid-based linkers. P7120PC00 37

[0224] In a particular embodiment, the polymeric backbone is a poly-lysine polymer.

[0225] Particularly, the capping agents are bound to the remaining monomeric units that are not bound to the recombinant peptide.

[0226] Particularly, the polymeric backbone has a total number of monomeric units comprised between 200 units and 600 units, and more particularly has a total number of 400 monomeric units.

[0227] Particularly, the percentage of the monomeric units bound to the recombinant peptide sequences is selected from the group of ranges consisting of: between 1 % and 45%, between 1 % and 40%, and between 1 % and 30%, and between 1 % and 25%, and more particularly is 5%, 10%, or 15%.

[0228] Particularly, the capping agent is selected from the group consisting of succinic acid, thioglycolic acid, cellobiose, and tri(ethylene glycol) functionalized with a maleimide.

[0229] Particularly, the linker is a maleimide-based linker or a thioester linker.

[0230] In a particular embodiment, the synthetic polymeric conjugate (conjugate of EXAMPLE 3.2.2, Scheme 2) comprises:

[0231] (i) a poly-lysine polymer having a total number of 400 monomeric units;

[0232] (ii) one or more recombinant peptides, wherein the recombinant peptides are bound to 15% of the total number of monomeric units, wherein each recombinant peptide is a linear sequence comprising between 8 and 35 amino acids which binds to and / or inhibits a target anti-AAV neutralizing antibody;

[0233] (iii) one or more capping agents, wherein the capping agents are bound to the remaining monomeric units that are not bound to the recombinant peptide, and wherein the capping agent is thioglycolic acid (TGA); and

[0234] (iv) a linker which binds each recombinant peptide to a single monomeric unit, wherein the linker is a thioester linker. Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 199 (conjugate of EXAMPLE 3.2.7, Scheme 7). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 191 (conjugate of EXAMPLE 3.2.8, Scheme 8). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 195 (conjugate of EXAMPLE 3.2.10). In a particular embodiment, the synthetic polymeric conjugate is that of Scheme 2, Scheme 7, or Scheme 8.

[0235] In a particular embodiment, the synthetic polymeric conjugate comprises:

[0236] (i) a poly-lysine polymer having a total number of 400 monomeric units;

[0237] (ii) one or more recombinant peptides, wherein the recombinant peptides are bound to 10% of the total number of monomeric units, wherein each recombinant peptide is a linear sequence comprising between 8 and 35 amino acids which binds to and / or inhibits a target anti-AAV neutralizing antibody;

[0238] (iii) one or more capping agents, wherein the capping agents are bound to the remaining monomeric P7120PC00 38 units that are not bound to the recombinant peptide, and wherein the capping agent is thioglycolic acid (TGA); and

[0239] (iv) a linker which binds each recombinant peptide to a single monomeric unit, wherein the linker is a thioester linker. Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 193 (conjugate of EXAMPLE 3.2.6, Scheme 6). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 192 (conjugate of EXAMPLE 3.2.9, Scheme 9). In a particular embodiment, the synthetic polymeric conjugate is that of Scheme 6 or Scheme 9.

[0240] In a particular embodiment, the synthetic polymeric conjugate (conjugate of EXAMPLE 12.1.6, Scheme 12) comprises:

[0241] (i) a poly-lysine polymer having a total number of 400 monomeric units;

[0242] (ii) one or more recombinant peptides, wherein the recombinant peptides are bound to 15% of the total number of monomeric units, wherein each recombinant peptide is a linear sequence comprising between 8 and 35 amino acids which binds to and / or inhibits a target anti-AAV neutralizing antibody;

[0243] (iii) one or more capping agents, wherein the capping agents are bound to the remaining monomeric units that are not bound to the recombinant peptide (i.e., 85%), and wherein the capping agent is succinic acid;

[0244] (iv) a linker which binds each recombinant peptide to a single monomeric unit, wherein the linker is a maleimide group; and

[0245] (v) a cysteine residue, wherein the cysteine is placed at the N-terminus of the recombinant peptide; and wherein the monomeric units bound to a recombinant peptide have the following structure: monomeric unit - linker - cysteine - recombinant peptide. In a particular embodiment, the synthetic polymeric conjugate has the following structure: recombinant peptide (15%) - cysteine - maleimide group - poly-lysine polymer (400 monomeric units) - succinic acid (85%). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 193 (conjugate of EXAMPLE 12). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 283 or SEQ ID NO: 304 (conjugate of EXAMPLE 13). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 299 (conjugate of EXAMPLE 13). In a particular embodiment, the synthetic polymeric conjugate is that of Scheme 12.

[0246] In a particular embodiment, the synthetic polymeric conjugate (conjugate of EXAMPLE 12.1.6, Scheme 13) comprises:

[0247] (i) a poly-lysine polymer having a total number of 400 monomeric units;

[0248] (ii) one or more recombinant peptides, wherein the recombinant peptides are bound to 15% of the total number of monomeric units, wherein each recombinant peptide is a linear sequence comprising between 8 and 35 amino acids which binds to and / or inhibits a target anti-AAV neutralizing antibody;

[0249] (iii) one or more capping agents, wherein the capping agents are bound to the remaining monomeric units that are not bound to the recombinant peptide (i.e., 85%), and wherein the capping agent is succinic acid; P7120PC00 39

[0250] (iv) a linker which binds each recombinant peptide to a single monomeric unit, wherein the linker is a maleimide group;

[0251] (v) a spacer which binds each recombinant peptide to a single monomeric unit and spatially separates each recombinant peptide from the polymeric backbone, wherein the spacer is a polyethylene glycol comprising 4 ethylene oxide units (i.e., PEG4); and

[0252] (vi) a cysteine residue; wherein the spacer is placed between the recombinant peptide and the cysteine, and wherein the cysteine residue is placed between the spacer and the linker; and wherein the monomeric units bound to a recombinant peptide have the following structure: monomeric unit - linker - Cys - spacer - recombinant peptide. In a particular embodiment, the synthetic polymeric conjugate has the following structure: recombinant peptide (15%) - PEG4 - cysteine - maleimide group - poly-lysine polymer (400 monomeric units) - succinic acid (85%). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 192 (conjugate of EXAMPLE 12). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 193 (conjugate of EXAMPLE 12). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 305 or SEQ ID NO: 197 (conjugate of EXAMPLE 12). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 199 (conjugate of EXAMPLE 12). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 283 or SEQ ID NO: 304 (conjugate of EXAMPLE 13). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 299 (conjugate of EXAMPLE 13). In a particular embodiment, the synthetic polymeric conjugate is that of Scheme 13.

[0253] In a particular embodiment, the synthetic polymeric conjugate (conjugate of EXAMPLE 12.1.7, Scheme 14) comprises:

[0254] (i) a poly-lysine polymer having a total number of 400 monomeric units;

[0255] (ii) one or more recombinant peptides, wherein the recombinant peptides are bound to 15% of the total number of monomeric units, wherein each recombinant peptide is a linear sequence comprising between 8 and 35 amino acids which binds to and / or inhibits a target anti-AAV neutralizing antibody;

[0256] (iii) one or more capping agents, wherein the capping agents are bound to the remaining monomeric units that are not bound to the recombinant peptide (i.e., 85%), and wherein the capping agent is succinic acid;

[0257] (iv) a linker which binds each recombinant peptide to a single monomeric unit, wherein the linker is a maleimide group;

[0258] (v) a spacer which binds each recombinant peptide to a single monomeric unit and spatially separates each recombinant peptide from the polymeric backbone, wherein the spacer is a polyethylene glycol comprising 4 ethylene oxide units (i.e., PEG4); and

[0259] (vi) a cysteine residue; wherein the spacer is placed between the linker and the monomeric unit, and wherein the cysteine residue is placed between the recombinant peptide and the spacer; and P7120PC00 40 wherein the monomeric units bound to a recombinant peptide have the following structure: monomeric unit - spacer - linker - cysteine - recombinant peptide. In a particular embodiment, the synthetic polymeric conjugate has the following structure: recombinant peptide (15%) - cysteine - maleimide group - PEG4 - poly-lysine polymer (400 monomeric units) - succinic acid (85%). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 192 (conjugate of EXAMPLE 12). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 305, or SEQ ID NO: 197 (conjugate of EXAMPLE 12). In a particular embodiment, the synthetic polymeric conjugate is that of Scheme 14.

[0260] In a particular embodiment, the synthetic polymeric conjugate (conjugate of EXAMPLE 12.1.7, Scheme 15) comprises:

[0261] (i) a poly-lysine polymer having a total number of 400 monomeric units;

[0262] (ii) one or more recombinant peptides, wherein the recombinant peptides are bound to 15% of the total number of monomeric units, wherein each recombinant peptide is a linear sequence comprising between 8 and 35 amino acids which binds to and / or inhibits a target anti-AAV neutralizing antibody;

[0263] (iii) one or more capping agents, wherein the capping agents are bound to the remaining monomeric units that are not bound to the recombinant peptide (i.e., 85%), and wherein the capping agent is succinic acid;

[0264] (iv) a linker which binds each recombinant peptide to a single monomeric unit, wherein the linker is a maleimide group;

[0265] (v) two spacers which bind each recombinant peptide to a single monomeric unit and spatially separates each recombinant peptide from the polymeric backbone, wherein each spacer is a polyethylene glycol comprising 4 ethylene oxide units (i.e., PEG4); and

[0266] (vi) a cysteine residue; wherein the first spacer is placed between the recombinant peptide and the cysteine, wherein the second spacer is placed between the linker and the monomeric unit, and wherein the cysteine residue is placed between the first spacer and the linker; and wherein the monomeric units bound to a recombinant peptide have the following structure: monomeric unit - spacer - linker - cysteine - spacer - recombinant peptide. In a particular embodiment, the synthetic polymeric conjugate has the following structure: recombinant peptide (15%) - PEG4 - cysteine - maleimide group - PEG4 - poly-lysine polymer (400 monomeric units) - succinic acid (85%). In a particular embodiment, the synthetic polymeric conjugate is that of Scheme 15.

[0267] In a particular embodiment, the synthetic polymeric conjugate (conjugate of EXAMPLE 14.1.5, Scheme 19) comprises:

[0268] (i) a poly-lysine polymer having a total number of 400 monomeric units;

[0269] (ii) one or more recombinant peptides, wherein the recombinant peptides are bound to 15% of the total number of monomeric units, wherein each recombinant peptide is a linear sequence comprising between 8 and 35 amino acids which binds to and / or inhibits a target anti-AAV neutralizing antibody;

[0270] (iii) one or more capping agents, wherein the capping agents are bound to the remaining monomeric P7120PC00 41 units that are not bound to the recombinant peptide (i.e., 85%), and wherein the capping agent is cellobiose;

[0271] (iv) a linker which binds each recombinant peptide to a single monomeric unit, wherein the linker is a maleimide group; and

[0272] (v) a cysteine residue, wherein the cysteine is placed at the N-terminus of the recombinant peptide; and wherein the monomeric units bound to a recombinant peptide have the following structure: monomeric unit - linker - cysteine - recombinant peptide. In a particular embodiment, the synthetic polymeric conjugate has the following structure: recombinant peptide (15%) - cysteine - maleimide group - poly-lysine polymer (400 monomeric units) - cellobiose (85%). In a particular embodiment, the synthetic polymeric conjugate is that of Scheme 19.

[0273] In a particular embodiment, the synthetic polymeric conjugate (conjugate of EXAMPLE 14.1.5, Scheme 20) comprises:

[0274] (i) a poly-lysine polymer having a total number of 400 monomeric units;

[0275] (ii) one or more recombinant peptides, wherein the recombinant peptides are bound to 15% of the total number of monomeric units, wherein each recombinant peptide is a linear sequence comprising between 8 and 35 amino acids which binds to and / or inhibits a target anti-AAV neutralizing antibody;

[0276] (iii) one or more capping agents, wherein the capping agents are bound to the remaining monomeric units that are not bound to the recombinant peptide (i.e., 85%), and wherein the capping agent is cellobiose;

[0277] (iv) a linker which binds each recombinant peptide to a single monomeric unit, wherein the linker is a maleimide group;

[0278] (v) a spacer which binds each recombinant peptide to a single monomeric unit and spatially separates each recombinant peptide from the polymeric backbone, wherein the spacer is a polyethylene glycol comprising 4 ethylene oxide units (i.e., PEG4); and

[0279] (vi) a cysteine residue; wherein the spacer is placed between the recombinant peptide and the cysteine, and wherein the cysteine residue is placed between the spacer and the linker; and wherein the monomeric units bound to a recombinant peptide have the following structure: monomeric unit - linker - Cys - spacer - recombinant peptide. In a particular embodiment, the synthetic polymeric conjugate has the following structure: recombinant peptide (15%) - PEG4 - cysteine - maleimide group - poly-lysine polymer (400 monomeric units) - cellobiose (85%). In a particular embodiment, the synthetic polymeric conjugate is that of Scheme 20.

[0280] In a particular embodiment, the polymeric backbone is a polyacrylic acid polymer.

[0281] In a particular embodiment, the synthetic polymeric conjugate (conjugate of EXAMPLE 3.2.5, Scheme 5) comprises:

[0282] (i) a polyacrylic acid polymer having a total number of 150 monomeric units;

[0283] (ii) one or more recombinant peptides, wherein the recombinant peptides are bound to 5% of the P7120PC00 42 total number of monomeric units, wherein each recombinant peptide is a linear sequence comprising between 8 and 35 amino acids which binds to and / or inhibits a target anti-AAV neutralizing antibody;

[0284] (iii) one or more capping agents, wherein the capping agents are bound to 15% of the total number of monomeric units, and wherein the capping agent is tri(ethylene glycol) functionalized with a ma- leimide, and wherein the remaining monomeric units (i.e., 80%) are not bound to either a recombinant peptide or a capping agent; and

[0285] (iv) a linker which binds each recombinant peptide to a single monomeric unit, wherein the linker is tri(ethylene glycol) functionalized with a maleimide; wherein the monomeric units bound to a recombinant peptide have the following structure: monomeric unit - capping agent - linker - recombinant peptide. Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 196 (conjugate of EXAMPLE 3.2.11). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 305 or SEQ ID NO: 197 (conjugate of EXAMPLE 3.2.12). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 191 (conjugate of EXAMPLE 3.2.13). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 192 (conjugate of EXAMPLE 3.2.14). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 193 (conjugate of EXAMPLE 3.2.15). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 194 (conjugate of EXAMPLE 3.2.16). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 193 (conjugate of EXAMPLE 3.2.15). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 194 (conjugate of EXAMPLE 3.2.16). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 195 (conjugate of EXAMPLE 3.2.17). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 198 (conjugate of EXAMPLE 3.2.18). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 199 (conjugate of EXAMPLE 3.2.19). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 303, SEQ ID NO: 200, or SEQ ID NO: 201 (conjugate of EXAMPLE 3.2.20). Particularly, the recombinant peptide has an amino acid sequence set forth in SEQ ID NO: 28 or SEQ ID NO: 202 (conjugate of EXAMPLE 3.2.21). In a particular embodiment, the synthetic polymeric conjugate is that of Scheme 5.

[0286] Pharmaceutical composition

[0287] A second object of protection is constituted by a pharmaceutical composition comprising a synthetic polymeric conjugate according to the present invention; and at least one pharmaceutically acceptable excipient, carrier or vehicle.

[0288] In the context of the present invention, appropriate amounts of the synthetic polymeric conjugate as described herein can be formulated with pharmaceutically acceptable excipients, vehicles and / or P7120PC00 43 carriers to obtain a pharmaceutical composition.

[0289] In the context of the present invention, the term “appropriate amount” refers to an effective quantity of the polymeric conjugate in the pharmaceutical composition. This quantity can vary within a wide range and, in general, will vary depending on the particular circumstances of application, duration of the exposure and other considerations. In a particular embodiment, the dose ranges from 0.01 mg / kg to 100 mg / kg, particularly from 0.05 mg / kg to 50 mg / kg, more particularly from 0.1 mg / kg to 10 mg / kg, and more particularly from 0.1 mg / kg to 5 mg / kg.

[0290] In the context of a particular embodiment, the present invention relates to a pharmaceutical composition, such as a vaccine or a gene therapy composition, comprising the polymeric conjugate defined herein and further comprising an AAV vector or an AdV vector and optionally at least one pharmaceutically acceptable excipient. Particularly, this pharmaceutical composition is for use in vaccination or gene therapy and / or for use in prevention or inhibition of an undesirable immune reaction against the viral vector.

[0291] The term “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable excipients, vehicles and / or carriers” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the agents according to the invention.

[0292] A pharmaceutical composition that includes a synthetic polymeric conjugate as described herein can be delivered to a subject by a variety of routes including, without limitation, by local (e.g. topical, rectal, ocular, etc.) or systemic administration. Systemic delivery can include oral or parental (e.g., intravenous, subcutaneous, intramuscular, and intraperitoneal) administration. Additionally, it is also possible to administer the pharmaceutical composition comprising the synthetic polymeric conjugate intranasally or sublingually which allows systemic administration by a non-aggressive mode of administration. Also, intraventricular administration can be adequate. A particular route of delivery is intravascular (e.g. intraarterial or intravenous) or subcutaneous injection. In a particular embodiment, the agent for use according to the invention is administered to the subject subcutaneously or intravenously. Those skilled in the art are familiar with the principles and procedures discussed in widely known and available sources as Remington's Pharmaceutical Science (17th Ed.), 1985; and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics (8th Ed.), 1990 both of which are incorporated herein by reference. P7120PC00 44

[0293] The pharmaceutical composition can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids and the like, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, thriethylamine, 2-ethylamino ethanol, histidine, procaine or similar. In a particular embodiment, the salt is a sodium or potassium salt.

[0294] Solid dosage forms for oral administration can include conventional capsules, sustained release capsules, conventional tablets, sustained-release tablets, chewable tablets, sublingual tablets, effervescent tablets, pills, suspensions, powders, granules, and gels. Formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate, etc. Such dosage forms can also comprise, as in normal practice, additional substances other than inert diluents, such as dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. In the case of capsules, tablets, effervescent tablets and pills, the dosage forms can also comprise buffering agents. Tablets and pills can be prepared with enteric coatings.

[0295] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and combinations thereof.

[0296] Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and combinations thereof.

[0297] Exemplary surface active agents and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl al- P7120PC00 45 cohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters {e.g., polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrroli- done), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof.

[0298] Exemplary binding agents include, but are not limited to, starch (e.g., cornstarch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalac- tan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; and combinations thereof.

[0299] Exemplary preservatives can include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, brono- pol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and P7120PC00 46 phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, German 115, Germaben 11 , NeoIone, Kathon, and Euxyl. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0300] Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and combinations thereof.

[0301] Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0302] Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba , macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

[0303] Liquid dosage forms for oral administration can include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the technique, P7120PC00 47 such as water. Those compositions can also comprise adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening agents, flavoring and perfuming agents.

[0304] Various delivery systems are known in the art, including encapsulation in liposomes, microbubbles, emulsions, microparticles, microcapsules and the like.

[0305] Injectable preparations, for example, aqueous or oleaginous suspensions, can be formulated according to the known technique using suitable dispersing agents, wetting agents and / or suspending agents. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. Sterile oils are also conventionally used as solvents or suspending media.

[0306] Typically, compositions for intravenous, intramuscular, subcutaneous, intraperitoneal or intraventricular administration are solutions in sterile isotonic aqueous buffer. In some embodiments, the composition can contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Illustrative non-limiting examples of pH buffering agents include Tris-HCI buffer, acetate buffer, citrate and phosphate buffer or combinations thereof. The term “acetate buffer”, “citrate buffer” and “phosphate buffer” as used herein can referto a buffer system comprising an organic acid (acetic acid, citric acid, and phosphoric acid, respectively) and a salt thereof. Each of them can be added in a sufficient amount. The pH of the composition according to the present invention can be in the range from about 4 to about 9, particularly from about 5 to about 8, including pH 5, pH 5.5, pH 6, pH 6.5 and pH 7.

[0307] Where necessary, the polymeric conjugate is comprised in a composition also including a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0308] The effective quantity of the polymeric conjugate can vary within a wide range and, in general, will vary depending on the particular circumstances of application, duration of the exposure and other considerations. In a particular embodiment, the dose ranges from 0.01 mg / kg to 100 mg / kg, particularly from 0.05 mg / kg to 10 mg / kg, such as from 0.05 mg / kg to 2 mg / kg or 0.1 mg / kg to 5 mg / kg. In particular embodiments, the dose ranges from 0.01 to 5 mg / kg, particularly from 0.05 to 2.5 mg / kg and most particularly from 0.1 to 1 mg / kg. In other particular embodiments, said dose is less than 1 .0 mg / kg, such as from 0.1 to 0.5 mg / kg, including from 0.3 to 0.4 mg / kg. P7120PC00 48

[0309] The synthetic polymeric conjugate or a pharmaceutical composition comprising thereof, can be administered a single time. It typically will be administered regularly throughout the course of a treatment, for example, one, two, three, four, or more times a day, every other day, weekly, bi-weekly, every three weeks or monthly.

[0310] In the context of the present invention, the pharmaceutical composition is a non-immunogenic pharmaceutical composition. More particularly, a non-immunogenic pharmaceutical composition does not bind to any HLA and / or MHC molecule (e.g. in a mammal, particularly in a human, in a non-human primate, in a sheep, in a pig, in a dog or in a rodent; or of the individual to be treated) in vivo.

[0311] Medical uses

[0312] A third object of protection relates to a synthetic polymeric conjugate or a pharmaceutical composition as described herein, for use as a medicament. It should be understood that said object analogously refers to a method for treating and / or preventing a disease in a subject comprising administering the synthetic polymeric conjugate or the pharmaceutical composition as described herein.

[0313] Another aspect relates to a synthetic polymeric conjugate or a pharmaceutical composition as described herein for the treatment or prevention of one disease selected from the group consisting of: cancer, ocular disease, neurological disease, metabolic disease, hematological disease, neuromuscular disease, cardiovascular disease, autoimmune disease, and infectious disease. Alternatively, the invention also encompasses a method for treating and / or preventing a disease in a subject comprising administering the synthetic polymeric conjugate or the pharmaceutical composition as described herein, wherein the disease is selected from the group consisting of: cancer, ocular disease, neurological disease, metabolic disease, hematological disease, neuromuscular disease, cardiovascular disease, autoimmune disease, and infectious disease.

[0314] In the context of the present invention, any product defined for use in medicine, or for a specific treatment or prevention of illnesses should be understood also as a method of treatment for, and even as a compound for manufacturing a medicament for treating illnesses. The terms “treating”, “treatment”, “therapy” or “therapeutic” refer to the administration or consumption of an active ingredient, that is a synthetic polymeric conjugate according to the invention, or of a pharmaceutical composition comprising such an active ingredient for the purposes of curing, relieving, reducing or attenuating, or improving a disease or a pathological disorder, or one or several associated symptoms, or for preventing or slowing down the progression of this symptom or symptoms or this disease, or for stopping the development of this symptom or symptoms, or this disease or this pathological disorder, in a statistically significant manner. More particularly, “treating” or “treatment” includes any approach for obtaining a beneficial effect or a desired result with respect to a disease in an individual. The beneficial or desired clinical results can include, but are not limited to, the attenuation or improvement of the disease or one or several symptoms of such a disease; the diminution or reduction of the P7120PC00 49 extent of the disease, the stabilization, that is, the absence of worsening of a disease, or one or several symptoms of such a disease; the prevention of a disease, or one or several symptoms of such a disease; the prevention of the propagation of a disease, or one or several symptoms of such a disease; the slowing down of a disease, or one or several symptoms of such a disease or the progression of one or more symptoms of such a disease; the diminution of the recurrence of an associated disease, or one or several symptoms of such a disease; and the interruption of a disease, or one or several symptoms of such a disease. In other words, “treatment” as used herein comprises any recovery, improvement, reduction or interruption of a disease, or one or several symptoms of such a disease. A “reduction” of a symptom or a disease means a diminution of the severity or the frequency of the disease or the symptom, or the elimination of the disease or the symptom.

[0315] Another aspect relates to a synthetic polymeric conjugate or a pharmaceutical composition as defined herein for use in the treatment of a subject for the removal of one or more anti-AAV or anti-AdV neutralizing antibodies, wherein the treatment is intracorporeal or extracorporeal. In a particular embodiment, the synthetic polymeric conjugate or the pharmaceutical composition as defined herein is for use in the treatment of a subject for the removal of one or more anti-AAV neutralizing antibodies, wherein the treatment is intracorporeal. Alternatively, the invention also encompasses a method for removing of one or more anti-AAV or anti-AdV neutralizing antibodies in a subject, comprising administering to the subject a synthetic polymeric conjugate or a pharmaceutical composition as described herein, wherein the treatment is intracorporeal or extracorporeal.

[0316] In the context of the present invention, the term "removing" in relation to “removing one or more anti- AAV antibodies” is not limited to the complete elimination of such antibodies. Rather, it is intended to encompass any lowering, reduction, sequestration, or depletion of the corresponding neutralizing antibodies, to an extent sufficient to achieve the therapeutic or functional effect described in the invention.

[0317] In the context of the present invention, pre-existing humoral response against the AAV or AdV vector capsid, measured as anti-AAV or anti-AdV NAbs, likely comes from the immunological memory of past encounters with a wild-type virus. This is the single most detrimental burden to AAV or AdV- based gene therapy because even low levels of seropositivity in an individual can drastically reduce the efficacy of the gene therapy treatment with AAV or AdV vectors if the vector is given by intravenous administration to the individual. As for the AAV or AdV-based gene therapy, NAbs are also diminishing the efficacy of AAV or AdV-based vaccines in an individual.

[0318] According to a particular embodiment, the recombinant peptide loaded to the polymeric backbone is capable of recognizing NAbs that are in the system of an individual. Those NAbs appear in the individual after natural exposure to wild-type AAV or AdV serotypes or after receiving gene therapy with rAAV or rAdV vectors. According to a particular embodiment, the synthetic polymeric conjugate or the pharmaceutical composition comprising the synthetic polymeric conjugate is for use in the gene P7120PC00 50 therapy treatment of a subject for the removal of one or more anti-AAV or anti-AdV neutralizing antibodies.

[0319] According to a particular embodiment, the polymeric conjugate is for intracorporeal sequestration, or intracorporeal depletion, or intracorporeal removal of at least one neutralizing antibody against the AAV or AdV vector in an individual, particularly in the bloodstream of the individual and / or for reduction of the titer of at least one neutralizing antibody against the AAV or AdV vector in the individual, particularly in the bloodstream of the individual.

[0320] Immuno-apheresis is an extracorporeal medical procedure in which the blood of a patient is passed through a device that separates blood into different components, removes one or more constituents and returns the rest, with or without a replacement fluid. Selective immune-apheresis involves the filtration of the individual's plasma through an extracorporeal, selective antibody-adsorber cartridge that will deplete the undesired antibody such as NAbs based on selective binding to its antigen binding site. Although not yet established in the clinical routine, selective immune-apheresis has for instance been used for removing anti-A or anti-B antibodies from the blood prior to ABO-incompatible transplantation or with respect to indications in transfusion medicine. According to a particular embodiment, the polymeric conjugate is for extracorporeal sequestration or extracorporeal removal of at least one neutralizing antibody against an AAV or AdV vector used in the gene therapy of an individual.

[0321] According to another particular embodiment, one or more anti-AAV or anti-AdV neutralizing antibodies are present in the subject, said neutralizing antibodies being specific for at least one epitope comprised in the recombinant peptides present in the synthetic polymeric conjugate.

[0322] The terms "subject", "patient", and "individual", and variants thereof are used interchangeably herein and refer to any mammalian subject, including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like), and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like) for whom diagnosis, treatment, or therapy is desired, particularly humans. The uses and methods described herein are applicable to both human therapy and veterinary applications. In an embodiment, the subject is a human.

[0323] In the context of the present invention, the subject to be treated can be a non-human animal, particularly a non-human primate, a sheep, a pig, a dog or a rodent, in particular a mouse. In a particular embodiment, the individual to be treated is a human.

[0324] According to the particular embodiment, the treatment improves the efficacy of a therapy comprising an AAV vector or AdV vector, and the synthetic polymeric conjugate or pharmaceutical composition is administered prior to or concurrently with the AAV vector or AdV vector. P7120PC00 51

[0325] According to a particular embodiment, the synthetic polymeric conjugate or the pharmaceutical composition for use in a gene therapy treatment of a subject increases or improves the efficacy of the gene therapy comprising an AAV vector or AdV vector.

[0326] In the context of the present invention, the term "gene therapy" refers to a technique that uses a gene(s) to treat, prevent or cure a disease or medical disorder utilizing virus vectors as a platform for gene delivery. The virus vectors can particularly be an AAV vector or an AdV vector. In the context of the present invention, the term “gene therapy composition” refers to a composition comprising an AAV vector or an AdV vector.

[0327] In the context of a particular embodiment, the pharmaceutical composition comprising the polymeric conjugate is administered to the individual prior to the administration of the gene therapy or the gene therapy composition. In a particular embodiment, the pharmaceutical composition comprising the polymeric conjugate is administered to the individual concurrently or at the same time as the administration of the gene therapy or the gene therapy composition.

[0328] According to a particular embodiment, the pharmaceutical composition comprising the synthetic polymeric conjugate is for use in the gene therapy treatment of an individual. In the use of a gene therapy treatment comprising an AAV vector or AdV vector in an individual, the pharmaceutical composition is meant to encompass the administration of the referred pharmaceutical composition and the gene therapy composition to an individual, in the same pharmaceutical formulations or in separate pharmaceutical formulations, and at the same time or at different times. If both therapeutic compositions, the pharmaceutical composition and the gene therapy composition, are administered at different times, they should be administered sufficiently close in time to provide for a synergistic response to occur. In such instances, it is contemplated that one would typically administer both therapeutic compositions within about 12-24 hours of each other and, more particularly , within about 6-12 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1 , 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. In other situations, it might be desirable to reduce the time between administration, administering both therapeutic agents within seconds or minutes to hours, particularly within about 6 hours from each other, more particularly within about 1 or 3 hours.

[0329] In a particular embodiment, the therapy comprising an AAV vector or AdV vector is a vaccine, or an AAV-based vaccine, and the pharmaceutical composition is used to increasing the efficacy of the vaccine comprising an AAV vector or AdV vector.

[0330] Use for detection and method for obtaining a synthetic polymeric conjugate

[0331] The present invention also relates to a use of a synthetic polymeric conjugate or a pharmaceutical P7120PC00 52 composition of the present invention for the in vitro detection and / or quantification of an anti-AAV neutralizing antibody in a sample from a subject.

[0332] Another aspect relates to a method for obtaining a synthetic polymeric conjugate, comprising attaching a recombinant peptide selected from the group consisting of SEQ ID NO: 1 to 202 and SEQ ID NO: 283 to 305, to a polymeric backbone as described herein.

[0333] Another object of protection is a method for obtaining a synthetic polymeric conjugate of the present invention, comprising the following steps: a) isolating a human anti-AAV neutralizing antibody or a human anti-AdV neutralizing antibody from a human sample using cell-based assays and isolation procedures; b) evaluating the neutralization capacity of the isolated human anti-AAV neutralizing antibody or the human anti-AdV neutralizing antibody by cell-based assays; c) determining a recombinant peptide comprising an epitope targeted by the human anti-AAV neutralizing antibody or the human anti-AdV neutralizing antibody by printed peptide-arrays; d) synthesizing the recombinant peptide comprising the epitope; e) evaluating the capacity of the peptide comprising the epitope to bind and block / inhibit the target human anti-AAV neutralizing antibody or the human anti-AdV neutralizing antibody; and f) synthesizing the synthetic polymeric conjugate by covalently attaching the recombinant peptide comprising the epitope to the polymeric backbone.

[0334] In a particular embodiment, the term “epitope” is a neutralizing epitope that identifies, bins and blocks / inhibits the target human anti-AAV neutralizing antibody or the human anti-AdV neutralizing antibody.

[0335] In the context of the particular invention the term “neutralizing antibody” refers to an antibody that binds to the rAAV capsid or the rAdV capsid and can inhibit vector transduction.

[0336] For the identification of neutralizing AAV epitopes the inventors employed a multi-step process of antibody selection and isolation to obtain a precise and specific matrix of neutralizing anti-AAV antibodies (Example 2). First, NHS was exposed to each AAV serotype to isolate serotype-specific AAV- binding antibodies (BAb). These BAbs were characterized by isotyping (Figure 15) and then used in an in vitro AAV neutralization assay (Figure 14). During the assay, the BAbs were incubated with their corresponding AAV serotype to allow the formation of the BAb-AAV complexes. The mixture BAb-AAV was used to infect HEK293T cells. After 24h of infection, cell supernatant was collected, which likely contained AAV particles bound to BAbs that were unable to enter the cells, indicating neutralization by neutralizing antibodies (NAbs). The NAbs were then purified using magnetic beads, isotyped (Figure 15), and utilized as a refined and specific matrix of AAV-neutralizing antibodies for P7120PC00 53 the identification of neutralizing AAV epitopes by Peptide arrays (Table 1). Further bioinformatic analysis of the identified sequences revealed a list of consensus sequences and singletons conserved across serotypes (Table 2). A selection of 12 consensus and singletons sequences (Table 3) were used as free peptides as well as polypeptides conjugated to PLys or PAA for the in vitro proof of concept (PoC).

[0337] Another aspect relates to an in vitro method for detecting and / or quantifying an anti-AAV neutralizing antibody or an anti-AdV neutralizing antibody in a sample from a subject by means of a synthetic polymeric conjugate of the present invention, wherein the method comprises: a) contacting the sample with the synthetic polymeric conjugate and a detection agent, b) incubating the sample with the synthetic polymeric conjugate and a detection agent to form a detection-polymeric conjugate-neutralizing antibody complex; c) separating the detection-polymeric conjugate-neutralizing antibody complex from the remaining sample containing the unbound detection agent; and d) detecting the anti-AdV or anti-AAV neutralizing antibody.

[0338] In the context of the present invention, the term “sample", or “antibody-containing sample", or “anti- body-containing biological sample” as used herein includes biological fluids, but not limited to whole blood, serum, plasma, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural effusion, urine, as well as any tissue or any other bodily constituent that could contain antibodies. Particularly, said “antibody-containing sample” is whole blood, serum or plasma; more particularly serum. Particularly, the sample is obtained from a mammal, particularly a human. In a particular embodiment, the sample is obtained from a non-human animal, particularly a nonhuman primate, a sheep, a pig, a dog or a rodent, in particular a mouse.

[0339] In a particular embodiment, the synthetic polymeric conjugate employed for the in vitro method for detecting and / or quantifying an anti-AAV neutralizing antibody is a polyacrylic acid polymer, more particularly, the synthetic polymeric conjugate is that of Scheme 5.

[0340] Kit

[0341] Another object of protection is a kit for detecting and / or quantifying an anti-AAV neutralizing antibody or an anti-AdV neutralizing antibody in a sample from a subject, comprising: a) a capture agent comprising a solid support or carrier coated with a synthetic polymeric conjugate of the present invention or the recombinant peptide comprising an AAV epitope selected from the group consisting of Table 1 and / or Table 10 (SEQ ID NO: 1 to SEQ ID NO 202 and SEQ ID NO: 283 to SEQ ID NO: 305); b) a labeled detection agent comprising an antibody that binds the anti-AAV neutralizing antibody or an anti-AdV neutralizing antibody; c) an incubator configured to incubate the sample with the capture agent and detection agent P7120PC00 54 to form a polymeric conjugate-neutralizing antibody-detection agent complex or a peptide- neutralizing antibody-detection agent complex; and d) means for detecting and / or quantifying the presence of the polymeric conjugate-neutralizing antibody-detection agent complex or the peptide-neutralizing antibody-detection agent complex; wherein the sample is cell supernatant, blood, serum, plasma or any other organic fluid.

[0342] In the context of the present invention, the means for detection and / or quantifying the presence of the polymeric conjugate-neutralizing antibody-detection agent complex or the peptide-neutralizing antibody-detection agent complex can be made but not limited to colorimetric assay, luminescence and fluorescence.

[0343] EXAMPLES

[0344] EXAMPLE 1. Examples referred to HART (Harmful Antibody Removal Technology)

[0345] EXAMPLE 1 summarizes the polymeric conjugates used by the applicant prior to the present invention. These compounds are exemplified herein to demonstrate that such polymeric conjugates are capable of removing harmful antibodies and do not exhibit toxicity. In the present invention, the inventors present improved polymeric conjugates which are, for the first time, conjugated with a recombinant polypeptide (AAV epitope).

[0346] RemAb Therapeutics developed a relevant proof of concept in Cynomolgus monkeys (2.5-3 years old) to study the efficacy of HART for selectively removing a specific autoantibody. In this case, naturally produced anti-aGal antibodies (Galili U et al., 1984) were removed from animals by administering a polymeric scaffold developed in HART, where varying loads of the aGal antigen (Gala1 ,3- Galpl ,4-GlcNAc) were covalently conjugated to the structure (RA01 compounds, see e.g., compound RA0127 in Scheme 11 , EXAMPLE 1.1).

[0347] The scaffold or polymeric backbone is a synthetic homo-poly amino acid, particularly formed by L- lysine monomeric units. The scaffold's degree of polymerization (DP) is from 25-1500, more preferable from 50-1200, and still more preferable from 100 to 1000. The loads of the aGal antigen in the final compound are from 1-100%, more preferable from 5-50%, and still more preferable from 10- 30%. The glycan (aGal antigen, i.e., Galal ,3-Galp1 ,4-GlcNAc) is separated from the scaffold with convenient linkers elsewhere described. The linker group of formula -R4-Z, which is attached at the reducing end of the aGal antigen contains a functional group -Z, which is either a -NH2, or -COOH, or -SH group, which allows to covalently bind the active carbohydrate moiety to a suitable support, said functional group being linked to the carbohydrate though a spacer group R4. (i) In one embodiment, Z is -NH2. (ii) In another embodiment, Z is -SH. (iii) In another embodiment, Z is -COOH. (iv) P7120PC00 55

[0348] Spacer, (v) The spacer (R4) is an arm connecting the oxygen atom at the reducing end of the carbohydrate moiety with the functional group Z. This linking arm maintains the active carbohydrate epitope spatially separated at a suitable distance from the polymeric support in the glyco conjugate. In this regard, therefore, the selection of a particular spacer is not critical insofar as it is suitable for this function.

[0349] Typically, the spacer R4 is a linear chain, for example, of 2 to 20 atoms, which is not immunogenic and also not reactive. R4 is typically a linear alkylene, for example, a C2-C20 linear alkylene, particularly a C2-C10 linear alkylene, which is unsubstituted or 15 substituted, for example, mono-, di- or trisubstituted, for example, by groups such as alkyl, alkylamino, hydroxyl, alkoxy, hydroxyalkyl, halogen, thiol, alkylthio, or cyano, among others; and wherein some non-consecutive -CH2- groups in the alkylene chain, for example, up to 5 non-consecutive -CH2- groups, particularly up to 3 non- consecutive -CH2- groups can be replaced by other groups, by 20 interrupting the alkylene chain with, for example, ether, thioether, ketone, amine, and / or an amide groups, among others.

[0350] In one embodiment, the spacer R4 is a linear C2-C10 alkylene group, particularly unsubstituted, and wherein up to 3 nonconsecutive -CH2- groups can be replaced by a group selected from -O-, -S-, - N(H)-, -N(R5)-, -N(OR5)-, -N(H)-CO-, -CO-N(H)-, -N(R5)-CO- and -CO-N(R5)-, wherein R5 is a C1- C3 alkyl group.

[0351] In one particular embodiment, the spacer R4 is a group of formula - 30 (CH2)2-8-NH2, for example, a group of formula -(CH2)3-NH2, which is the one used in the preparative examples, but other spacers, as defined above, can be equally suitable.

[0352] The remaining functional groups are reacted with suitable non-immunogenic synthetic capping substance, with the purpose of suppressing the reactivity of the remaining free functional groups on the polymer. The capping agent to be used depends on the particular functional group on the polymer. The selection of the specific capping agent is not critical, as long as it can be easily bound with high yield to the free reactive sites on the polymer and it produces stable, non-reactive capped ends. The capping molecule is particularly hydrophilic to avoid loss of solubility in water, not antigenic, and not highly charged. It is therefore well within the capabilities of the skilled in the art to choose a suitable capping agent in each case. Commonly used capping agents are, for example, glycolic acid, gluconic acid, succinic acid, ethanol amine or other amine derivatives, thio-compounds such as thioglycerol, among many others.

[0353] 1.1 Selective intracorporeal removal of anti-aGal autoantibodies in non-human primates with RemAb technology (HART).

[0354] Figure 1 shows the efficacy of HART for RA0127 (DP 1000, 27% aGal load). Figure 1(A). Scheme of the RA0127 intravenous administration in Cynomolgus monkeys and the different time points of blood collection. Animals received 50 mg / kg through a 30-minute intravenous infusion three times a P7120PC00 56 week for four weeks. The blood samples were taken before administration (B. inf), at 24 hours (day 1) after the first administration, 72 hours after the last administration (day 29), 7 days after the last administration (day 33), and 18 days after the last administration (day 44). Control animals only received the vehicle (n=2). Figure 1(B). Baseline anti-aGal antibody levels varied among individuals and by isotype (IgG or IgM). 50 mg / kg administration resulted in a removal of 90% (IgG) and 97% (IgM) within 24 hours, demonstrating the high potentiality of HART in terms of efficacy in vivo. After 18 days following the final administration (day 44), antibody levels showed minimal recovery, proving a long-lasting anti-aGal antibody intracorporeal inhibition. The control animals (vehicle) showed stable levels of anti-aGal antibodies during the procedure. Therefore, HART could offer immediate intracorporeal removal of undesirable circulating antibodies and open safe windows (weeks) with a minimum return of antibodies depending on the dosing scheme. This is particularly advantageous when safe windows are needed to administer specific therapies affected by circulating harmful antibodies, like the case of gene therapy, cancer therapies, and vaccines using adenovirus or adeno- associated adenovirus.

[0355] Scheme 11. RA0127 chemical structure, n represents the average degree of polymerization, x represents the fraction of glycosylated monomer; and 1-x represents the fraction of thioglycerol-capped monomer.

[0356] 1.2 HART compounds have a fast pharmacokinetic (PK), with no systemic accumulation

[0357] Cynomolgus monkeys were administered with increased doses of RA0127 (5-15-50 mg / Kg) through a 30-minute intravenous infusion three times a week for four weeks. Blood samples were taken on days 1 (first dose) and 26 (last dose) before administration (Figure 2(B)), just after finishing the infusion (TO), and at 2, 4, 8, 24, 36, and 48 hours after the administration.

[0358] The compound was followed in monkey serum by ELISA using a specific monoclonal antibody de P7120PC00 57 veloped by RemAb Therapeutics (clone 34D11). As shown in Figure 2(B), the compound has maximal exposition almost immediately after the first administration on day 1. The compound is then cleared very quickly from circulation. Depending on the dosing scheme, there was nearly no compound in circulation between 4-10 hours after the first administration. A second PK study confirmed these results. Similarly, there was a maximum peak almost immediately after the last administration on week 4 (day 26), with nearly no compound accumulation from the previous doses. Then, the compound was rapidly cleared from circulation following a similar kinetic pattern to the one obtained after the first administration (day 1). There was no compound accumulation in circulation despite administering three doses of the drug product per week for 4 weeks. This fact is also a relevant advantage of HART. The example demonstrated the fast clearing of the compound from circulation with no systemic accumulation, reducing potential side effects. This is particularly advantageous when quick responses are needed to administer certain therapies affected by circulating harmful antibodies, like the case of gene therapy, cancer therapies, and vaccines using adenovirus or adeno- associated adenovirus.

[0359] 1 .3 HART scaffolds are not toxic

[0360] Another advantage of the scaffolds used for improving the efficacy of viral vectors in gene therapy, vaccines, etc, is the low risk of toxicity associated with the polymeric scaffold developed by HART. The systemic activation of complement (C3a) in Cynomolgus monkeys after the administration of RA0127 was followed by ELISA. Figure 3 shows animals treated with the highest dose evaluated (50 mg / Kg) and control (vehicle). There was no systemic activation of complement, demonstrating the lack of immunotoxicity of scaffolds developed by HART (n=10). Importantly, we also observed no anti-drug antibodies and noted that the total Ig and white blood cell levels remained stable during the procedure. Indeed, it confirms the non-immunogenic nature of these compounds and the safety of the scaffolds. This is particularly advantageous for certain therapies affected by circulating harmful P7120PC00 58 antibodies, such as gene therapy, cancer therapies, and vaccines using adenovirus or adeno-asso- ciated adenovirus.

[0361] EXAMPLE 2. Neutralizing AAV epitopes methodology description

[0362] For the identification of neutralizing AAV epitopes we employed a multi-step process of antibody selection and isolation to obtain a precise and specific matrix of human anti-AAV neutralizing antibodies (Figure 13). First, normal human serum (NHS) was exposed to each AAV serotype to isolate serotype-specific AAV-binding antibodies (BAb). These BAbs were characterized by isotyping (Figure 15) and then used in an in vitro AAV neutralization assay (Figure 14). During the assay, the BAbs were incubated with their corresponding AAV serotype to allow the formation of the BAb-AAV complexes. The mixture BAb-AAV was used to infect HEK293T cells. After 24h of infection, cell supernatant was collected, which likely contained AAV particles bound to BAbs that were unable to enter the cells, indicating neutralization by neutralizing antibodies (NAbs). The NAbs were then purified using magnetic beads, isotyped (Figure 15), and utilized as a refined and specific matrix of AAV-neutralizing antibodies for the identification of neutralizing AAV epitopes by Peptide arrays (Table 1). Further bioinformatic analysis of the identified sequences revealed a list of consensus sequences and singletons conserved across serotypes (Table 2). A selection of 12 consensus and singletons sequences (Table 3) were used as free peptides as well as polypeptides conjugated to PLys or PAA for the in vitro PoC.

[0363] Table 1. Unique Epitope Sequence (204 sequences). The “X” in the sequence means that the amino acid can change between serotypes and could be any. P7120PC00 59 P7120PC00 60 P7120PC00 61

[0364] Table 2. List of consensus and singletons of all peptides identified in peptide arrays. The “X” in the sequence means that the amino acid can change between serotypes and could be any. The fifth column contains information on the other serotypes in which the sequence can be identified. P7120PC00 62

[0365] Table 3. List of the consensus and singletons sequences selected for the antibody neutralizing studies. A total of 12 consensus peptide sequences, derived from Table 2, were chosen due to their P7120PC00 63 conserved nature across various serotypes and their potential capacity to bind AAV-NAbs. The “X” in the sequence means that the amino acid can change between serotypes and could be any.

[0366] Free peptides are referred to in the present application by a number, i.e., peptide 19.1 (SEQ ID NO: 24). When such free peptides are conjugated to the polymeric conjugate, they are, in some cases, designated by the same number followed by a "C", i.e., peptide 19.1 C (SEQ ID NO: 200), indicating the presence of a cysteine introduced as a conjugation handle. It is noted that this cysteine is not necessarily located at the N-terminal of the recombinant peptide; its position can vary depending on the desired orientation of the peptide upon conjugation (see Section "Linker and spacer" of the De- tailed Description).

[0367] In some cases, one or more internal cysteine residues naturally present within the recombinant peptide sequence are substituted with methionine or other non-reactive amino acids to prevent undesired side reactions during conjugation. Recombinant peptides in which such internal cysteines have been P7120PC00 64 substituted are herein referred to by the original peptide number followed by “CM” or"M" e.g., peptide 19.1 CM (SEQ ID NO: 201).

[0368] All peptides of Table 3, both as free peptides or conjugated peptides were successfully synthesized (EXAMPLE 3). All peptides of Table 3 were tested as free peptides in EXAMPLE 4. Some peptides of Table 3 were tested as conjugated peptides in both EXAMPLE 5 and EXAMPLE 6.

[0369] EXAMPLE 3. Material and Methods of Synthesis of Peptides and Polymeric conjugates

[0370] 3.1 Synthesis and control of free peptides

[0371] Peptides were synthesized by Fmoc / t- Butyl solid-phase strategy on Wang (4-(Hydroxymethyl)phe- noxymethyl]polystyrene) polymer. All acylation reactions were carried out using Fmoc-amino acids activated with 0-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (1 eq.) in the presence of N,N-diisopropylethylamine (DIPEA) (1.1 eq.). (PEG)n (Polyethylene glycol)) was incorporated into the peptides using Fmoc-NH-(PEG)n-CH2COOH. The peptides were deprotected and cleaved from the polymer with trifluoroacetic acid (TFAybW / anisole / ethanedithiol 90 / 4 / 4 / 2. After precipitation with ice-cold diethyl ether the deprotected peptides were purified by preparative RP-HPLC in a FW-acetonitrile (0.1 % TFA) system. The purified peptides were characterized by liquid chromatography-mass spectrometry (LC-MS) method.

[0372] 3.1.1 Chemical synthesis of peptide 17.1 (SEQ ID NO: 22): AspValPheMetlleProGInTyrGlyTyr- LeuThrLeuAsnAsp (Figure 4) and other free peptides.

[0373] Attaching the first amino acid to Wang-polymer (P):

[0374] 200 mg Wang-polymer containing 1.0 mmole hydroxyl groups per gram was washed 3x15 ml with dry methylene chloride. 412 mg (1.0 mmole) Fmoc-Asp(OBut)-OH and 135 mg HOBt (1 mmol) were dissolved in 5 ml methylene chloride, the solution was stirred 5 min and added to the polymer. 157 pl DIC (1 mmol) was added to the polymer mixture, then solution 2.4 mg DMAP (0.02 mmole) in 100 pl dimethylformamide (DMF) was added. The reaction was carried out for one hour at room temperature and stirring. After the end of the reaction, the polymer was filtered and washed with methylene chloride.

[0375] Description of one synthetic cycle for elongation of peptide chain. Preparation of Fmoc- Asn(Trt)Asp(OBut)-P:

[0376] 1.1 the peptidyl-polymer obtained as described above was treated with 20% solution of piperidine in dimethylformamide for 20 min. Then the polymer was washed for 2 min with 5 ml dimethylformamide two times, for 5 min with 5 ml dioxane-water mixture (2:1) and five times for 2 min with 5 ml dimethylformamide again.

[0377] 1 .2 597 mg (1 .0 mmole) Fmoc-Asn(Trt)-OH, 380 mg HBTU (1 .0 mmoles) were dissolved in 5 ml DMF, then 192 pl (1.1 mmoles) DIPEA was added, the solution was stirred for 10 min and P7120PC00 65 added to the polymer. The reaction was carried out for 4 hours with regular stirring. At the end of the reaction process the polymer was filtered off, washed with dimethylformamide and treated with 5 ml mixture Ac20-pyridine-dimethylformamide (20:20:60) for 15 min, then the polymer was washed with dimethylformamide, isopropanol and again dimethylformamide.

[0378] The synthesis of polypeptide chains was carried out manually in a glass flow reactor (2x20 cm) according to the following protocol, requiring 8-10 ml of solvent for 200 mg of starting polymer for each synthesis cycle. The volume of the reaction mixture was 5-7 ml:

[0379] 1. 20% piperidine in DMF (15 min);

[0380] 2. DMF (3 x 2 min);

[0381] 3. dioxane-water, 2:1 (2 x 5 min);

[0382] 4. DMF (5 x 2 min);

[0383] 5. Condensation reaction: 5 molar equivalents of activated Fmoc-amino acid (2 h);

[0384] 6. DMF (3 x 2 min);

[0385] 7. acetylation: Ac20-pyridine-dimethylformamide, 20:20:60, (15 min);

[0386] 8. DMF (5x2min).

[0387] For activation of Fmoc amino acid derivatives by HBTU / DIPEA, 1.0 mmol (5 equivalents) of Fmoc- protected amino acid and 380 mg of HBTU (1 .0 mmol, 5 equivalents) were dissolved in 4 ml of DMF, then 192 pl (1.1 mmol) of DIPEA was added to the solution, the solution was stirred for 10 min. The completeness of condensation reaction after operation 5 of synthetic protocol was controlled by ninhydrin test; in case of N-terminal proline, Isatin Test was used.

[0388] Cleavage of peptide from polymer:

[0389] For the reaction of cleavage of the peptide from the polymer and simultaneous removal of the blocking side chain groups, 800 mg of peptidyl polymer was used. 15 ml of TFA / FW / anisole / ethanedithiol mixture (90 / 4 / 4 / 2 v / v) was added to the peptidyl polymer and the suspension was stirred for 1 hour. The resulting peptide solution was then filtered off from the polymer, the polymer was washed with 5 ml TFA and the excess TFA was removed from the combined solution by evaporation under reduced pressure. The peptide was precipitated by adding 100 ml ethyl ester, filtered and washed with the ester (5 x 20 ml). The precipitate was dissolved in 150 ml of 50% acetonitrile and freeze-dried. The peptide was purified by reversed-phase HPLC on column C8, 20x250mm using a gradient of acetonitrile in water (from 10% to 70% in 60 min) in the presence of 0.1 % TFA at the flow rate 10 ml / min, eluate absorbance was detected at 226 nm and molecular weight was determined by ESI mass-spectrometry.

[0390] Synthesis of other peptides:

[0391] The chemical synthesis of peptide 1.1 (SEQ ID NO: 1 , wherein X is alanine), peptide 2.1 (SEQ ID

[0392] NO: 4, wherein X is alanine), peptide 3.1 (SEQ ID NO: 7, wherein X is alanine), peptide 4.1 (SEQ ID P7120PC00 66

[0393] NO: 9, wherein X is alanine), peptide 5.1 (SEQ ID NO: 10, wherein X is alanine), peptide 8.1 (SEQ ID NO: 13), peptide 13.1 (SEQ ID NO: 18), peptide 14.1 (SEQ ID NO: 19, wherein X is alanine), peptide 19.1 (SEQ ID NO: 24), peptide 19.1 M (SEQ ID NO: 303), and peptide 23.1 (SEQ ID NO: 28) was carried out using the same methodology as that employed for peptide 17.1 (SEQ ID NO: 22). Peptide 17.1 is provided as a representative example, since the same synthetic strategy was applied to all peptides. HPLC and MS spectra confirming the identity and purity of each peptide are available but not shown in the present application.

[0394] Peptides were synthesized by Fmoc / t- Butyl solid-phase strategy on Wang SS, 1973 and Wang SS, 1976 (4 (Hydroxymethyl) phenoxymethyl polystyrene) polymer.

[0395] The biological activity of all these free peptides, including also peptide 17.1 , was tested in the following EXAMPLE 4.

[0396] 3.2 Synthesis and control of polymeric conjugates

[0397] NMR spectra were recorded on NMR spectrometers Bruker Avance 700 or 800 (Bruker BioSpin MRI GmbH). Thin layer chromatography (TLC) was performed using precoated aluminum sheets Kie- selgel 60 F254 (Merck, Darmstadt, Germany); after applying the samples in DMF or DMSO, the plates were dried in vacuum; visualization was performed by charring with 7% H3PO4, absorption in ultraviolet or treatment with 2% ninhydrin solution in 2-propanol (amines).

[0398] The value of pH was measured on 2 pL of reaction mixture diluted with 6 pL of water using pH indicator strips Pehanon (Macherey-Nagel, Germany) and Panpeha (Sigma-Aldrich, Germany).

[0399] 3.2.1 Chemical synthesis of polymeric conjugate intermediate PLys400-NH-(CO)CH2CI. (Figure 5, section 2 (middle); Scheme 1).

[0400] To a stirred solution of PLys_DP400xHBr (50.9 mg, 243 micromole of NH2 groups) in DMF (1 .7 mL) chloroacetic anhydride (125 mg, 731 micromole) in DMF (0.624 mL) and then 1 ,8-Diazabicy- clo[5.4.0]undec-7-ene (DBU) (55 pL, 368 micromole) were added. After 30 min the solution was diluted with ethyl acetate + 1 % AcOH to the volume 45 mL, thoroughly mixed and centrifuged (5000 g for 8 min). The precipitate was suspended in methanol + 5% AcOH (3 mL), diluted with ethyl acetate to the volume 45 mL, thoroughly mixed and centrifuged (5000 g for 8 min). The last procedure was repeated. The precipitate was dried in vacuum (water jet pump), suspended in water and freeze- dried. Yield 43.2 mg (87%) as white solid. TLC (CHCh / MeOH 10:1 +2 % AcOH): PLys_DP400xHBr Rf = 0, ninhydrine-positive; PLys-NH-(CO)CH2CI Rf = 0, ninhydrine-negative.1H NMR spectrum of PLys400xHBr (D2O, 303K, 700 MHz) 6 4.750 (s; HOD), 4.371 (m, 400H; CH-alpha), 3.058 (m, 800H; CH2N), 1.837-1.465 (m, 2400H; CH2CH2CH2) ppm.1H NMR spectrum of PLys400-NH-(CO)CH2CI (D7-DMF, 303K, 700 MHz) 6 8.395 (broad, 400H; NH at C-alpha), 8.214 (s, 400H; NHCOCH2CI), 8.023 (s; HCO of DMF), 4.149 (s, 800H; CH2CI), 3.996 (broad m, 400H; CH-alpha), 3.436 (s, HOD), 3.246 (s, 800H; CH2N), 2.914 (m; CDH2 of DMF), 2.744 (m; CDH2 of DMF), 1.702 (m, 2400H; P7120PC00 67

[0401] CH2CH2CH2) ppm.

[0402] Scheme 1. Synthesis of chloroacetyl derivative of PLys_DP400 (polymeric conjugate intermediate).

[0403] 3.2.2 Chemical synthesis of polymeric conjugate PLys_DP400-TGA (thioglycolate) and PLys_DP400-TGA control (Figure 5, section 1 (bottom); Scheme 2).

[0404] To a stirred solution of PLys-NH-(CO)CH2CI (4.37 mg, 21.35 micromole of chloride) (intermediate obtained in EXAMPLE 3.2.1 , Scheme 1) in DMF (0.874 mL), thioglycolic acid (85.4 micromole, 9.17 pL of 70% aq. thioglycolic acid) and DBU (120 micromole, 18 pL, pH was 9.0) were added. After 5 minutes, the reaction mixture became cloudy, and precipitate was formed. The mixture was stirred for 40 min and after addition of AcOH (6.8 pL) was diluted to volume 13 mL with ethyl acetate, thoroughly mixed and centrifuged (5000 g for 8 min). The precipitate was dried in vacuum (water jet pump), dissolved in 4 mL of water + 64 pL 1 M NaHCOs (pH of solution 6.9). Ultrafiltration on Amicon® Ultra-15 Centrifugal Filter Units (50 kDa), with subsequent washing with PBS (5 mL) and water (2 x 5 mL) gave small residue, that was dissolved in water (4 mL), filtered (filter# 2) and freeze- dried. Yield of PLys_DP400-N-(CO)CH2SCH2COONa 5.52 mg (92%).1H NMR spectrum of PLys_DP400-N-(CO)CH2SCH2COONa (D2O, 303K, 800 MHz) 6 4.750 (s; HOD), 4.320 (br.s, 400H; CH-alpha), 3.294 (br.s, 800H; SCH2COO-), 3.262 (s, 800H; NCOCH2S), 3.207 (m, 800H; CH2N), 1.610 (m, 2400H; CH2CH2CH2) ppm.

[0405] Scheme 2. Synthesis of Peptide-PLys_DP400-TGA and PLys_DP400-TGA control.

[0406] 3.2.3 Chemical synthesis of polymeric conjugate intermediate PLys_DP400-OEGi2-NH2 amine (Figure 6, section 1 (bottom); Scheme 3).

[0407] 1. Fmoc-OEGi2-ONSu. To a stirred solution / suspension of H2N-dPEGi2-COOH (200 mg, 0.324 mmole) in 1 ,2-dichloroethane (0.5 mL) a solution of Fmoc-ONSu (115 mg, 0.34 mmole) in DMF (0.5 P7120PC00 68 mL) was added. The reaction mixture became clear after a few minutes and was stirred for 40 minutes. After addition of dicyclohexylcarbodimide (67 mg, 0.325 mmole) in DMF (0.15 mL) the reaction mixture was stirred for 15 h, dicyclohexylurea was filtered off and Fmoc-OEGi2-ONSu was isolated on Sephadex LH-20 column (105 mL) in CHCh / MeOH 1 :1 + 1 % AcOH. The residue after evaporation fractions was thoroughly dried in vacuum. Yield (very viscous colorless liquid) was 274 mg (90%). TLC (CHCh / MeOH 10:1 + 1 % AcOH): H2N-dPEGi2-COOH Rf=0.04 (ninhydrin-positive), Fmoc-NH-dPEGi2-COOH Rf=0.19, Fmoc-OEGi2-ONSu Rf=0.48.

[0408] 2. PLys_DP400-OEGi2-NH2. To a stirred solution of PLys_DP400xHBr (18 mg, 86.1 micromole of NH2 groups) in DMSO (0.9 mL) a solution of Fmoc-OEGi2-ONSu (121 mg, 129 micromole) in DMSO (0.9 mL) and i-Pr2NEt (22.5 pL, 129 micromole) were added. The solution was stirred for 40 min, then ethanolamine (10.4 pL, 172 micromole) was added to deactivate excessive Fmoc-OEGi2-ONSu active ester. 20 minutes later pyperidine (100 pL) was added and solution was stirred for 2 h. After addition of AcOH (300 pL) the solution with precipitate of Fmoc-pyperidine was diluted with water to 10 mL volume and filtered. PLys_DP400-OEGi2-NH2 was isolated by ultrafiltration on Amicon® Ultra- 15 Centrifugal Filter Unit (50 kDa) in 0.2% AcOH. The residue on the filter was dissolved in water and after addition of 2M HCI (43 pL) freeze-dried. Yield of PLys_DP400-OEGi2-NH2*HCI was 58.6 mg (89%). TLC (MeOH / 1 M Py x HOAc 4:1): PLys_DP400-OEGi2-NH2Rf = 0, ninhydrine-positive; OEG12 amine Rf = 0.37, ninhydrine-positive; pyperidine Rf = 0.45, ninhydrine-positive.

[0409] 1H NMR spectrum of PLys_DP400-OEGi2-NH2(D2O, 303K, 800 MHz) 6 4.750 (s, HOD), 4.310 (m, 400H; CH-apIha of Lys), 3.706 (m, 19200H; 24 CH2of OEG), 3.221 (m, 800H; CH2N of OEG), 3.166 (m, 800H; CH2N of Lys), 2.514 (m, 800H; COCH2 of OEG), 2.057 (p, d-AcOH), 1 .576 (m, 2400H; 3 CH2 of Lys) ppm.

[0410] Scheme 3. Synthesis of PLys400-OEGi2 amine (polymeric conjugate intermediate).

[0411] 3.2.4 Chemical synthesis of polymeric conjugate intermediate Plys400-OEGi2-NH-(CO)CH2CI. (Scheme 4).

[0412] To a stirred solution / suspension of PLys_DP400-OEGi2-NH2*HCI (23 mg, 0.0752 micromole, 30.1 micromole of NH2 groups) (intermediate obtained in EXAMPLE 3.2.3, Scheme 3) in DMF (1.15 mL) a solution of chloroacetic anhydride (15.4 mg, 90.3 micromole) in DMF (86 pL) and DBU in DMF (5 P7120PC00 69 pL, 33.1 micromole in 95 pL of DMF) were added. After 10 minutes, the reaction mixture became homogeneous and was stirred for additional 20 minutes (TLC showed a negative reaction of ninhydrin to amine). After addition of AcOH (67 pL) and MeCN / water 1 :2 + 1 % AcOH (1 mL) the solution was centrifuged to remove little precipitate and applied to Sephadex LH-20 column (105 mL in MeCN / water 1 :2 + 1 % AcOH). The fractions containing the product were evaporated, the residue was dissolved in water and lyophilized. Yield of Plys400-OEGi2-NH-(CO)CH2CI was 22.6 mg (93%).

[0413] After lyophilization from water, Plys400-OEGi2-NH-(CO)CH2CI showed very low solubility in DMF, MeOH, water and other solvents. Such a low solubility of Plys400-OEGi2-NH-(CO)CH2CI was completely unexpected, since Plys400-NH-(CO)CH2CI not containing OEG is well soluble in DMF.

[0414] The reaction was carried out again and an attempt was made to isolate the product in an eluent that does not contain AcOH. The eluate was evaporated for removing MeCN and Plys400-OEGi2-NH- (CO)CH2CI was obtained in an aqueous solution. However under these conditions Plys400-OEGi2- NH-(CO)CH2CI was strongly retained on the column, and only ~ 1 / 3 of the substance was isolated (defined by lyophilization of aliquot). The aqueous solution (with a concentration of Plys400-OEGi2- NH-(CO)CH2CI ~ 2 mg / ml) was quite stable, it was even possible to obtain the1H NMR spectrum of the substance (in H2O + 5% D2O).

[0415] 1H NMR spectrum of Plys400-OEGi2-NH-(CO)CH2CI (H2O + 5% D2O, 303K, 800 MHz; due to the intense suppression of the H2O signal, all integrals are greatly understated in the direction to the water signal) 6 4.32 (CH-apIha of Lys), 4.04 (COCH2CI), 3.80-3.50 (24 CH2of OEG), 3.46 (CH2N of OEG), 3.26 (CH2N of Lys), 2.53 (COCH2 of OEG), 2.09-1 .62 (3 CH2of Lys) ppm.

[0416] PLys400-OEG12amine PLys400-OEG12-WH-(CO)CH2CI

[0417] Scheme 4. Synthesis of PLys400-OEGi2-NH-(CO)CH2CI (polymeric conjugate intermediate).

[0418] An attempt to conjugate the peptide 17.1 C with an aqueous solution of the Plys400-OEGi2-NH- (CO)CH2CI at pH 9.0 was performed. It turned out that the PLys-OEG bond, namely -NH-CO- CH2CH2-O- is rapidly (10-20 min) broken in water due to retro-Michael reaction (p-elimination). For peptide 17.1 C sequence see SEQ ID NO: 199 on Table 1 .

[0419] 3.2.5 Chemical synthesis of polymeric conjugate PAA-OEGs-Maleimide (20 mole %) and control- PAA (Scheme 5).

[0420] 1. PAA-OEG3-NH2 (20 mole %). To a solution of poly(4-nitrophenyl acrylate) with average Mw 30 P7120PC00 70 kDa (83.1 mg, 0.430 mmole by 4-nitrophenyl acrylate) in DMSO (1 .04 mL), a solution of CF3(CO)NH- (CH2CH2O)2-CH2CH2NH2 x CF3(CO)OH (30.8 mg, 86 pmol) in DMSO (0.31 mL) and Et3N (24 pL, 172 pmol) were added. The solution was kept for 4 h at room temperature; TLC showed full binding of amine. After addition of ethanolamine (104 pL, 1 .722 mmol) the solution was kept for 2 h at 40 °C, and after dilution with water (3.1 mL) and addition of EtsN (140 pL), the solution was kept for 17 h at room temperature. The resulting polymer was isolated on a Sephadex LH-20 column (240 mL) in MeCN / water 1 :2 + 1 % AcOH, the residue after evaporation was freeze-dried from water. Yield 57.8 mg (93%). Calculated content of NH2 groups in the polymer is 1 .383 pmol / mg.

[0421] 2. PAA-OEGs-Maleimide (20 mole %). To a solution of PAA-OEG3-NH2 (20 mole %) (57.8 mg, 80 pmol of NH2 groups) in DMSO (1.45 mL), a solution of N-(3-maleimidopropionic acid) NHS ester (42.6 mg, 160 pmol) in DMSO (530 pL) was added, and the solution was kept for 4 h at room temperature.

[0422] Scheme 5. Synthesis of Peptide-PAA and control-PAA.

[0423] In this particular example, the degree of polymerisation (n) of the synthetic polymeric conjugate of

[0424] Scheme 5 is 150 units.

[0425] TLC showed absence of free amine. After addition of AcOH (100 pL) the resulting polymer was isolated on Sephadex LH-20 column (140 mL) in MeCN / water 1 :2 + 1 % AcOH, the residue after P7120PC00 71 evaporation was freeze-dried from water. Yield 60 mg (92%). Calculated content of maleimide groups the in polymer is 1 .229 pmol / mg.

[0426] 3.2.6 Chemical synthesis of 3.1C (10%)-PLys400-TGA conjugate (Figure 7, section 1 (bottom); Scheme 6), and 3.1 C (5%)-PLys400-TGA conjugate. For peptide 3.1 C sequence see SEQ ID NO: 193 (wherein X is alanine) on Table 1.

[0427] To a stirred solution of PLys-N-(CO)CH2CI in DMF (2.95 mg in 590 mL; 0.036 micromole, 14.4 micromole of chloride), a solution of 3.1 C peptide (5.65 mg, 2.158 micromole mono-CFsCOOH salt) in DMF (283 pL) and DBU (10% solution by volume in DMF, 26.4 pL ~ 8 mole / mole of peptide) were added (2 pL of reaction mixture diluted with 6 pL of water has pH 8.5-9.0) and the mixture was stirred for 1 h. The binding of peptide to polylysine was controlled by TLC. Then thioglycolic acid (57.6 micromole, 4 mole / mole of total chloride, 6.2 pL of 10% solution in DMF of 70% aq. TGA) and DBU (12.4 pL) were added. After 5 minutes of stirring the reaction mixture became cloudy and precipitate was formed. The mixture was stirred for 30 min and after addition of AcOH (to pH 7) was diluted with water or methanol\water 3:1 to ~ 10 mL. The conjugate was isolated by ultrafiltration of the solution on Amicon® Ultra-15 Centrifugal Filter Units (100 kDa) with subsequent washing with PBS and water (twice). The remainder on the filter was dissolved / suspended in water and evaporated. The pH of aqueous solution / suspension of conjugate (2-5 mL) was adjusted to 6.6-6.8 with an aqueous Na- HCO3 and ultrasound treatment was done. The solution was filtered (glass filter #2), evaporated and freeze-dried from water.

[0428] Scheme 6. Peptide 3.1 C conjugated to PLys-TGA.

[0429] According to TLC data only ~2 / 3 of the peptide was bound, that is ~10% molar of its content in conjugate. The conjugate does not contain free peptides. Yield of conjugate was 6.80 mg (calculated on PLys 91 % for 3.1 C(10%)-PLys). Analysis of1H NMR spectrum of 3.1 C (10%)-PLys gave molar content of peptide ~ 9%. TLC (2-propanol / MeCN / water 4:3:2): peptide 3.1 C Rf = 0.79; 3.1 C-PLys conjugate Rf = 0.

[0430] The chemical synthesis of 3.1 C (5%)-PLys400-TGA conjugate was carried out using the same methodology as that employed for 3.1 C (10%)-PLys400-TGA conjugate. HPLC and MS spectra confirming the identity and purity of the conjugate with 5% of conjugated peptide are available but not shown in the present application. P7120PC00 72

[0431] 3.2.7 Chemical synthesis of 17. 1C (15%)-PLys400-TGA conjugate (Figure 8, section 1 (bottom); Scheme 7). For peptide 17. 1C sequence see SEQ ID NO: 199 on Table 1.

[0432] To a stirred solution of PLys-N-(CO)CH2CI in DMF (2.94 mg in 588 mL; 0.0359 micromole, 14.35 micromole of chloride), a solution of 17.1 C peptide (4.32 mg, 2.153 micromole mono-CFsCOOH salt) in DMF (360 pL) and DBU (10% solution by volume in DMF, 35.7 pL ~ 11 mole / mole of peptide) were added (2 pL of reaction mixture diluted with 6 pL of water has pH 8.5-9.0) and the mixture was stirred for 1 h. The binding of peptides to polylysine was controlled by TLC. Then thioglycolic acid (57.4 micromole, 4 mole / mole of total chloride, 6.2 pL of 10% solution in DMF of 70% aq. TGA) and DBU (12.4 pL) were added. After 5 minutes of stirring the reaction mixture became cloudy and precipitate was formed. The mixture was stirred for 30 min and after addition of AcOH (to pH 7) was diluted with water or methanol / water 3:1 to ~ 10 mL. The conjugate was isolated by ultrafiltration of the solution on Amicon® Ultra-15 Centrifugal Filter Units (100 kDa) with subsequent washing with PBS and water (twice). The remainder on the filter was dissolved / suspended in water and evaporated. The pH of aqueous solution / suspension of conjugate (2-5 mL) was adjusted to 6.6-6.8 with an aqueous Na- HCO3 and ultrasound treatment was done. The solution was filtered (glass filter #2), evaporated and freeze-dried from water.

[0433] According to TLC data, the binding of the peptide was almost quantitative. The conjugate does not contain free peptides. Yield of conjugate was 5.96 mg (calc, on peptide 79% for 17.1 C(15%)-PLys). Analysis of1H NMR spectrum of 17.1 C(15%)-PLys gave molar content of peptide ~ 15%. TLC (MeOH / water 2:1): peptide 17.1 C Rf = 0.67; 17.1 C-PLys conjugate Rf = 0.

[0434] Scheme 7. Peptide 17.1 C conjugated to PLys-TGA.

[0435] 3.2.8 Chemical synthesis of 1.1C (15%)-PLys400-TGA conjugate. (Figure 9, section 1 (bottom);

[0436] Scheme 8). For peptide 1. 1C sequence see SEQ ID NO: 191 (wherein X is alanine) on Table 1. P7120PC00 73

[0437] Yield of conjugate was 90 % of the calculated one. Analysis of1H NMR spectrum of 1.1C (15%)- PLys gave molar content of peptide ~ 14%. TLC (CHCh / MeOH / water 2:6:1): peptide 1 ,1C Rf = 0.61 ; 1.1C-PLys conjugate Rf = 0.

[0438] Scheme 8. Peptide 1.1C conjugated to PLys-TGA.

[0439] 3.2.9 Chemical synthesis of 2.1C (10%)-PLys400-TGA conjugate. (Figure 10, section 1 (bottom); Scheme 9). For peptide 2.1C sequence see SEQ ID NO: 192 (wherein X is alanine) on Table 1.

[0440] According to TLC data only ~2 / 3 of the peptide was bound, that is ~10% molar of its content in conjugate. Yield of conjugate was 89 % (calculated on PLys). Analysis of1H NMR spectrum of 2.1 C (10%)-PLys gave molar content of peptide ~ 9%. TLC (2-propanol / MeCN / water 1 :1 :1): peptide 2.1 C

[0441] Rf = 0.76; 2.1C-PLys conjugate Rf = 0.

[0442] Scheme 9. Peptide 2.1 C conjugated to PLys-TGA.

[0443] 3.2.10 Chemical synthesis of 5.1 C (15%)-PLys400-TGA conjugate (Figure 11; section 1 (bottom); Scheme 10). For peptide 5.1C sequence see SEQ ID NO: 195 (wherein X is alanine) on Table 1.

[0444] Yield of conjugate was 87 % of the calculated one. Analysis of1H NMR spectrum of 5.1C (15%)- PLys gave molar content of peptide ~15%. TLC (2-propanol / MeCN / water 4:3:2): peptide 5.1 C Rf = 0.65; 5.1C-PLys conjugate Rf = 0. P7120PC00 74

[0445] Scheme 10. Peptide 5.1 C conjugated to PLys-TGA.

[0446] 3.2.11 Chemical synthesis of 8.1 C (5%) PAA conjugate. For peptide 8.1C sequence see SEQ ID NO: 196 on Table 1.

[0447] To a stirred solution of PAA-OEGs-Maleimide (2.34 mg) in DMF (78 pL), a solution of peptide 8.1 C (1 .86 mg, 0.718 micromole of di-CFsCOOH salt) in DMF (124 pL) and i-Pr2NEt (10 pL of 10% solution by volume in DMF, 8 mole / mole of peptide) were added (2 pL of reaction mixture diluted with 6 pL of water have pH 6.6-6.8) and the mixture was stirred for 40 min at room temperature. The binding of peptide to PAA-OEGs-Maleimide was controlled by TLC. Then thioglycolic acid (6.2 pL of 10% solution in DMF of 70% aq. TGA, 2 mole / mole of total maleimide) and i-Pr2NEt (10% solution in DMF, 10.5 pL, to pH 6.8-7.0) were added. The mixture was stirred for 20 min and then was diluted with water to 0.4 mL. Pept-PAA conjugate was isolated by ultrafiltration on Microcon-10kDa Centrifugal Filter Unit: washing with 1x PBS then with water (two times). The residue on membrane was dissolved in water, the pH was adjusted to 6.6-6.8 with an aqueous NaHCOs, the solution was treated with ultrasound, filtered (filter #2) and freeze-dried. Yield of 8.1 C (5%)-PAA conjugate was 2.98 mg (69% of the calculated one). The conjugate does not contain free peptides. TLC (2-propa- nol / MeCN / water 4:3:2): peptide 8.1 C Rf = 0.24; 8.1 C-PAA conjugate Rf ~ 0.

[0448] 3.2. 12 Chemical synthesis of 13. 1CM (5%)-PAA conjugate. For peptide 13. 1CM sequence see SEQ ID NO: 197 on Table 1.

[0449] To a stirred solution of PAA-OEGs-Maleimide (2.97 mg) in DMF (99 pL), a solution of peptide 13.1 CM (1.98 mg, 0.911 micromole of di-CF3COOH salt) in DMF (132 pL) and i-Pr2NEt (12.7 pL of 10% solution by volume in DMF, 8 mole / mole of peptide) were added (2 pL of reaction mixture diluted with 6 pL of water have pH 6.6-6.8) and the mixture was treated with ultrasound for 1 h at room temperature. The binding of peptide to PAA-OEGs-Maleimide was controlled by TLC. Then thioglycolic acid (7.9 pL of 10% solution in DMF of 70% aq. TGA, 2 mole / mole of total maleimide) and i- Pr2NEt (10% solution in DMF, 13.4 pL, to pH 6.8-7.0) were added. The mixture was treated with ultrasound for 20 min and then was diluted with water to 0.4 mL. Pept-PAA conjugate was isolated by ultrafiltration on Microcon-10kDa Centrifugal Filter Unit: washing with 1x PBS then with water (two P7120PC00 75 times). The residue on membrane was dissolved / suspended in water, the pH was adjusted to 6.6- 6.8 with an aqueous NaHCCh, the solution (total volume 3.5 mL) was treated with ultrasound, centrifuged to remove very little precipitate and freeze-dried. Yield of 13.1 CM (5%)-PAA conjugate was 3.29 mg (65% of the calculated one). The conjugate does not contain free peptides. TLC (MeOH / wa- ter 1 :2 + 5% 12M NH3): peptide 13.1 CM Rf = 0.48; 13.1 CM-PAA conjugate Rf ~ 0.

[0450] 3.2.13 Chemical synthesis of 1.1C (5%)-PAA conjugate. For peptide 1.1C sequence see SEQ ID NO: 191 (wherein X is alanine) on Table 1.

[0451] Yield of 1.1C (5%)-PAA conjugate was 86% of the calculated one. TLC (CHCh / MeOH / water 2:6:1): peptide 1 ,1 C Rf = 0.61 ; 1 .1 C-PAA conjugate Rf ~ 0.

[0452] 3.2.14 Chemical synthesis of 2.1C (5%)-PAA conjugate. For peptide 2.1C sequence see SEQ ID NO: 192 (wherein X is alanine) on Table 1.

[0453] Yield of 2.1 C (5%)-PAA conjugate was 76% of the calculated one. TLC (2-propanol / MeCN / water 1 :1 :1): peptide 2.1 C Rf = 0.79; 2.1 C-PAA conjugate Rf ~ 0.

[0454] 3.2.15 Chemical synthesis of 3.1C (5%)-PAA conjugate. For peptide 3.1C sequence see SEQ ID NO: 193 (wherein X is alanine) on Table 1.

[0455] Yield of 3.1 C (5%)-PAA conjugate was 82% of the calculated one. TLC (2-propanol / MeCN / water 4:3:2): peptide 3.1 C Rf = 0.79; 3.1 C-PAA conjugate Rf ~ 0.

[0456] 3.2.16 Chemical synthesis of 4.1C (5%)-PAA conjugate. For peptide 4.1C sequence see SEQ ID NO: 194 on Table 1.

[0457] Yield of 4.1 C (5%)-PAA conjugate was 61 % of the calculated one. TLC (MeOH / water 1 :1 + 2% EtsN): peptide 4.1 C Rf = 0.84; 4.1 C-PAA conjugate Rf ~ 0.

[0458] 3.2.17 Chemical synthesis of 5.1C (5%)-PAA conjugate. For peptide 5.1C sequence see SEQ ID NO: 195 (wherein X is alanine) on Table 1.

[0459] Yield of 5.1 C (5%)-PAA conjugate was 81 % of the calculated one. TLC (2-propanol / MeCN / water 4:3:2): peptide 5.1 C Rf = 0.65; 5.1 C-PAA conjugate Rf ~ 0.

[0460] 3.2. 18 Chemical synthesis of 14. 1C (5%)-PAA conjugate. For peptide 14.1 C sequence see SEQ ID NO: 198 on Table 1.

[0461] Yield of 14.1 C (5%)-PAA conjugate was 68% of the calculated one. TLC (2-propanol / MeCN / water 4:3:2): peptide 14.1 C Rf = 0.25; 14.1 C-PAA conjugate Rf ~ 0.

[0462] 3.2. 19 Chemical synthesis of 17. 1C (5%)-PAA conjugate. For peptide 17. 1C sequence see SEQ ID NO: 199 on Table 1.

[0463] Yield of 17.1 C (5%)-PAA conjugate was 66% of the calculated one. TLC (MeOH / water 2:1): peptide 17.1 C Rf = 0.67; 17.1 C-PAA conjugate Rf ~ 0. P7120PC00 76

[0464] 3.2.20 Chemical synthesis of 19.1CM (5%)-PAA conjugate. For peptide 19.1CM sequence see SEQ ID NO: 201 on Table 1.

[0465] Yield of 19.1 CM (5%)-PAA conjugate was 66% of the calculated one. TLC (2-propanol / MeCN / water 4:3:2): peptide 19.1 CM Rf = 0.19; 19.1 CM-PAA conjugate Rf ~ 0.

[0466] 3.2.21 Chemical synthesis of 23.1 C (5%)-PAA conjugate. For peptide 23.1 C sequence see SEQ ID NO: 202 on Table 1.

[0467] Yield of 23.1 C (5%)-PAA conjugate was 64% of the calculated one. TLC (MeCN / water / HCOOH 1 :8: 1 ): peptide 23.1 C Rf = 0.33; 23.1 C-PAA conjugate Rf ~ 0.

[0468] 3.2.22 Chemical synthesis of polymeric conjugate Control-PAA.

[0469] The Control-PAA was obtained similarly without binding of peptide from 5.55 mg of PAA-OEGs-Ma- leimide in DMF (185 pL), thioglycolic acid (14.8 pL of 10% solution in DMF of 70% aq. TGA, 2 mole / mole of total maleimide) and i-Pr2NEt (10% solution in DMF, 25 pL). Yield of Control-PAA conjugate was 3.44 mg (50% of the calculated one).

[0470] EXAMPLE 4: Neutralization Capacities Exerted by the Free Peptides

[0471] A total of 12 consensus peptide sequences were chosen due to their conserved nature across various serotypes and their potential capacity to bind AAV-NAbs: peptide 1 .1 (SEQ ID NO: 1 , wherein X is alanine), peptide 2.1 (SEQ ID NO: 4, wherein X is alanine), peptide 3.1 (SEQ ID NO: 7, wherein X is alanine), peptide 4.1 (SEQ ID NO: 9, wherein X is alanine), peptide 5.1 (SEQ ID NO: 10, wherein X is alanine), peptide 8.1 (SEQ ID NO: 13), peptide 13.1 (SEQ ID NO: 18), peptide 14.1 (SEQ ID NO: 19, wherein X is alanine), peptide 17.1 (SEQ ID NO: 22), peptide 19.1 (SEQ ID NO: 24), peptide 19.1 M (SEQ ID NO: 303), and peptide 23.1 (SEQ ID NO: 28). These peptide sequences correspond to the selection of sequences as set forth in Table 3, but without being conjugated, i.e., they do not have an additional Cys (see Table 2). These free peptides were chemically synthesized according to Example 3.1 .

[0472] For the evaluation of the AAV-NAbs binding activity of the Free Peptides by suspension treatment, HEK 293T cells were seeded and incubated overnight. The Free Peptides were mixed with NHS at various concentrations and incubated with different AAV serotypes.

[0473] Free peptides demonstrated varying degrees of neutralization capacity (Figure 18), effectively blocking AAV-NAbs in NHS, leading to a statistically significant improvement of the AAV infection compared with the control of infection. Notably, all peptides were efficient in blocking AAV2-NAbs. Some peptides also exhibited the ability to neutralize AAV8-NAbs and AAV9-NAbs, indicating a broadspectrum profile for these sequences. This broad neutralization capacity makes them particularly interesting for potential wide use with different AAV serotypes. P7120PC00 77

[0474] NHS dilutions were treated with free peptides for specific antibody neutralization, after which, a specific AAV serotype was added and used to infect HEK 293T cells. The percentage of AAV infection was calculated by measuring the concentration of GFP by ELISA. The results show the mean of three independent experiments, except for 23.1 (SEQ ID NO: 28) in AAV8 where n=1 . #, statistical significance found, see Figure 18.

[0475] Table 4. List of the significance degree level for each virus and peptide. Crossed-box: not tested; I-, statistics could not be performed; ns, no significance; *, p<0.05; **, p<0.01 ; ***, p<0.001 ; ****

[0476] EXAMPLE 5: Neutralization Capacities Exerted by PLys Conjugates - Suspension Treatment

[0477] For the inhibition of anti-AAV antibodies by the conjugated peptides, both suspension (EXAMPLE 5) and solid-phase (EXAMPLE 6) NHS treatments were employed to evaluate the neutralizing capacity of said polymeric conjugates.

[0478] PLys conjugates were synthesized as defined previously: EXAMPLE 3.2.8 for peptide 1 ,1 C (15%), EXAMPLE 3.2.9 for peptide 2.1 C (10%), EXAMPLE 3.2.6 for peptide 3.1 C (5%), EXAMPLE 3.2.6 for peptide 3.1 C (10%), EXAMPLE 3.2.10 for peptide 5.1 C (15%), and EXAMPLE 3.2.7 for peptide 17.1 C (15%).

[0479] NHS dilutions were treated with PLys Conjugates in suspension for specific antibody neutralization, after which a specific AAV serotype was added and used to infect HEK 293T cells. The percentage of AAV infection was calculated by measuring the concentration of GFP by ELISA. The results show the mean of three independent experiments, see Figure 19. The concentration of the PLys-conju- gates used for blocking human AAV2-NAbs was 7.5 pg / ml for conjugates 2.1 C 10% and 3.1 C 5%, and 10pg / ml for the rest, meanwhile for blocking human AAV8-NAbs the concentrations used were 75 pg / ml for 2.1 C 10% and 3.1 C 5% and 100 pg / ml for the rest. P7120PC00 78

[0480] In the suspension treatment, PLys-based conjugates effectively block human AAV-NAbs and result in improved AAV infection rates for AA2 and AA8 serotypes (Figure 19). PLys-conjugates 1 ,1 C and 3.1 C improved the infectivity of AAV2 as shown in Figure 19, although not significant (Table 5), meanwhile, all the conjugates tested gave positive results for AAV8.

[0481] Table 5. List of the significance degree level for each virus and conjugated peptide. Crossed-box: not tested; ns, no significance; *, p<0.05; ***, p<0.001 ; ****, p<0.0001.

[0482] EXAMPLE 6: Neutralization Capacities Exerted by PLys Conjugates - Solid-Phase Treatment

[0483] Solid-phase treatment was implemented to mimic in vivo conditions, where removal of NAbs must be completed prior to viral vector administration. This protocol involved two main steps (Figure 20): immunoadsorption to remove anti-AAV antibodies and an in vitro neutralization assay with the anti- body-depleted NHS.

[0484] In this setup, conjugated peptides were used to coat multi-well plates to specifically remove anti- AAV2, anti-AAV8, or anti-AAV9 antibodies from the NHS. PLys conjugates were synthesized as defined previously: EXAMPLE 3.2.8 for peptide 1.1 C (15%), EXAMPLE 3.2.9 for peptide 2.1 C (10%), EXAMPLE 3.2.6 for peptide 3.1 C (5%), EXAMPLE 3.2.6 for peptide 3.1 C (10%), EXAMPLE 3.2.10 for peptide 5.1 C (15%), and EXAMPLE 3.2.7 for peptide 17.1 C (15%).

[0485] NHS was subjected to a solid-phase treatment with PLys Conjugates for specific anti-AAV antibody capture and elimination. Followed by the addition of AAV2, AAV8, or AAV9 to the antibody-depleted NHS and used to infect HEK 293T cells. The percentage of AAV infection was calculated by measuring the concentration of GFP by ELISA. The results show the mean of two or three independent experiments. The concentration of Plys-conjugates coating used for solid-phase antibody neutralizing assays was 10 pg / ml. The subsequent neutralization assay with anti-AAV2, anti-AAV8 or anti- AAV9 Abs-depleted NHS demonstrated an increase in the infection independently of serum dilution, confirming the enhanced neutralization capacity of the conjugated peptides compared to control conditions (Figure 21).

[0486] Antibody removal was then verified by applying the depleted NHS to an AAV-serotype-specific coating to measure antibody binding before and after immunoadsorption (Figure 22). The percentage of anti-AAV IgG removal was calculated from the basal antibody measurements from non-depleted P7120PC00 79

[0487] NHS. The efficiency of antibody removal varied depending on the conjugate and AAV serotype used.

[0488] Table 6. List of the significance degree level for each virus and conjugated peptide. Crossed-box: not tested; ns, no significance; *, p<0.05; ***, p<0.001 ; ****, p<0.0001.

[0489] EXAMPLE 7: Validation of the activity of polymeric conjugates using AAV chimeras

[0490] Currently, many AAV-based gene therapy studies focus on developing strategies to evade the immune system (IS), often through the design of AAV chimeras. AAV chimeras are adeno-associated viruses whose capsid proteins have been modified — either synthetically or naturally — to alter their immunogenicity or tropism. These modifications can include amino acid substitutions, peptide insertions, or the exchange of capsid regions between different AAV serotypes. The goal is to incorporate features from less immunogenic or less pre-existing immunity-associated serotypes to reduce recognition by the IS. Such chimeric AAVs have shown improved immune evasion and, consequently, enhanced transduction efficiency and therapeutic success rates in some preclinical and clinical contexts.

[0491] Due to the clinical relevance of AAV chimeras, we aimed to validate our polymeric conjugates to test their ability to remove AAV-neutralizing antibodies from normal human serum (NHS) samples, which could interfere with the infection of chimeric AAVs. For this purpose, we used two different chimeras: AAV-DJ and AAV9P31 . AAV-DJ is a hybrid vector generated by DNA shuffling of capsid sequences from multiple serotypes, including AAV1 , AAV2, AAV3B, AAV8, AAV9, AAVrh8, and AAVrhI O, resulting in a vector with high transduction efficiency and reduced susceptibility to pre-existing neutralizing antibodies. In contrast, AAV9P31 is derived from AAV9, in which specific capsid regions have been substituted by sequences from other serotypes such as AAV2 or AAV3B, aiming to preserve the systemic tropism of AAV9 while improving immune evasion.

[0492] The tested conjugates were synthesized as defined previously: EXAMPLE 3.2.8 for peptide 1.1 C (15%), EXAMPLE 3.2.9 for peptide 2.1 C (10%), EXAMPLE 3.2.6 for peptide 3.1 C (5%), EXAMPLE 3.2.6 for peptide 3.1 C (10%), EXAMPLE 3.2.10 for peptide 5.1 C (15%), and EXAMPLE 3.2.7 for peptide 17.1 C (15%). P7120PC00 80

[0493] 7.1 Materials and method for neutralizing assays

[0494] HEK293T cells were seeded into a transparent Nunc 96-well plate at a density of either 3x104or 1 x10scells per well, depending on the AAV serotype used. The plate was incubated overnight at 37°C in a humidified atmosphere with 5% CO2.

[0495] On the following day, free or conjugated peptides (depending on the Example) were mixed in an Eppendorf tube with DMEM (Dulbecco's Modified Eagle Medium) and either NHS or monoclonal neutralizing antibody at the selected concentrations, and the mixture was incubated for 1 h at 37°C. Subsequently, the AAV was diluted in 1 x PBS and added to the peptide-serum mixture at the selected MOI, followed by incubation for an additional 1 h at 37°C.

[0496] After incubation, the cell plate was washed once with 1 x PBS, and 100 pL per well of the peptide- serum-AAV mixture was added to the cells and incubated for 24 h at 37°C to allow AAV entry and expression of GFP.

[0497] AAV infection efficiency was evaluated by ELISA, quantifying GFP expression 24 h post-infection. Briefly, a Maxisorp ELISA plate was coated overnight at 4°C with anti-GFP antibody at 0.25 pg / mL in coating buffer. The wells were washed three times with washing buffer and blocked for 2 h with blocking buffer. Meanwhile, cells were lysed with 100 pL per well of lysis buffer and subjected to three freeze-thaw cycles. Then, 50 pL of each lysate was transferred to the coated plate and incubated overnight at 4°C. After washing, wells were incubated for 2 h with 100 pL of biotinylated anti- GFP antibody, followed by incubation with a streptavidin-enzyme conjugate for 1 h. Finally, GFP was detected by incubating with 3,3',5,5'-tetramethylbenzidine (TMB) solution at room temperature for 5-15 min, and the reaction was stopped with 50 pL of stop buffer. Optical density was measured at 450 nm using a Biotek 800 TS spectrophotometer.

[0498] Table 7. List of buffers for the GFP-detection ELISA procedure.

[0499] 7.2 Results

[0500] Our results demonstrate that the conjugates efficiently inhibit AAV-neutralizing antibodies present in NHS (Figure 23). This effective depletion of neutralizing antibodies suggests that the conjugates can prevent immune interference during AAV infection. Consequently, these findings indicate that such conjugates could be employed to enhance the success rate of gene therapies mediated by chimeric AAV vectors by improving their ability to evade pre-existing immunity and achieve efficient transduction. P7120PC00 81

[0501] EXAMPLE 8. Characterization of commercial mouse-derived anti-AAV monoclonal antibodies

[0502] To further improve the previous results, it was decided to modify the experimental strategy by adopting a simpler and more controlled model, in which NHS was replaced with mouse-derived anti-AAV monoclonal antibodies. The choice of mouse as the host species was based on the goal of applying this system in subsequent in vivo assays to be conducted in this animal model and therefore avoiding an immunological response. A panel of eight monoclonal antibodies was selected, each recognizing either linear or conformational epitopes from different AAV serotypes (Table 8).

[0503] The strategy used for these monoclonal antibodies was analogous to that used for identifying neutralizing sequences from NHS-purified antibodies, consisting of AAV neutralization analysis, peptide array screening, sequence sorting, and sequence validation. The neutralization assay was carried out as explained in EXAMPLE 7.1.

[0504] Table 8. List of the selected mouse-derived anti-AAV monoclonal antibodies.

[0505] The selected antibodies were tested against the specific AAV serotypes they recognize, as well as the chimeric vectors included in this study, AAV-DJ and AAV9P31 .

[0506] Results showed that antibodies recognizing conformational epitopes were highly effective at neutralizing their corresponding serotypes (Figure 24). Notably, ADK9 partially neutralized AAV9P31 , which was expected given the vector’s origin, while ADK1 a also neutralized AAV6, and ADK8 partially neutralized AAV5. In contrast, antibodies recognizing linear epitopes failed to achieve complete neutralization of any serotype, although the highest neutralization effects (25-50% decrease) were observed for A1 and B1 against AAV8 and AAV9. A69 antibody did not neutralize any AAV serotype. P7120PC00 82

[0507] Given these results, antibodies A1 , B1 , A20, ADK1 a, ADK6, ADK8, and ADK9 were characterized using a peptide array (EXAMPLE 9).

[0508] EXAMPLE 9. Linear epitope identification for antibodies A1 , B1 , A20, ADK1a, ADK6, ADK8 and ADK9

[0509] 9.1 Identification of peptide epitopes by peptide arrays

[0510] Printed peptide arrays used in these studies have the following features:

[0511] PEPperCHIP® Linear Pan-AAV Capsid Protein Microarray

[0512] 1. Capsid proteins of AAV1 (protein ID NP_049542.1), AAV2 (protein ID P03135), AAV3b (protein ID 056139), AAV4 (protein ID 041855), AAV5 (protein ID Q9YIJ1), AAV6 (protein ID 056137), AAV7 (protein ID Q8JQG0), AAV8 (protein ID Q8JQF8), AAV9 (protein ID AAS99264.1), AA- Vrh.10 (protein ID AAO88201.1), AAVpo.1 (protein ID ACN42940.1), AAV10 (protein ID AAT46337.1), AAV11 (protein ID AAT46339.1), AAV12 (protein ID ABI16639.1) and AAV13 (protein ID ABZ10812) as overlapping peptides.

[0513] 2. 5,519 different linear peptides were printed in duplicate.

[0514] 3. 15 amino acids with 13 amino acids peptide-peptide overlap.

[0515] 4. Including HA and polio as positive control peptides.

[0516] Table 9. List of buffers for the peptide array procedure.

[0517] Refrigerated PEPperCHIP® Peptide Microarrays slides were pre-warmed for 10 min at room temperature. Slides were equilibrated in B3 for 15 minutes at RT on an orbital shaker at 140 rpm. Slides were blocked using B2 for 30 minutes at RT with mild shaking (20 rpm). Later, pre-warmed and centrifuged (3500 rpm, 8 min) sample diluted in B1 was added to the slide. For each slide, we used 2 pg / ml of the antibodies in Table 8 and 0.5 ug / ml of HA-Cy5 IgG diluted in a total of 850 pL of B1 . Then slides were incubated for 1 h at 37°C with mild shaking (50 rpm). After incubation, slides were washed twice for 5 minutes in B3 buffer. Next, slides were incubated in darkness with 1 .25 ug / ml of Cy™3Affinity pure Goat Anti-Mouse IgG (#115-165-009) as secondary antibody for 1 h at RT (20 rpm). After incubation, the slides were washed with B3 buffer for 5 minutes, 3 times, and later another wash in B4 for 5 min. Finally, slides were dipped twice in DB (Tris pH 7.4) for 5 min and centrifuged at 500 rpm until they dried completely. Slides can be stored for up to one week until scanning. Finally, slides were determined using the InnoScan 700 scanner. P7120PC00 83

[0518] 9.2 Results

[0519] A sequence screening was performed based on the scores obtained from the peptide arrays. The top 5% of sequences were identified and selected. Consecutive sequences were then grouped, and the sequence with the highest score within each group was selected for peptide synthesis (Table 10). Most of the sequences obtained for antibodies A1 and B1 contained the binding motifs previously reported in the literature, LEPLGLV and IGTRYLTR, respectively. In contrast, for A20, ADK1 a, ADK6, ADK8, and ADK9, antibodies known to recognize conformational epitopes according to the literature, multiple linear sequences were unexpectedly identified as potential binding sites. A total of 20 sequences were synthesized for their validation in cell-based assays (EXAMPLE 10).

[0520] Table 10. List of the top-ranked antibody-inhibitory sequences selected for peptide synthesis.

[0521] EXAMPLE 10. Linear epitope validation for antibodies A1 , B1 , A20, ADK1a, ADK6, ADK8 and ADK9

[0522] The neutralization assay for the free linear peptides was performed as described in EXAMPLE 7.1.

[0523] From the list of 21 sequences selected for synthesis and cell-based validation, two — A20.2.2 (SEQ ID NO: 283) and AD6.1 .3 (SEQ ID NO: 299) — demonstrated excellent performance in neutralizing their respective antibodies, A20 and ADK6 (Figure 25). Pretreatment of the antibodies with their corresponding peptides effectively prevented viral inhibition, resulting in the expected increase in infectivity after treatment. These two peptides were selected for conjugation to PLys. P7120PC00

[0524] These findings are particularly noteworthy, not only because they identified two outstanding candidates for subsequent studies, but also because they reveal that antibodies recognizing conformational epitopes can be effectively blocked by linear peptides.

[0525] EXAMPLE 11. Comparative analysis between peptide sequences in human serum and those identified using mouse Anti-AAV monoclonal antibodies

[0526] Additionally, a comparative study was conducted between the mouse antibody peptide array results and the sequences previously identified in EXAMPLE 2, particularly in Table 3, which can neutralize anti-AAV antibodies in human serum. The analysis revealed that some of the sequences previously obtained and validated fall within the top 5% of the peptide array scores for the conformational antibodies A20, ADK1a, ADK6, ADK8, and ADK9 (Table 11). Notably, sequence 13.1 (SEQ ID NO: 18), which is identical to sequence A20.2.2 (SEQ ID NO: 283) except for the absence of the terminal phenylalanine, was highlighted. These findings enabled to assay the first interactions between the antibodies and the conjugates, aimed at validating their specific binding to this new molecule.

[0527] Table 11. List of the sequences identified in EXAMPLE 2 (Table 1) that can inhibit human anti-AAV neutralizing antibodies, ranked based on the mouse-derived antibody peptide array. The symbols represent the percentile in which the sequence was found.

[0528] EXAMPLE 12. In vitro characterization of A20 monoclonal antibody interactions with PLys conjugates and antibody clearance

[0529] 12.1 Synthesis of PLys conjugates

[0530] 12.1.1 Peptide-PLys-DP400 conjugates and PLys-DP400 controls P7120PC00 85

[0531] Polylysine hydrobromide DP_400 was from Sigma-Aldrich. DMF (N,N-dimethylformamide from Sigma-Aldrich) was distilled, the first 10% were discarded, b.p. 85 °C (75 mm Hg), stored with 4A molecular sieves. DMSO was from Merck, stored with 4A molecular sieves. i-Pr2NEt (N,N-diisopro- pylethylamine from Sigma-Aldrich) was dried with NaOH and distilled, stored over NaOH. Acetic acid, 99.8% was from Acros Organics.

[0532] TLC was performed using precoated aluminum sheets Kieselgel 60 F254 (Merck, Darmstadt, Germany); after applying the samples in DMF or DMSO, the plates were dried in vacuum; visualization was performed by charring with 7% H3PO4, absorption in ultraviolet or treatment with 2% ninhydrine solution in 2-propanol (to observe amines). The value of pH was measured on 2 pL of reaction mixture diluted with 6 pL of water using pH indicator strips Pehanon (Macherey-Nagel, Germany) or Panpeha (Sigma-Aldrich, Germany).

[0533] NMR spectra were recorded on NMR spectrometers Bruker Avance 700 or 800 (Bruker BioSpin MRI GmbH).

[0534] 3-(Maleimido)propionic acid NHS ester Mal-pAla-ONSu was synthesized from 3-(maleimido)propi- onic acid, N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide in MeCN, recrystallized from MeCN / i-PrOH, it contains 94% of active ester by weight.

[0535] 12.1.2 Mal-pAla-ONSu

[0536] To a solution of 3-(maleimido)propionic acid (2709 mg, 16.02 mmole) in dry MeCN (78 mL) N-hy- droxysuccinimide (1880 mg, 16.34 mmole) and a solution of N,N'-dicyclohexylcarbodiimide (3322 mg, 16.1 mmole) in DMF (17 mL) were added. The reaction was kept for 4 h at r.t. Dicyclohexylurea was filtered off and washed with MeCN (4 x 20 mL). Filtrate was evaporated (30 °C in a water bath) to the MeCN volume ~ 25 mL (control by weight). After evaporation, 100 mL of 2-propanol containing 0.5% AcOH was added to the residue, stirred for 0.5 hours at r.t. and kept for 3 hours at 0 °C. The crystalline precipitate was filtered, washed with heptane (2 x 20 mL) and dried in vacuum. Yield 3708 mg (87%), the content of active ester estimated by NMR data was ~ 94% by weight.

[0537] 1H NMR spectrum in D6-DMSO + 0.3% CD3COOD (303 K, 800 MHz): 7.036 (s, 2H; CH=CH of ma- leimide), 3.740 (t, J = 6.9, 6.9 Hz, 2H; CH2N of pAla), 3.040 (t, J = 6.9, 6.9 Hz, 2H; CH2CO of pAla), 2.789 (s, 4H; CH2CH2 of succinimide), 2.500 (p; D5-DMSO), 2.070 (s; traces of AcOH), 1.883 (p; D4- AcOH) 6, ppm. TLC: Rf = 0.75 (CHCh / MeOH 10:1 + 2% AcOH).

[0538] NHS ester of Mal-pAla-PEG4-CH2COOH acid was synthesized from N3-PEG4-CH2COOH acid: reduction to H2N-PEG4-CH2COOH with dithiothreitol, maleimidation with Mal-pAla-ONSu, activation of acid with N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide in DMF, was used without isolation (content of the active ester >94% by TLC data). P7120PC00 86

[0539] 12.1.3 H2N-PEG4-CH2COOH

[0540] 1H NMR spectrum in D6-DMSO (303 K, 800 MHz): 3.646 (m, 2H; CH2O), 3.615 (s, 2H; OCH2COO), 3.556 (m, 10H; 5 CH2O), 3.468 (m, 2H; CH2O), 2.866 (m, 2H; CH2N), 2.500 (p, D5-DMSO) 6, ppm. TLC: Rf = 0.41 (CHCI3 / MeOH / H2O 5:5:1 , ninhydrine-positive.

[0541] 12.1.4 Mal- / 3Ala-PEG4-CH2COOH

[0542] Amino acid H2N-PEG4-CH2COOH (283 mg, 1.126 mmole) was suspended (incomplete dissolution) in 1 .5 mL of DMF, then dry Mal-pAla-ONSu (330 mg,1 .239 mmole) was added. After stirring for 3.5 h (within 20 min the mixture became homogeneous) the mixture was diluted with AcOH (75 pL) and water (300 pL). The product was isolated on Sephadex G-10 column (160 mL) in MeCN / water 1 :1 + 0.8% AcOH with admixture of Mal-pAla-ONSu (it separates poorly). During the evaporation of the eluate, Mal-pAla-ONSu precipitated in the form of crystals. An aqueous solution (2 mL) with a suspension of crystals was cooled to 0 °C, filtered, and the precipitate was washed with 2 mL of cold (0 °C) 0.5% AcOH. According to TLC data, the resulting solution of the product contains negligible traces of Mal-pAla-ONSu. The aqueous solution was evaporated on a rotor at 30 °C, coevaporated with MeCN (20 mL), then with a mixture of MeCN and heptane (20 + 10 mL). The remainder in the form of a thin film was thoroughly dried in vacuum. Yield of Mal-pAla-PEG4-CH2COOH was 372 mg (84%).

[0543] 1H NMR spectrum in D6-DMSO + 0.4% of CD3COOD (303 K, 800 MHz): 12.030 (broad s; COOH), 7.982 (t, J = 5.6, 5.6 Hz; NH), 6.993 (d, J = 1.4 Hz, 2H; CH=CH of maleimide), 4.015 (s, 2H; OCH2COO), 3.550 (m, 14H; 7 CH2O), 3.364 (t, J = 5.9, 5.9 Hz, 2H; CH2N of pAla), 3.147 (m, 2H; CH2N of PEG), 2.500 (p, D5-DMSO), 2.328 (t, J = 7.3, 7.3 Hz, 2H; CH2CO of pAla), 1.881 (p; D4- AcOH) 6, ppm. TLC: Rf = 0.61 (CHCI3 / MeOH / H2O 5:5:1).

[0544] 12. 1.5 NHS ester of Mal- / 3Ala-PEG4-CH2COOH acid

[0545] Initially, an attempt was made to carry out maleimidation and preparation of NHS ester in a single reaction solution. However, during isolation on Sephadex G-10 column in a mixture MeOH / water 2:1 +1 % AcOH, the unstable product turned out into a mixture of methyl ester and acid. Therefore, it was decided to use the Mal-pAla-PEG4-CH2COONSu obtained in solution without isolation.

[0546] Maleimide derivative Mal-pAla-PEG4-CH2COOH (124.1 micromoles) was dissolved in solution of N- hydroxysuccinimide (130.3 micromoles) in DMF (0.2 mL), then a solution of N,N'-dicyclohexylcar- bodiimide (136.5 micromoles) in DMF (0.5 mL) was added. The reaction mixture was kept for 4 h at 40 °C, TLC showed ~full conversion of the acid into NHS ester. After cooling to room temperature, dicyclohexylurea was removed by centrifugation, precipitate was washed with DMF (0.9 mL), and the volume of the resulting solution was measured. Based on the amount of loaded matter and the volume of the solution, the concentration of the Mal-pAla-PEG4-CH2COONSu was estimated as 77.7 micromoles / mL with ~94% content of the active ester. TLC: Rf = 0.63 (CHCI3 / MeOH / H2O 20:6:1 + 2% AcOH). P7120PC00 87

[0547] 12.1.6 Preparation of Mal(15%)-PLys400-Suc(85%) solution (Figure 29, section 1, top) for syntheses by Scheme 12 and Scheme 13 (Figure 12(A))

[0548] To an intensively stirred solution of PLys400«HBr (25.7 mg, it contains 122.9 micromole of Lys) in DMSO (2570 pL) a solution of NHS ester of 3-maleimidopropionic acid Mal-pAla-ONSu (19.36 micromole, 5% excess, 5.46 mg for 94% content of active ester) in DMSO (182 pL) and then i-Pr2EtN (21.6 micromole, 75.3 pL of 5% v / v solution in DMF / DMSO 1 :1 , containing 2 eq. of AcOH per i- Pr2EtN) were added. The solution was stirred for 25 min at r.t. (the completeness of the maleimide binding was checked using TLC), then succinic anhydride (209 micromole, 20.9 mg in 140 pL of DMF) and i-Pr2EtN (157 micromole, 547 pL of 5% v / v solution in DMF / DMSO 1 :1 , containing 2 eq. of AcOH per i-Pr2EtN) were added with intensive stirring. The reaction mixture became slightly cloudy, but after 2 minutes it became transparent again. After 40 min at r.t. MeOH (840 micromole, 34 pL, ~ 1 % of reaction volume) was added to transform excessive succinic anhydride into methyl ester, the solution was kept for 3 h at r.t. The reaction solution was diluted with 315 pL of DMF to obtain DMSO / DMF ratio ~4.

[0549] Based on the measured volume of the solution and the amount of PLys introduced into the reaction, the concentration of the resulting maleimide was 6.65 mg / mL by the initial PLys«HBr or 31.82 micromole of Lys per 1 mL. It is better to conjugate the prepared maleimide Mal(15%)-PLys400- Suc(85%) solution with thiol peptides immediately. However, when stored at -18 °C, it is suitable for the reaction for 3 days, and when stored at -70 °C, it is suitable for the reaction at least for a month. When the resulting maleimide was isolated from the reaction mixture by precipitation with 25 volumes of ethyl acetate / heptane 1 :1 (+0.2% AcOH) and then dried in vacuum, it becomes poorly soluble in DMSO, DMF, alcohols, water and their mixtures. Sample for NMR (low concentration) was obtained by freeze-drying of the precipitate suspension in water.

[0550] 1H NMR spectrum of Mal(15%)-PLys400-Suc(85%) in D6-DMSO / D2O 4 / 1 + 1 % CD3COOD (303 K, 800 MHz): 6.870 (s, 120 H; CH=CH of 60 Mai), 4.039 (s: HOD and CH of 400 Lys), 3.618 (broad, 120 H; CH2N of 60 p-Ala), 3.601 (h, J = 6.7 Hz, 80 H; CH of 40 i-Pr2EtN), 3.106 (q, J = 7.4 Hz, 80 H; CH2of 40 i-Pr2EtN), 3.010 (broad s, 800 H; CH2N of 400 Lys), 2.553 (p, J = 2.0 Hz; D5-DMSO), 2.445 (broad m, 680H; 340 CH2COO of succinate), 2.333 (s, 800H; 340 CH2(CO)N of succinate and 120 H of CH2CO of p-Ala), 2.045-1.102 (broad m„ 2400 H; 400 CH2CH2CH2of 400 Lys), 1.947 (s; residual AcOH), 1.922 (p; CD2HCOOD), 1.268 (d, J = 6.6 Hz, 480 H; 4CH3of 40 i-Pr2EtN), 1 .259 (t, J = 7.4 Hz, 120 H; CH3 of 40 i-Pr2EtN), (traces of heptane and ethyl acetate) 6, ppm.

[0551] TLC: Mal-pAla-ONSu: Rf = 0.75 (CHCh / MeOH 10:1 + 2% AcOH). GalNAca1-3(Fuca1-2)Galp- O(CH2)3NH2Rf = 0.07 (2-propanol / MeCN / H20 4:3:2), ninhydrine-positive. GalNAca1-3(Fuca1- 2)Galp-O(CH2)3NH-CO(CH2)2COOH Rf = 0.55 (2-propanol / MeCN / H20 4:3:2), ninhydrine-negative. Mal(15%)-PLys400-Suc(85%) Rf = 0 (2-propanol / MeCN / H20 4:3:2), ninhydrine-negative. P7120PC00 88

[0552] Scheme 13. Peptide-PEG4-Cys(15%)-PLys-Suc(85%), also shown in Figure 12(A).

[0553] 12.1.7 Preparation of Mal-PEG4(15%)-PLys400-Suc(85%) solution (Figure 29, section 2, bottom) for syntheses by Scheme 14 (Figure 12(B)) and Scheme 15

[0554] To an intensively stirred solution of PLys400«HBr (25.7 mg, 122.9 micromole of Lys) in DMSO (2570 pL) a solution of NHS ester of Mal-pAla-PEG4-CH2COOH acid (19.36 micromole, 5% excess, 265 pL of 77.7 micromole / mL solution in DMF for 94% content of active ester) and then i-Pr2EtN (21 .6 micromole, 75.3 pL of 5% v / v solution in DMF / DMSO 1 :1 , containing 2 eq. of AcOH per i-Pr2EtN) were added. The solution was stirred for 25 min at r.t. (the completeness of the maleimide binding was checked using TLC), then succinic anhydride (209 micromole, 20.9 mg in 140 pL of DMF) and i-Pr2EtN (157 micromole, 547 pL of 5% v / v solution in DMF / DMSO 1 :1 , containing 2 eq. of AcOH per i-Pr2EtN) were added with intensive stirring. After 40 min at r.t. MeOH (840 micromole, 34 pL, ~1 % of reaction volume) was added to transform excessive succinic anhydride into methyl ester, the solution was kept for 3 h at r.t. Complete methanolysis of anhydride was confirmed by TLC.

[0555] Based on the measured volume of the solution (solvents DMSO / DMF ratio ~4) and the amount of PLys introduced into the reaction, the concentration of the resulting maleimide was 7.081 mg / mL by the initial PLys«HBr or 33.87 micromole of Lys per 1 mL. It is better to conjugate the prepared maleimide Mal-PEG4(15%)-PLys400-Suc(85%) solution with thiol peptides immediately. However, when P7120PC00 89 stored at -18 °C, it is suitable for the reaction for 3 days, and when stored at -70 °C, it is suitable for the reaction at least for month.

[0556] When the resulting maleimide was isolated from the reaction mixture by precipitation with 25 volumes of ethyl acetate / heptane 1 :1 (+0.2% AcOH) and then dried in vacuum, it becomes poorly soluble in DMSO, DMF, alcohols, water and their mixtures. Sample for NMR (low concentration) was obtained by dilution of the precipitate suspension in methanol (+5% AcOH) with 25 volumes of ethyl acetate / heptane 1 :1 , drying of precipitate and freeze-drying of its suspension in water.

[0557] 1H NMR spectrum of Mal-PEG4(15%)-PLys400-Suc(85%) in D6-DMSO / D2O 4 / 1 + 1 % CD3COOD (303 K, 800 MHz): 6.891 (s, 120 H; CH=CH of 60 Mai), 4.027 (s: HOD and CH of 400 Lys), 3.881 (broad, 120H; OCH2CO of 60 PEG4), 3.623 (t, J = 7.1 Hz, 120H; CH2N of 60 pAla), 3.564-3.379 (m, 844 H; OCH2 of 60 PEG4 and CH of 4 i-Pr2EtN), 3.165 (t, J = 5.6 Hz, 120H; CH2N of 60 PEG4), 3.105 (m, 8H; CH2of 4 i-Pr2EtN), 3.013 (broad, 800 H; CH2N of 400 Lys), 2.553 (p, J = 2.0 Hz; D5- DMSO), 2.449 (broad m, 680H; 340 CH2COO of succinate), 2.354 (s, 800H; 340 CH2(CO)N of succinate and 120 H of CH2CO of p-Ala), 2.040-0.980 (broad m., 2400 H; 400 CH2CH2CH2 of 400 Lys), 1.949 (s; residual AcOH), 1.924 (p; CD2HCOOD), 1.263 (m, 60 H; 15 CH3of 4 i-Pr2EtN), (traces of heptane and ethyl acetate) 6, ppm.

[0558] TLC: Mal-pAla-PEG4-CH2COONSu: Rf = 0.63 (CHCI3 / MeOH / H2O 20:6:1 + 2% AcOH). GalNAcal- 3(Fuca1-2)Galp-O(CH2)3NH2 Rf = 0.07 (2-propanol / MeCN / H20 4:3:2), ninhydrine-positive. GalNAca1-3(Fuca1-2)Galp-O(CH2)3NH-CO(CH2)2COOH Rf = 0.55 (2-propanol / MeCN / H20 4:3:2), ninhydrine-negative. Mal-PEG4(15%)-PLys400-Suc(85%) Rf = 0 (2-propanol / MeCN / H20 4:3:2), nin- hydrine-negative.

[0559] Scheme 14. Peptide-Cys-PEG4(15%)-PLys-Suc(85%), also shown in Figure 12(B). P7120PC00 90

[0560] Scheme 15. Peptide-PEG4-Cys-PEG4(15%)-PLys-Suc(85%).

[0561] 12. 1.8 Synthesis of Peptide-PLys400-Suc conjugates (1 eq. of peptide per male imide)

[0562] To a solution (in a 14 mL centrifuge tube) of Mal(15%)-PLys400-Suc(85%) or Mal-PEG4(15%)- PLys400-Suc(85%) in DMSO / DMF (12 micromole by Lys, 31.82 or 33.87 micromole of Lys per 1 mL, see above) a solution of cysteine-containing peptide in DMSO (1.8 micromole, 15 mole % on Lys, 10-15 mg / mL; the molecular weights of the peptides were calculated based on the assumption that amino groups, guanidines and imidazoles of the peptides are in the form of CF3COOH salts) and the solution was kept for 40 min at r.t. According to TLC data, the reaction proceeds without the addition of a base (pH = 4.5-4.7 is sufficient for the reaction of N-terminal cysteine with maleimide), however, to complete conjugation, i-Pr2EtN (10% v / v solution in DMF, 187 or 192 | iL per 1 mL of maleimide solution) was added (pH = 6.4-6.7) and the solution was kept for additional 20 minutes. In order to ensure the complete absence of maleimide in the product, mercaptoethanol was added to the reaction (0.9 micromole, 6.3 pL of 1 % v / v solution in DMF) and the solution was kept for 3 minutes. After addition of sodium acetate (96 micromole, 24 pL of 4 M aqueous solution) and thorough mixing, 20 volumes of ethyl acetate / heptane 1 :1 mixture was added to the solution. The mixture was shaken intensively and after 20 minutes the precipitate was separated by centrifugation (5000 g / 10 min at 17 °C). The supernatant was separated by decantation, and the precipitate was dried in a vacuum of a waterjet pump. The precipitate was dissolved (or suspended) in 2 mL of water and 1 M NaHCCh (36 micromoles, 36 pL) was added, pH of the mixture was 7.2-7.6. If the conjugate was poorly soluble in water, the mixture was treated with ultrasound. The solution (suspension) and flushings from the tube were placed into Amicon® Ultra-15 Centrifugal Filter Unit (100 kDa) and centrifuged 4000 g / 45- 90 minutes at 20 °C. The residue on the filter was dissolved (suspended) in 7 mL of 0.01 M NaHCCh, centrifuged, and then the solution (suspension) was centrifuged in 6 mL of pure water. The residue was washed off with water into a clean tube (up to 10 ml of solution, if necessary, the solution was treated with ultrasound), centrifuged for 3000 g / 4 minutes, decanted from insignificant (if any) sediment and the solution was freeze-dried.

[0563] The yields of conjugates were 75-85 %. The molar content of peptides (mole %) in conjugates was estimated by NMR spectra. Integration of signals of aromatic amino acids and histidine in a weak field, CHs-groups of leucine and isoleucine in strong field, and middle area allow to evaluate the ratio of the polylysine matrix and the peptide. In addition, it is possible to integrate lysine CH2N signals and succinate signals, taking into account the signals of the parent peptide falling into these regions. P7120PC00 91

[0564] The unreacted maleimide in this case was modified by mercaptoethanol.

[0565] 12. 1.9 Synthesis of Peptide-PLys400-Suc conjugates (1.2-1.3 eq. of peptide per maleimide)

[0566] All reagents are introduced into the reaction in the same amount, except for the peptide, which is taken in 20-30% excess. In this case, there is no need to carry out treatment with mercaptoethanol. The resulting conjugate is isolated similarly.

[0567] 12. 1. 10 Preparation of PLys400-Suc (Figure 30, section 2, middle-top), ME-PLys400-Suc (Figure 30, section 3, middle-bottom) and ME-PEG4-PLys400-Suc (Figure 30, section 4, bottom) controls

[0568] PLys400-Suc, succinylated polylysine (Control 1 , Scheme 16) was prepared by treatment of PLys400«HBr (Figure 30, section 1, top) with succinic anhydride as described above, but without conjugation with the maleimide. The resulting conjugate was isolated similarly to peptide derivatives but on Amicon® Ultra-4 Centrifugal Filter Unit (50 kDa). The yield of Control 1 was 89%.

[0569] ME-PLys400-Suc (Control 2, Scheme 16) and ME-PEG4-PLys400-Suc (Control 3, Scheme 16) were prepared by treatment of Mal(15%)-PLys400-Suc(85%) or Mal-PEG4(15%)-PLys400-Suc(85%) with mercaptoethanol (8 eq. per maleimide) for 30 min. The resulting conjugates were isolated similarly to peptide derivatives but on Amicon® Ultra-4 Centrifugal Filter Unit (50 kDa). The yields of Control 2 and Control 3 were 79% and 93%.

[0570] 1H NMR spectrum of PLys400-Suc in D2O (303 K, 800 MHz): 4.750 (s, HOD), 4.321 (s, 400H; 400 a-CH of Lys), 3.158 (m, 800H; 400 CH2N of Lys), 2.455 (s, 1600H; succinate 400 COCH2CH2CO), 1 .764-1 .339 (m, 2400H; 400 CH2CH2CH2of Lys) 6, ppm.

[0571] 1H NMR spectrum of ME-PLys400-Suc in D2O (303 K, 800 MHz): 4.750 (s, HOD), 4.319 (s, 400H; 400 a-CH of Lys), 4.078 (m, 60H; 60 CHSCH2), 3.813 (m, 240H; 60 NCH2of pAla and 60 CH2OH), 3.325 (dd, J = 19, 9 Hz, 60H; 60 COCHCHS), 3.162 (m, 800H; 400 CH2N of Lys), 2.999 (dt, J = 13, 6, 6 Hz, 60H; 60 SCHCH2OH), 2.883 (dt, J = 13, 6, 6 Hz, 60H; 60 SCH'CH2OH), 2.730 (m, 60H; 60 COCH'CHS), 2.517 (t, J = 7.0 Hz, 120H; 60 CH2CO of pAla), 2.454 (s, 1360H; succinate 340 COCH2CH2CO), 1.760-1.343 (m, 2400H; 400 CH2CH2CH2of Lys) 6, ppm.

[0572] 1H NMR spectrum of ME-PEG4-PLys400-Suc in D2O (303 K, 800 MHz): 4.750 (s, HOD), 4.319 (s, 400H; 400 a-CH of Lys), 4.081 (m, 180H; 60 OCH2CO and 60 CHSCH2), 3.820-3.604 (m, 1080H; 60 CH2N of pAla, 60x7 OCH2of PEG4 and 60 CH2OH), 3.348 (m, 180H; 60 CH2N of PEG4 and 60 COCHCHS), 3.244 (broad s, 120H; 60 CH2N of PEG-linked Lys), 3.160 (m, 680H; 340 CH2N of succinylated Lys), 2.997 (dt, J = 13.7, 6.1 , 6.1 Hz, 60H; 60 SCHCH2OH), 2.886 (dt, J = 13.7, 6.1 , 6.1 Hz, 60H; 60 SCH'CH2OH), 2.736 (dt, J = 18.8, 3.8, 3.8 Hz, 60H; 60 COCH'CHS), 2.537 (t, J = 6.7, 6.7 Hz, 120H; 60 CH2CO of PAla), 2.453 (s, 1360H; succinate 340 COCH2CH2CO), 1.771-1.342 (m, 2400H; 400 CH2CH2CH2of Lys) 6, ppm. P7120PC00 92

[0573] Control#1 Control#2 Control#3

[0574] Scheme 13. Structures of PLys400-Suc, ME-PLys400-Suc and ME-PEG4-PLys400-Suc (controls).

[0575] 12.2 Antibody binding by ELISA

[0576] The specific binding of the anti-AAV antibodies to the PLys conjugates was quantified by ELISA.

[0577] A 96-well plate was first coated with the selected PLys conjugate (10 pg / mL) and incubated overnight at 4 °C. After washing, the plate was blocked at 4 °C for 1 h and subsequently incubated with varying concentrations of the selected antibody for 1 h at room temperature. Next, wells were incubated for 1 h with either an anti-IgG-HRP or anti-lgA-HRP secondary antibody, depending on the isotype of the primary antibody. The bound anti-AAV antibodies were then detected using TMB substrate, incubated at room temperature for 5-15 min. The reaction was stopped with 50 pL of 2 N H2SO4, and optical density was measured at 450 nm using a BioTek 800 TS spectrophotometer.

[0578] Table 12. List of buffers for the Ab-conjugate binding ELISA procedure.

[0579] 12.3 Results

[0580] As above-mentioned, all peptides of Table 3 were conjugated to the different polymeric structures of Schemes 12 to 15. Various anti-AAV-specific antibodies were evaluated against these polymeric structures.

[0581] Results of the specific binding are shown herein for conjugates 13.1 M-Scheme 13 and 17.1 -Scheme 13 (see Figure 26(A)) and 13.1 M-Scheme 13, 13.1 M-Scheme 14, 2.1-Scheme 13, 2.1 C-Scheme 14, 3.1-Scheme 13, 3.1 C-Scheme 12, PLys-Scheme 13, PLys-Scheme 14, 17.1-Scheme 13 (see Figure 26(B)). The NMR spectra for all these conjugates are shown in Figure 31 . P7120PC00 93

[0582] Varying concentrations of A20 were tested against three different molecules: conjugate 13.1 M, which was predicted by the peptide array to bind with high affinity; conjugate 17.1 , whose peptide array score indicated no expected interaction with the antibody; and PLys, the polylysine scaffold without any conjugated peptide, serving as a control to assess potential nonspecific binding.

[0583] The results revealed a clear and specific binding of A20 to conjugate 13.1 M, whereas the interactions with conjugate 17.1 or PLys were minimal and attributable to background signal (Figure 26(A)).

[0584] According to Figure 26(B), the results showed that ADK9 exhibited very high affinity for both the

[0585] 13.1 and 2.1 conjugates. Antibody A20 also demonstrated substantial binding to these molecules, as well as to 3.1 conjugate. In contrast, ADK6 and ADK8 displayed low binding levels across all molecules. Interactions observed with conjugate 17.1 or PLys were minimal and attributed to background signal.

[0586] EXAMPLE 13. In vitro characterization of A20 clearance after PLys conjugate treatment

[0587] Having established that the antibody binds specifically to conjugate 13.1 M, the next step was to evaluate the in vitro removal of the antibody following treatment with the conjugate. These assays were performed using both conjugate 13.1 M (SEQ ID NO: 305) and A20.2.2M (SEQ ID NO: 304).

[0588] 13.1 Synthesis of PLys conjugates

[0589] Synthesis of conjugate 13.1 M-PEG4-Cys-PLys400-Suc was performed as explained in EXAMPLE 12, and its NMR spectra shown in Figure 31 (section 2, bottom).

[0590] Peptide A20.2.2M (SEQ ID NO: 304) was conjugated to the polymeric structure of Scheme 12 (A20.2.2-Cys-PLys-Suc) and Scheme 13 (A20.2.2-PEG4-Cys-PLys-Suc), the synthesis of which is similar to that of peptide 13.1 M (SEQ ID NO: 305) explained in EXAMPLE 12. Results of Section 13.3 below are only shown for conjugate A20.2.2M-PEG4-Cys-PLys-Suc. NMR spectra for both conjugates are shown in Figure 32.

[0591] Peptide AD6.1.3 was synthetized and conjugated with Scheme 12 and Scheme 13. NMR spectra for both conjugates are shown in Figure 34.

[0592] 13.2 Antibody removal: in vitro treatment

[0593] To validate the antibody-blocking effect mediated by the PLys conjugates, an in vitro treatment was first performed, followed by quantification of the remaining free antibodies using ELISA. The buffers used in this procedure are described in Table 12.

[0594] For the in vitro treatment, 0.5-2 pg / mL of the selected antibody was incubated with 1-250 pg / mL of P7120PC00 94 the corresponding PLys conjugate for 45-60 min at room temperature to allow Ab-conjugate binding.

[0595] For the ELISA determination, a 96-well plate was coated with the same PLys conjugate (10 pg / mL) as in Scheme 13 and incubated overnight at 4 °C. On the following day, while the in vitro treatment was being carried out, the plate was washed and blocked at 4 °C for 1 h. After blocking, the samples from the in vitro treatment were added to the wells and incubated for 1 h at room temperature. The plate was then incubated with either an anti-IgG-HRP or anti-lgA-HRP secondary antibody, depending on the isotype of the primary antibody, for 1 h at room temperature. The binding of the remaining free antibodies was detected using TMB substrate incubated for 5-15 min at room temperature. Optical density was measured at 450 nm using a BioTek 800 TS spectrophotometer.

[0596] 13.3 Results

[0597] The results demonstrated high levels of A20 antibody removal after treatment with 13.1 M conjugate, reaching up to 80% removal at the highest concentration tested (Figure 27). When using the A20- specific sequence in conjugate A20.2.2M, removal levels increased to nearly 100%, with approximately 75% removal achieved even at a low concentration of 1 pg / mL (Figure 28).

[0598] EXAMPLE 14. Material and methods of synthesis of polymeric conjugates with cellobiose

[0599] In this example, the inventors successfully synthesized peptide-polylysine conjugates using disaccharides, particularly cellobiose, for increasing solubility in water.

[0600] 14.1 Synthesis of polymeric conjugates with cellobiose

[0601] To obtain a maleimide derivative of polylysine containing glycan as a hydrophilic substituent, disaccharide cellobiose was selected. The disaccharide fragment should contain a carboxyl group on a relatively short linker. A scheme was chosen consisting of the synthesis of glycosylamine and its acylation with glutaric anhydride (Scheme 17). The synthesis of cellobiose glycosylamine carbamate was carried out according to the described procedure (L. M. Likhosherstov et al., 2002) with some modifications. The glycosylamine carbamate itself was acylated. From synthesized cellobiose-NH- glutarate active hydroxybenzotriazole (HOBt) ester was obtained and was used without isolation. P7120PC00 95

[0602] Scheme 17. Synthesis of Cellobiose-NH-glutarate HOBt active ester.

[0603] The synthesis of a maleimide derivative of polylysine containing cellobiose as an electroneutral and hydrophilic fragment was carried out in two stages (Scheme 18). First, maleimide was conjugated with the polylysine by the reaction with activated ester of maleimidopropionic acid. Then all the remaining amino groups of polylysine were completely acylated with an excess of activated ester of cellobiose-NH-glutarate (compound 4). After separating the product from DMSO and DMF by precipitating it from the reaction mixture with an ethyl acetate / heptane mixture, the precipitate was dried, dissolved in 1 % AcOH, and purified from low-molecular-weight components by ultrafiltration. Lyophilization of the final solution yielded Maleimide-PLys400-Cellobiose as a dry, finely porous substance. Maleimide-PLys400-Cellobiose is soluble in DMSO, DMF and water, which allows its conjugation with thiol-containing compounds (including peptides) in these solvents, or mixtures thereof.

[0604] Mal(15%)-PLys-Cellobiose(85%) P7120PC00 96

[0605] Mal(n)-PLys400-Cellobiose(400-n)

[0606] Scheme 18. Synthesis of Maleimide-PLys400-Cellobiose. Maleimide % mole = 100n / 400 Cellobiose % mole = 100(400-n) / 400.

[0607] 14. 1. 1 Synthesis of Cellobiose-fi-NI- carbamate (compound 2)

[0608] Cellobiose Glcp1-4Glc (1) (3000 mg, 8.764 mmol) and grounded into powder ammonium carbamate (1368 mg, 17.53 mmol) were dissolved in 6 mL of 12.5 M aqueous NH3 (~ 15 min). The resulting solution was diluted with 24 mL of MeOH, mixed and kept for 27 h at 40 °C. The solution with precipitate formed was kept 17 h at 4 °C. The precipitate was filtered out (filter #4) and washed on a filter with 28 mL MeOH in three portions. After drying in vacuum (0.05 mBar) for 24 h at r.t. 2855 mg of Glcp1-4Glcp-NH3+O(CO)NH2(2) (81 %) was obtained.

[0609] TLC: Cellobiose Rf = 0.47 and 0.41 (alpha and beta anomers); Cellobiose-NH2 carbamate Rf = 0.35 (eluent 2-propanol / MeCN / water 4:3:2 + 2% 12.5 M aq. NH3).

[0610] 14. 1.2 Synthesis of Cellobiose-fi-NH-glutarate (compound 3)

[0611] To an intensive stirred solution of Cellobiose-p-NH2 carbamate (2) (2767 mg, 6.877 mmol) in a cold 0.5 M NaHCOs (62 mL) crystalline glutaric anhydride (1962 mg, 17.19 mmol) was added in three portions for 10 min. The mixture was stirred for 1 h, then after addition of AcOH (0.86 mL) the mixture was stirred for additional 1 h to remove CO2 (pH < 6). The solution was applied onto 75 mL column with AmberChrome 50WX4 in pyridinium form Fast elution with water gave solution of product, glutaric acid, AcOH and pyridine. The solution was evaporated as much as possible and a mixture of MeCN (60 mL) and AcOH (4 mL) was added to the residue. The resulting crystalline precipitate was carefully grounded, filtered, washed on a filter three times with a mixture of MeCN and AcOH (40+4 mL), and then two times with MeCN (20 mL). The weight of the vacuum-dried product was 2910 mg (~93%); according to NMR data, along with the target Glcp1-4Glcp-NH-CO(CH2)3COOH (3) it contains about 2 mole% of Glcp1-4Glca-NH-CO(CH2)3COOH and about 4 mole% of cellobiose. The product (2910 mg) was dissolved in water (11 mL) at 60 °C, the solution was diluted with AcOH (22 P7120PC00 97 mL) and then with MeCN (33 mL). After the beginning of crystallization, the mixture was kept for 3 hours at r.t., crystalline precipitate was filtered off, washed on a filter with MeCN (2 x 15 mL) and dried in vacuum. To minimize the quantity of residual AcOH, the crystals were dissolved in 40 mL of water and the solution was freeze-dried. Yield of pure (purity >99%) Glcp1-4Glcp-NH- CO(CH2)3COOH (3) was 2564 mg (82% on (2) or 68% on (1)).

[0612] TLC: Cellobiose Rf = 0.57; Cellobiose-NH-glutarate (3) Rf = 0.45 (eluent CHCh / MeOH / water 4:6:1).

[0613] 1H NMR spectrum of (3) in D2O (30 °C, 700 MHz) (Figure 31(A), section 2, middle): 5.009 (d, J = 9.2 Hz, 1 H; H-1 of Glcp-N), 4.750 (s, HOD), 4.540 (d, J = 8.0 Hz, 1 H; H-1 of GIcp-O), 3.947 (m, 2H), 3.840 (dd, J = 12.3, 3.8 Hz, 1 H), 3.759 (dd, J = 12.4, 5.9 Hz, 1 H), 3.699 (m, 3 H), 3.513 (m, 2 H), 3.448 (m, 2 H), 3.346 (dd, J = 9.4, 7.9 Hz, 1 H), 2.461 (t, J = 7.4 Hz, 2 H; CH2COO of glutarate), 2.412 (m, 2 H; CH2CON of glutarate), 1 .935 (p, J = 7.4 Hz, 2 H; CH2CH2CH2of glutarate) 6, ppm.

[0614] 14. 1.3 Synthesis cellobiose-fi-NH-glutarate HOBt ester (compound 4)

[0615] To a stirred solution of Cellobiose-p-NH-glutarate (3) (347 mg, 0.7619 mmol) in DMF (2.96 mL) a solution of Hydroxybenzotriazole monohydrate (HOBt) (128.4 mg, 0.8381 mmol) in DMF (0.642 mL) and a solution of N,N'-Dicyclohexylcarbodiimide (DCC) (149.4 mg, 0.724 mmol) in DMF (0.747 mL) were added. The solution was kept for 7 h at 30 °C and after cooling was filtered to remove precipitate of dicyclohexylurea. TLC data showed conversion of (3) to (4) ~ 85-90%. Based on the volume of the solution, the content of (4) can be estimated to be at least 0.149 mmol / mL. The solution can be stored for 2-3 days at -18 °C.

[0616] TLC: (4) Rf = 0.62; (3) Rf= 0.45 (eluent CHCh / MeOH / water 4:6:1 + 2% AcOH).

[0617] 14.1.4 Synthesis of Mal(15%)-PLys400-Cellobiose (85%)

[0618] To an intensively stirred solution of PLys400«HBr (25 mg, 0.2989 micromole, 119.6 micromole of Lys) (see Figure 33(A), section 1 , top for the NMR spectra of PLys400 x HBr in D2O) in DMSO (2500 pL) a solution of NHS ester of 3-maleimidopropionic acid Mal-pAla-ONSu (21 .5 micromole, 6.1 mg for 94% content of active ester) in DMSO (203 pL) and then / -Pr2EtN (22.4 micromole, 39 pL of 10% v / v solution in DMF) were added. The solution was stirred for 25 min at r.t., then solution of (4) (142 micromole, 953 pL of solution in DMF with cone. ~ 149 micromole / mL of active ester, see above) and / -Pr2EtN (127 micromole, 221 pL of 10% v / v solution in DMF) were added with intensive stirring. After 40 min at r.t. AcOH (78 pL, ~ 2% of reaction volume) was added and the solution was divided into two equal portions in 50 mL centrifuge tubes. After dilution with ~ 20 volumes of ethyl ace- tate / heptane 1 :1 + 1 % AcOH mixture (to 40 mL in centrifuge tubes) the mixture was shaken intensively and after 20 minutes the precipitate was separated by centrifugation (5000 g / 15 min at 17°C). The supernatant was separated by decantation, and the precipitate was dried in a vacuum of a water jet pump. Precipitates were dissolved in 1 % AcOH (a total of 8 mL) and the solution was placed into Amicon® Ultra-15 Centrifugal Filter Unit (30 kDa) and centrifuged 3500g / 40 minutes at 20 °C. The P7120PC00 98 residue was dissolved in 8 mL of 1 % AcOH and centrifuged again. The ultrafiltration procedure was performed two more times. The residue was dissolved in 8 mL of 1 % AcOH, the solution was filtered (filter #3) and freeze-dried. Yield of Mal(15%)-PLys400-Cellobiose (85%) was 50.7 mg (81 %),, white fine-pored substance.

[0619] 1H NMR spectrum of Mal(15%)-PLys400-Cellobiose(85%) in D2O + 1 % CD3COOD (30 °C, 700 MHz) (Figure 33(A), section 3, bottom, Figure 33(B)): 6.889 (s, 120 H; CH=CH of 60 Mai), 4.996 (d, J = 8.9 Hz, 340 H; H-1 of 340 Glcp-N), 4.750 (s, HOD), 4.534 (d, J = 7.9 Hz, 340 H; H-1 of 340 Glcp-O), 4.085 (broad, 400 H; CH of 400 Lys), 3.945-3.338 (total 4200 H; 12x340 H of cellobiose + 120 H of 60 CH2N of PAla), 3.170 (broad s, 800 H; CH2N of 400 Lys), 2.496 (s, 120 H; 60 CH2CO of PAla),

[0620] 2.361 and 2.270 (s, total 1360 H; 2x340 CH2CO of glutarate), 2.048-1.335 (total 3080 H; 2400 H of 400 CH2CH2CH2 of Lys and 680 H of 340 CH2 of glutarate) 6, ppm.

[0621] 14.1.5 Synthesis of conjugates: peptide-Cys(15%)-PLys-Glyc(85%) (Scheme 19) and peptide- PEG4-Cys(15%)-PLys-Glyc(85%) (Scheme 20).

[0622] Compound Mal(15%)-PLys400-Cellobiose (85%) was conjugated either with peptide-Cys or peptide- PEG4-Cys to obtain the synthetic polymeric conjugates of Scheme 19 and Scheme 20.

[0623] P7120PC00 99

[0624] Scheme 19 (whithout PEG)

[0625] Schemes 19 and 20. Synthesis of peptide-Cys(15%)-PLys-Glyc(85%) and peptide-PEG4- Cys(15%)-PLys-Glyc(85%). 14.2 Results and conclusion

[0626] Inventors observed that these new conjugates have a high degree of hydrophilisation. Mal(15%)- PLys-Glyc(85%) was isolated in its pure form by ultrafiltration under slightly acidic conditions followed by lyophilization. This structure -the starting one for the synthesis of peptide conjugates (Schemes 19 and 20)- dissolves well in DMSO, as do all the peptides. The resulting conjugates retain the acidbase balance of the original peptide. P7120PC00 100 P7120PC00 101 P7120PC00 102 P7120PC00 103 P7120PC00 104 P7120PC00 105 P7120PC00 106

[0627] P7120PC00 107

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Claims

P7120PC00 110CLAIMS1 . A synthetic polymeric conjugate comprising:(i) a polymeric backbone made of a plurality of monomeric units, each monomeric unit being bound to the following consecutive monomeric unit forming a chain, wherein the polymeric backbone has a total number of monomeric units between 2 and 1200 units, and wherein the polymeric backbone is an homologous a-amino acid polymer or an acrylic acid polymer;(ii) one or more recombinant peptides, wherein the recombinant peptides are bound to from 1 % to 50% of the total number of monomeric units, wherein each recombinant peptide is a linear sequence comprising between 8 and 35 amino acids which binds to and / or inhibits a target anti-adeno-associ- ated virus (AAV) neutralizing antibody;(iii) one or more capping agents, wherein the capping agents are bound to a plurality of monomeric units that are not bound to the recombinant peptide, and wherein the capping agent is selected from the group consisting of glycolic acid, gluconic acid, succinic acid, ethanol amine or other amine derivatives, a thio-compound, a disaccharide, a polyethylene glycol, and any combination thereof; and(iv) a linker which is positioned between each recombinant peptide and a single monomeric unit, wherein the linker is selected from the group consisting of thiol-reactive linkers, carbonyl-reactive linkers, disulfide-based linkers, PEG-based linkers, click-chemistry linkers, Michael-acceptor linkers, and diacid-based linkers.

2. The synthetic polymeric conjugate according to claim 1 , wherein the polymeric backbone is a polyacrylic acid polymer or a poly-lysine polymer.

3. The synthetic polymeric conjugate according to claim 1 , wherein the capping agents are bound to the remaining monomeric units that are not bound to the recombinant peptide.

4. The synthetic polymeric conjugate according to claim 3, wherein the polymeric backbone is a polylysine polymer.

5. The synthetic polymeric conjugate according to claim 1 , wherein:- the recombinant peptides are bound to from 1 % to 10% of the total number of monomeric units;- the capping agents are bound to from 5% to 20% of the total number of monomeric units; and- the remaining monomeric units are not bound to either a recombinant peptide or a capping agent.

6. The synthetic polymeric conjugate according to claim 5, wherein the polymeric backbone is a polyacrylic acid polymer.

7. The synthetic polymeric conjugate according to any one of claims 1 to 6, wherein the polymeric backbone has a total number of monomeric units comprised between 2 units and an upper limit selected from the group consisting of 1200 units, 1000 units, 600 units, 400 units, 200 units and 100P7120PC00 111 units.

8. The synthetic polymeric conjugate according to claim 7, wherein the polymeric backbone has a total number of monomeric units of 400 units.

9. The synthetic polymeric conjugate according to any one of claims 1 to 8, wherein the recombinant peptide is selected from the group consisting of: SEQ ID NO: 1 to 202, and SEQ ID NO: 283 to SEQ ID NO: 305.

10. The synthetic polymeric conjugate according to any one of claims 1 to 9, wherein all the recombinant peptides have the same sequence.11 . The synthetic polymeric conjugate according to any one of claims 1 to 10, wherein the percentage of the monomeric units bound to the recombinant peptide sequences is selected from the group of ranges consisting of: between 1 % and 45%, between 1 % and 40%, and between 1 % and 30%, and between 1 % and 25%.

12. The synthetic polymeric conjugate according to claim 11 , wherein the percentage of the monomeric units bound to the recombinant peptide sequences is 5%, 10%, or 15%.

13. The synthetic polymeric conjugate according to any one of claims 1 to 12, wherein the capping agent is selected from the group consisting of succinic acid, thioglycolic acid, cellobiose, lactose, and tri(ethylene glycol) functionalized with a maleimide.

14. The synthetic polymeric conjugate according to any one of claims 1 to 13, wherein the linker is a maleimide-based linker or a thioester linker.

15. The synthetic polymeric conjugate according to any one of claims 1 to 14, further comprising at least one spacer which binds each recombinant peptide to a single monomeric unit and spatially separates each recombinant peptide from the polymeric backbone.

16. The synthetic polymeric conjugate according to claim 15, wherein the spacer is a PEG-based spacer.

17. The synthetic polymeric conjugate according to claim 16, wherein the PEG-based spacer comprises 4 or 5 ethylene oxide units.

18. The synthetic polymeric conjugate according to any one claims 1 to 17, further comprising an additional cysteine residue within the synthetic polymeric conjugate.P7120PC00 11219. The synthetic polymeric conjugate according to claim 18, wherein the monomeric units bound to a recombinant peptide have one of the following structures:(i) monomeric unit - linker - cysteine - recombinant peptide;(ii) monomeric unit - linker - cysteine - spacer - recombinant peptide;(iii) monomeric unit - linker - spacer - cysteine - recombinant peptide;(iv) monomeric unit - spacer - linker - cysteine - recombinant peptide; or(v) monomeric unit - spacer - linker - cysteine - spacer - recombinant peptide.

20. A pharmaceutical composition comprising a synthetic polymeric conjugate according to any one of claims 1 to 19, and at least one pharmaceutically acceptable excipient, carrier or vehicle.

21. A synthetic polymeric conjugate according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 20, for use as a medicament.

22. A synthetic polymeric conjugate according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 20, for use in the treatment of a subject for the removal of one or more anti-AAV neutralizing antibodies, wherein the treatment is intracorporeal.

23. The synthetic polymeric conjugate for use according to claim 22 or the pharmaceutical composition for use according to claim 22, wherein the treatment improves the efficacy of a therapy comprising an AAV vector, and wherein the synthetic polymeric conjugate or the pharmaceutical composition is administered prior to or concurrently with the AAV vector.

24. Use of a synthetic polymeric conjugate according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 20 for the in vitro detection and / or quantification of an anti-AAV neutralizing antibody in a sample from a subject.

25. An in vitro method for detecting and / or quantifying an anti-AAV neutralizing antibody in a sample from a subject by means of a synthetic polymeric conjugate according to any of claims 1 to 19, wherein the method comprises: a) contacting the sample with the synthetic polymeric conjugate and a detection agent, b) incubating the sample with the synthetic polymeric conjugate and a detection agent to form a detection-polymeric conjugate-neutralizing antibody complex; c) separating the detection-polymeric conjugate-neutralizing antibody complex from the remaining sample containing the unbound detection agent; and d) detecting and / or quantifying the anti-AAV neutralizing antibody.

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