WO2025083119A1 - Assessment of aav-mediated complement activation potential - Google Patents

Abstract

Present invention relates to a novel method of predicting the complement activation potential in a subject.

Description

Title: Assessment of AAV-mediated complement activation potential

TECHNICAL FIELD

Present invention relates to a novel method of measuring the adeno-associated viral vector (AAV)-mediated complement activation. Specifically, present invention relates to measuring said activity in e.g. a patient selected for undergoing treatment related to AAV gene transfer therapy or treatment based thereon. The novel method presented herein is also applicable during the process of clinical development and trials relating to AAV gene transfer therapy in order to detect unwanted immune response to the employed AAV vector. Moreover, present invention also relates to a kit or a kit of parts comprising one or more AAV serotype particles, a conjugated antibody, wherein the antibody is conjugated to any means for detection such as e.g. a fluorescent protein or the likes which may be activated by a cleaving enzyme. In particular, present invention enables detection of AAV-mediated complement activation not only by the classical pathway but also via the antibody-independent activation of the complement system or alterative pathway. Present invention also enables a very sensitive method for detection of even small amounts of anti-AAV antibody driven complement activation.

BACKGROUND ART

Adeno-associated viruses (AAV) are small viruses that infect humans and some other primate species. They belong to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. They are small (approximately 25 nm in diameter) replication-defective, nonenveloped viruses and have linear single-stranded DNA (ssDNA) genome of approximately 4.7 kilobases (kb). Several features make AAV an attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell. In the native virus, however, integration of virally carried genes into the host genome does occur. Integration can be important for certain applications, but can also have unwanted consequences. In later decades the employment of AAV-gene therapy has attracted much attention. Promising results have been obtained in clinical trials for a number of diseases, including e.g. Leber's congenital amaurosis, hemophilia, congestive heart failure, spinal muscular atrophy, lipoprotein lipase deficiency, and Parkinson's disease. In 2012, the first AAV-mediated gene therapy was approved in Europe (Glybera, withdrawn 2017). Since then, 4 more gene therapy drugs using AAV as vectors (Luxturna (2017), Zolgensma (2019), Roctavian (2022) and Hemgenix (2023)) have been approved in Ell.

However, it is also important to investigate the risk of unwanted side effects such as e.g. unwanted immune response in the subject undergoing treatment. This is important both for individuals under on-going treatment but also e.g. in clinical trials. Specifically, host immune response to the vector poses a significant challenge for the durability and safety of AAV- mediated-gene therapy. In one aspect, complement activation has been observed in different clinical trials and patients using gene therapy products (e.g. Zolgensma (Onasemnogene abeparvovec)). These adverse effects have initially been observed in high-dose gene therapy administration, but also in clinical trials with doses that are not considered as extremely high (5x10¹³ vg/kg, SUNRISE Trial, LogicBio Therapeutics). Therefore, its prediction is not only related to the dose but other key factors. Among them, presence of AAV binding antibodies can trigger AAV response. Although low titers (<1 :10) of AAV neutralizing antibodies (NAb) in blood apparently have no profound effects on the innate immune response to AAV, higher NAb titers (>1 :100) significantly increased pro-inflammatory cytokine/chemokine secretion, vector uptake by antigen presenting cells (APCs) and complement activation. However, not all the severe effects had occurred in presence of anti-AAV antibodies. There are cases in which the AAV administrations has end with the decease of a patient due to complement activation even in absence of anti-AAV antibodies and using complement inhibitors (Lek et al., 2023).

AAV-mediated complement activation has initially been discussed using in vitro approximations, where both the role of the classical and the alternative pathways (AP) were highlighted. The activation of the classical pathway was thought to occur in the presence of anti-AAV antibodies which then activated through C1q the formation of C3 convertase. The AP pathway was discussed as some co-immunoprecipitations were showing C3b and factor H attached to the AAV capsid.

Later on, studies using high doses administered in non-human primates and piglets (AAV- PHP.B, AAVhu68) were showing important adverse effects in these animals mainly linked to complement activation. These were the first observations suggesting in vivo AAV-mediated complement activation. Unfortunately, these studies have been followed by the observation of several adverse effects that are linked to complement activation in AAV gene therapy clinical trials and even in approved products.

In the art, West Cara et al., "Complement Activation by Adeno-Associated Virus-Neutralizing Antibody Complexes", HUMAN GENE THERAPY, vol. 34, no. 11-12, 2023, pages 554-566 relates to treatment of monogenetic disorders using vectors based on adeno-associated virus (AAV). The document mentions that AAV is non-pathogenic human virus, and preexisting capsid antibodies are prevalent in the population posing a challenge to the safety and efficacy of AAV-mediated gene therapies. In the document, seropositive donor sera carrying neutralizing antibodies from a previous environmental exposure activated complement when admixed with AAV9 capsids and complement activation was associated with donors who had higher levels of anti-AAV lgG1 antibodies. It was also found that complement activation was abrogated after IgG-depletion using affinity columns or serum pretreatment with an IgG degrading enzyme. These results in the document demonstrate the role of preexisting neutralizing antibodies in activating complement; a risk that can be mitigated by using adequate immunosuppression strategies when dosing seropositive patients with vector.

Further art is e.g. Smith Corinne J. et al.: "Pre-existing humoral immunity and complement pathway contribute to immunogenicity of adeno-associated virus (AAV) vector in human blood", Frontiers in Immunology, vol. 13, 2022 relating to AAV gene transfer as a promising treatment for many patients with life-threatening genetic diseases. The document mentions that host immune response to the vector poses a significant challenge for the durability and safety of AAV-mediated gene therapy. In the document it was found that although low titers (<1 :10) of AAV neutralizing antibodies (NAb) in blood did not have profound effects on the innate immune response to AAV, higher NAb titers (>1 :100) significantly increased pro- inflammatory cytokine/chemokine secretion, vector uptake by antigen presenting cells (APCs) and complement activation. It was also found that both full and empty viral particles were equally potent in inducing complement activation and cytokine secretion. By using a compstatin-based C3 and C3b inhibitor, APL-9, it is demonstrated that complement pathway inhibition lowered CD86 levels on APCs, AAV uptake, and cytokine/chemokine secretion in response to AAV. Together these results suggest that the pre-existing humoral immunity to AAV may contribute to trigger adverse immune responses observed in AAV-based gene therapy, and that blockade of complement pathway may warrant further investigation as a potential strategy for decreasing immunogenicity of AAV-based therapeutics.

However, the two documents commented upon above conclude that AAV-mediated complement activation would occur through the complement classical pathway. In the art, several assays are mainly focus on testing that approach, i.e. the classical pathway, completely overlooking the need for testing seronegative patients that could trigger complement activation via alternative pathway

Consequently, present invention provides for methods and kits enabling elucidation and prediction of the risk in individual patients of having any of the above unwanted side effects such as e.g. the AAV-mediated complement activation in therapy or drug development. Above all, present invention provides for a more sensitive method able to detect low levels of anti- AAV antibodies as well as also being able to test not only activation via the classical pathway but also the alternative pathway. Also, and importantly, present invention provides for a method entailing less false positive hits due to the detection of de novo complement activation. Present invention also ensures that the detection is owing to specifically AAV-mediated activation of complement system and not other interfering factors such as unspecific activation of the alternative pathway.

SUMMARY OF THE INVENTION

Present invention was developed in light of the aforementioned prior art, aiming to provide a predictive method for enhancing the safety of AAV administration by identifying patients with an elevated risk of experiencing AAV-mediated complement activation. To the inventors’ knowledge, there is no existing method capable of predicting the complete risk of triggering complement activation in patients receiving AAV vectors. The early detection of this risk presents several benefits, including the avoidance of complex secondary effects (if used as exclusion criterion) and improved safety assessment that may lead to enhanced monitoring for patients at an increased risk. In the art, seropositive samples are being employed. However, present invention also relates to detection of adverse effects to an AAV-mediated therapy not only in seropositive patients, but also in seronegative ones, which consequently, are not carrying any anti-AAV antibodies from a previous environmental exposure. Prior art thus relates to known pre-existing humoral immunity and complement pathway. However, present invention is able to detect not previously known immune responses to an AAV- mediated therapy.

Currently, there is a wide range of kits available for detecting complement activation, including SVAR Life Science Complement Activity Biomarkers (C4d, TCC), the Whole blood complement activation assay from Sanquin or MicroVue Complement Multiplex from Quidel, among others. However, these assays are limited to detecting complement activation that has already occurred in patients, lacking the ability to predict such activation in advance. The present invention provides a method for assessing the potential for an AAV-derived triggering of the complement cascade, thus predicting the risk for an adverse event for a patient following gene therapy treatment. This complement activation potential is represented by a low, medium, or high degree, corresponding to a plausible low, medium or high risk for complement activation by the specific AAV particles upon treatment. If the test result for a patient indicates a medium or high risk for complement activation by the AAV particles used for treatment, then that patient should be closely monitored during therapy. In spite of this, severe symptoms can occur unexpectedly in any patient, and signs of complement activation should always be monitored during gene therapy treatment as recommended by FDA.

Thus, present invention has the capacity to detect the potential for activation of any of the complement activation pathways. On one hand, this assay provides the advantage of detecting susceptibility to complement activation due to presence of antibodies. The complement activation triggering antibodies is not limited to IgG (the presence of which is normally assessed before AAV-administration) but may also be IgM or IgA. On the other hand, the design of this assay enables detection of susceptibility to complement activation from all three pathways. This susceptibility may arise from abnormalities within the various components of the complement system pathway, potentially amplifying its activation.

In one aspect, and importantly, present invention enables detection of proteins typically deposited on the surface of pathogens or surrounding membranes/capsids/compounds as a result of the activation of the host's complement system, and as such is an effector of the immune system and thus consequently, detection of the membrane attack complex (MAC) or terminal complement complex (TCC).

BRIEF DESCRIPTION OF THE DRAWINGS

Fig.1. Illustrates the results from the AAV-complement assay using Kiovig (ivIG) at different concentrations as source of antibodies. The complement system source was coming from a positive control (PC) of pooled human serum. The negative control (NC) was produced by heat inactivating the serum, which blocks complement system activation. The result from this assay shows a correlation between the levels of ivIG (antibodies) and the OD signal obtained. In absence of ivIG or complement system, or with a high PC dilution the assay presents negative results.

Fig.2. Illustrates the results from the AAV-complement assay using different amounts of 4 different serum samples. The complement system source was coming from a positive control (PC) of pooled human serum diluted 1/25. The result from this assay shows a correlation between the amount of serum in the positive samples (sample 1 and 3) and the signaling obtained.

Fig.3. Illustrates the evaluation of the presence of (A) total antibodies (TAbs) and (B) neutralizing antibodies (NAbs) against AAV2. TAbs were measured using an ELISA, while NAbs were measured using the i Lite AAV NAb platform (and the percentage of inhibition based calculated based on Kiovig). The results from these assays indicated that samples 7 and 10 present a high amount of anti-AAV2 antibodies with neutralizing capacity.

Fig.4. Illustrates the results from the AAV-complement assay for 10 individual human serum samples using as complement source (A) pooled human serum or (B) the same individual sera. First, human serum diluted 1/25 in EDTA-containing buffer was added to AAV-coated plates and incubated for 60 minutes at 21 °C. Then, the plates were washed 3 times, and the same human serum diluted 1/25 or a pool of serum samples was added to the plate and incubated for 60 minutes at 37°C. Lastly, signal development was performed, and the output read after 90 minutes.

Fig. 5 Illustrates the assay approach effectiveness across multiple AAV serotypes. Plates coated with the tested serotypes — (A) AAV2, (B) AAV8, and (C) AAV9 — successfully inducing specific complement activation mediated by anti-AAV antibodies.

Fig. 6 illustrates Quantification of anti-AAV IgG antibodies on the 24 samples used in Figure 5 against (A) AAV2, (B) AAV8, (C) AAV9. The results indicate a correlation between complement activation and presence of IgG. Notably, complement activation does not always correlate with the quantity of anti-AAV IgG antibodies, reflecting the known influence of antibody isotype and other antibody-dependent characteristics on complement activation.

Fig. 7 Illustrates the quantification of anti-AAV9 IgM antibodies on the 24 samples used in Figure 5 and 6. As is apparent from the figure sample 12, which is anti-AAV9 IgG negative (Figure 6C) but exhibits high complement activation (Figure 5C), shows the highest levels of anti-AAV9-lgM antibodies compared to other samples. This clearly evidences a high sensitivity to even small amounts of anti-AAV IgM-driven complement activation.

Fig. 8 Illustrates the determination of AAV9-mediated complement activation in presence or absence of Anti-AAV antibodies (using Intravenous Immunoglobulins (IVIg)). The results shows baseline overactivation of the complement system driven by the absence of Factor H (FH), the main regulator of the alternative pathway. The addition of IVIg further accelerates the complement response.

Fig. 9 Illustrates the determination of AAV9-mediated complement activation in presence or absence of different complement proteins (Factor H and C3). The results show either baseline overactivation or complete absence/lack of the complement system activation, either driven by the absence of Factor H (FH) or Complement 3 (C3) proteins, respectively. The addition of sufficient serum (containing all complement system components) in the 2^nd step can rescue the phenotype, highlighting the versatility of the assay to detect effects of individual serums.

Fig. 10 Illustrates the determination of AAV-mediated complement activation in presence of increasing amounts of IVIg, naturally containing anti-AAV antibodies. For comparison purposes background at 0 mg/ml of Kiovig was subtracted for each detection antibody. As can be observed, all tested antibodies against (B) C5bC9, (C) C3b, (D) C4d, (E) C5b yielded similar signals.

Fig. 11 Illustrates the tick-over complement activation caused by incubation of serum at 37°C. Measurement of the complement system end product, soluble TCC, in samples either incubated at 37°C (activated sera) or stored in ice (non-activated sera).

Fig. 12 Illustrates measurement of de novo formation of MAC (solid-phase TCC) in samples (samples same as experiment for Fig. 11) incubated in the AAV-Complement solid-phase ELISA assay, demonstrating that the AAV-complement assay detects only de novo MAC/TCC formation independently of the amount of TCC already present initially in the test sample. The figure illustrates that the AAV-complement assay detects only de novo MAC/TCC formation independently of the amount of TCC already present in the test sample.

DETAILED DESCRIPTION

Certain aspects of the present disclosure are directed to methods of identifying a subject suitable for a safer AAV gene therapy administration, comprising the assessment of the potential of AAV-mediated complement activation in a biological sample obtained from the subject using an enzyme - linked immunosorbent assay (ELISA).

Definitions

In order to enhance the comprehension of the present disclosure, certain terminology is initially elucidated. Except where expressly specified otherwise in this application, each term below is assigned the defined meaning. Additional definitions are interspersed throughout the application.

The term "and/or" as employed in this document is understood to denote a distinct representation of each of the two outlined features or components, irrespective of the presence or absence of the other. Hence, "and/or" when used in a context such as "A and/or B" is designed to encompass "A and B," "A or B," "A" (individually), and "B" (individually). Similarly, "and/or" within a context like "A, B, and/or C" is intended to cover each of the following scenarios: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (individually); B (individually); and C (individually).

It is recognized that when elements are characterized in this document utilizing the term "comprising," parallel aspects articulated as "consisting of" and/or "consisting essentially of" are likewise included. Unless otherwise stipulated, all technical and scientific terminology employed in this document carry the same denotation as typically comprehended by a practitioner with standard proficiency in the field to which this disclosure is connected. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

The term "inhibition," along with its grammatical derivatives such as "inhibiting", "inhibited" or “inhibits” is intended to encompass not only the complete cessation of a process or activity but also any reduction, suppression, attenuation, diminution, dilution, hindrance, or limitation thereof. Thus, "inhibition" may refer to any degree of decrease in the rate, extent, or efficacy of a biological, chemical, or physical process, ranging from a slight reduction to full cessation. This definition includes, but is not limited to, processes wherein the activity is diminished, slowed, obstructed, repressed, impeded, or otherwise rendered less effective or efficient.

Units, prefixes, and symbols are presented in their officially recognized Systeme international d'unites (SI) format. Numerical ranges include the numbers demarcating the range. The headings presented herein do not confine the diverse aspects of the disclosure, which should be comprehended through reference to the entire specification. The terms immediately defined below should be interpreted in the context of the entire specification.

As employed herein, the term "adeno-associated virus", "adeno-associated viral vector" or "AAV" encompasses, but is not restricted to, AAV of serotype 1 , AAV of serotype 2, AAV of serotype 3 (including of serotypes 3A and 3B), AAV of serotype 4, AAV of serotype 5, AAV of serotype 6, AAV of serotype 7, AAV of serotype 8, AAV of serotype 9, AAV of serotype 10, AAV of serotype 11 , AAV of serotype 12, AAV of serotype 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, those AAV serotypes and clades disclosed by Gao et al. (Gao et al., 2004) and Mietzsch et al. (Mietzsch et al., 2021) and any other AAV currently recognized or subsequently identified including any AAV with a custom/synthetic capsid. See, for instance, Fields et al., Virology, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). AAV pertains to the Parvoviridae family of viruses. For instance, the AAV could be an AAV derived from a naturally occurring "wild-type" virus, an AAV derived from a recombinant AAV (rAAV) genome packaged into a capsid produced from capsid proteins encoded by a naturally occurring cap gene and/or a rAAV genome packaged into a capsid produced from capsid proteins encoded by a non-natural capsid cap gene. In this context, "AAV" may be employed to denote the virus itself or its derivatives. The term encompasses all subtypes and both naturally occurring and recombinant forms, except where explicitly stated otherwise. AAV comprises AAV of serotype 1 (AAV-1), AAV of serotype 2 (AAV-2), AAV of serotype 3 (AAV-3), AAV of serotype 4 (AAV-4), AAV of serotype 5 (AAV-5), AAV of serotype 6 (AAV-6), AAV of serotype 7 (AAV-7), AAV of serotype 8 (AAV-8), AAV of serotype 9 (AAV-9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, nonprimate AAV, and ovine AAV. "Primate AAV" denotes AAV that infect primates, "non-primate AAV" signifies AAV that infect non-primate mammals, "bovine AAV" indicates AAV that infect bovine mammals, etc. See, for instance, BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (3d edition Lippincott-Raven Publishers), which is incorporated herein by reference in its entirety.

The term "rAAV" denotes a "recombinant AAV." In certain embodiments, a recombinant AAV possesses an AAV genome where a portion or the entirety of the rep and cap genes have been substituted with heterologous sequences. An "rAAV vector" as utilized herein denotes an AAV vector incorporating a polynucleotide sequence not derived from AAV (i.e. , a polynucleotide heterologous to AAV), typically a sequence of significance for the genetic modification of a cell. Typically, the heterologous polynucleotide is bordered by at least one, and usually by two AAV inverted terminal repeat sequences (ITRs). The term rAAV vector includes both PAAV vector particles and rAAV vector plasmids.

An "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein (typically all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the virus or particle contains a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be transported to a mammalian cell), it may be referred to as an "rAAV vector particle." Therefore, the production of an rAAV particle intrinsically involves the creation of an rAAV vector, as such a vector is included within an rAAV particle.

As employed herein, the term "Adeno-associated Virus-Like Particles", "AAV-VLP” or “VLP” denotes multi-protein assemblies that are designed to mirror the structure and conformation of AAV, albeit in the partial or total absence of the viral genomic content. These entities faithfully mimic the native three-dimensional configuration of AAV, which is capable of triggering robust immune responses. The term "AAV-VLP", as used within the context of this disclosure, embodies a spectrum of adeno-associated virus-like particles, and is not limited to, but does indeed encompass, all adeno-associated virus serotypes and capsid types presented herein. Such inclusivity is not restricted to the particular instances described in this patent, thereby extending the scope of "AAV-VLP" to potentially include additional serotypes and capsid types not explicitly mentioned within this document, yet still adhering to the fundamental characteristics of adeno-associated virus-like particles. Given the pronounced structural resemblance between AAV-VLP and AAV, the designation "AAV" may, in certain instances, serve as a substitute for "AAV-VLP". For instance, in references such as "anti-AAV antibodies," this nomenclature might also imply antibodies that target AAV-VLP.

The term "inverted terminal repeat" (or "ITR") is applied to denote a particular subsequence of nucleic acid situated at either the 5' or 3' terminus of a single stranded nucleic acid sequence. The ITR embodies a distinct collection of nucleotides (hereafter referred to as the "initial sequence") that is succeeded in the downstream direction by its reverse complement, thereby forming a palindrome-like sequence. It should be noted that the quantity of nucleotides constituting the intervening sequence that separates the initial sequence and its reverse complement is not fixed and may assume any length.

As utilized in this context, the term "tropism" is indicative of the capability of an AAV vector or virion to infect a single or multiple predetermined cell types. Tropism may also extend to the functionality of the vector in transducing these specific cell types. In other words, tropism is characterized by a preference for the AAV vector or virion to gain entry into certain cell or tissue types and/or an interaction bias with the cell surface that aids in its entry into these specific cell or tissue types. Following entry, the expression of sequences borne by the AAV vector or virion within the cell is expected, and typically preferred, which may include transcription and, optionally, translation. For instance, in the case of a recombinant virus, this would involve the expression of the heterologous nucleotide sequences.

The term "transduction", as used here, pertains to the capacity of an AAV vector or virion to infect one or more specific cell types. Specifically, transduction involves the penetration of the AAV vector or virion into the cell and the subsequent transfer of genetic material encapsulated within the AAV vector or virion into the cell, resulting in expression from the vector genome. It should be noted that while transduction and tropism may show a correlation in some instances, this is not universally applicable.

The term "administering" as used herein encompasses the act of physically delivering a therapeutic agent into a subject, by employing any of the diverse methods and delivery systems that are well known in the field. Illustrative methods of administration, for instance, in the case of an AAV therapy, can include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral modes of delivery, typically through injection or infusion.

The term "antibody" or “Ab” as used herein signifies, but is not restricted to, an immunoglobulin that binds specifically to an antigen. It is composed of a minimum of two heavy (H) chains and two light (L) chains linked by disulfide bonds or in certain cases of only one heavy chain or one chain consisting of a recombinant associated heavy (H) and light (L) chain (referred to as single-chain (scFv) antibodies). Each H chain includes a heavy chain variable region (herein abbreviated as VH) and a heavy chain constant region. The heavy chain constant region contains at least three constant domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (herein abbreviated as VL) and a light chain constant region. The light chain constant region encompasses one constant domain, CL. The VH and VL regions can be further split into hypervariable regions, known as complementarity determining regions (CDRs), interspersed with more conserved regions, referred to as framework regions (FRs). Both VH and VL comprise three CDRs and four FRs, organized from the amino-terminus to the carboxy-terminus in the following sequence: FR1 , CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains incorporate a binding domain that interacts with an antigen. The constant regions of the antibodies may facilitate the binding of the immunoglobulin to host tissues or factors, which can include various cells of the immune system (for instance, effector cells) and the first component (C1q) of the classical complement system.

The term "antibody" (Ab) is inclusive, but not limited to, any antigen-binding fragment of an immunoglobulin that binds specifically to an antigen as defined in this description. An immunoglobulin can originate from any of the commonly recognized isotypes, inclusive but not limited to IgA, secretory IgA, IgG, and IgM. Subclasses of IgG include but are not limited to human lgG1, lgG2, lgG3 and lgG4. The term "isotype" pertains to the antibody class or subclass (e.g., IgM or lgG1, respectively) encoded by the heavy chain constant region genes. The term "antibody" includes, for instance, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; entirely synthetic antibodies; and single chain antibodies. A nonhuman antibody can be humanized using recombinant methods to decrease its immunogenicity in humans. Unless expressly stated otherwise, and except where the context suggests differently, the term "antibody" also includes an antigen-binding fragment or an antigen-binding portion of any of the aforementioned immunoglobulins. This covers a monovalent and a divalent fragment or portion, as well as a single chain antibody. The term "isolated antibody" refers to an antibody that is substantially devoid of other antibodies with different antigenic specificities (for example, an isolated antibody that specifically binds to an AAV is largely free from antibodies that specifically bind to antigens other than the AAV). Furthermore, an isolated antibody may be substantially free from other cellular materials and/or chemicals.

The term "monoclonal antibody" (mAb) refers to a preparation of antibody molecules, not naturally occurring, of a single molecular composition. That is, antibody molecules that are essentially identical in their primary sequences and exhibit a single binding specificity and affinity for a specific epitope. An example of an isolated antibody is a monoclonal antibody. Monoclonal antibodies can be generated by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the field.

The term "total antibodies" (TAb) refers to the total number of antibodies that bind to a given target, such as an AAV. These antibodies can bind to AAV's surface proteins and might potentially interfere with its ability to enter a cell and deliver its genetic payload. However, these antibodies can be either neutralizing or non-neutralizing. Patients with high levels of total antibodies against a particular AAV serotype may be less likely to respond to gene therapy using that serotype.

The term "neutralizing antibody" (NAb) refers to an antibody that attaches to a target and impairs or obstructs the function of that target. For instance, a neutralizing antibody that attaches to an AAV-2 is capable of binding AAV-2 and impairing the function of the AAV-2. In certain embodiments, a neutralizing AAV antibody attaches to an AAV viral particle prior to the viral particle binding and transduction of a target cell, hindering the viral particle from binding and/or entering the target cell and discharging a transgene within the cell.

An "anti-antigen antibody" refers to an antibody that specifically binds to the antigen. For instance, an anti-AAV antibody specifically attaches to an AAV or AAV-VLP viral particle.

An "antigen-binding portion" of an antibody (also referred to as an "antigen-binding fragment") refers to one or more fragments of an antibody that maintain the capacity to specifically bind to the antigen that the whole antibody binds to.

The term "titer," or equivalently "antibody titer," as employed herein, signifies the quantity of a specific antibody present in a sample, such as in a biological sample procured from an individual. The quantification of an antibody's titer can be achieved by employing any suitable techniques that are recognized in the field. In particular embodiments, the titer of an antibody is ascertained by performing serial dilutions of a sample harboring the antibody until the point where the antibody is indistinguishable from the baseline. Under this context, the titer of an antibody is depicted as the multiplication factor of the dilution beyond which the antibody's concentration falls to baseline levels. For instance, if an antibody remains discernible at a dilution of 1 :800, which is the maximum dilution in a series where it surpasses baseline levels, then the titer of the antibody is denoted as 800. If a dilution of 1 :40,000 is the maximum dilution in a series above baseline levels, then the titer of the antibody is denoted as 40,000. In specific embodiments, the titer of the anti-AAV antibody is roughly less than 40,000. In certain other embodiments, the titer of the anti-AAV antibody is approximately less than 800.

The term "complement activation potential" refers to the inherent ability or propensity of a substance or agent, including, but not limited to, agents like viral vectors such as AAV, to initiate or amplify the innate immune complement activation cascade. This activation pertains to the triggering or enhancement of the cascade of complement proteins, which typically function in conjunction with antibodies or other specific molecular structures in order to eliminate pathogens or support immune responses. The potential is indicative of the extent to which the substance or agent might lead to immune reactions or other related responses in the body due to interactions with the complement system.

In certain configurations, the complement activation potential is ascertained using an enzyme- linked immunosorbent assay (ELISA). Methods employing any recognized variant of ELISA can be utilized according to the current context. ELISA operates based on a sequence of antibody reactions designed to recognize the existence of a specific target in an in vitro sample. ELISA protocols can encompass a direct assay, an indirect assay, or a sandwich assay (also referred to as a capture assay). Within a direct assay, an antigen is affixed to a surface, followed by the addition of a primary antibody that's been linked to an enzyme; this antibody associates with the antigen. Subsequently, a substrate is introduced, which, under the enzyme's influence, yields a detectable outcome, signaling the antigen's presence on the given surface. In the context of an indirect assay, following the antigen's attachment to the surface, a primary antibody is added. Post its binding with the antigen, a secondary enzyme- linked antibody is applied, which has the ability to latch onto the primary antibody. The introduction of a substrate then culminates in a detectable outcome indicative of the antigen's presence on the surface. During a capture assay, an antibody is tethered to the surface prior to the application of a sample containing the antigen. Both primary and secondary antibodies are subsequently introduced in a manner parallel to the indirect assay.

In particular configurations, the ELISA executed is termed as a "chemiluminescent ELISA". Such an ELISA, also known as a "luminescent assay" or "chemiluminescent assay", employs light emission as a marker for target detection. The magnitude of the light released can be gauged and equated with the target's concentration within a sample. In this specific ELISA variant, a particular enzyme acts upon a substrate to form a reaction byproduct that radiates light. Luminescence is typified as the light emanation from a substance transitioning from an excited electronic phase back to its fundamental phase. Chemiluminescence involves light generation through a chemical transformation. When the stimulated intermediates revert to their fundamental stable state, a photon gets emitted, which is sensed by an appropriate luminescent detector. The potency of the emitted light signal might be represented in terms of chemiluminescence units. A higher count of these units usually corresponds to a higher target titer in the examined biological sample.

For the disclosed procedures, any recognized chemiluminescent enzyme and its corresponding substrate are deemed appropriate. In certain configurations, the chemiluminescent enzyme may be, but not limited to, alkaline phosphatase (AP), horse radish peroxidase (HRP), or a combination of beta-galactosidase and beta-lactamase.

In one aspect of the invention, antibodies that detects a product of complement activation may be employed. This can include, but are not limited to, monoclonal antibodies (aE11) reacting with a C9 neoantigen of the terminal complement complex (TCC). The binding site of aE11 is created by two distinct areas on the outer part of the C9 structure, making it a non-continuous epitope in the quaternary structure. Moreover, the adjacent TSP1 , LDLRA, and MACPF domains of two neighbouring C9 protomers contribute to these surfaces (Bayly-Jones et al., 2023).

Consequently, in one aspect, the secondary antibody or detection antibody present 6 different CDR named depending on the chain they are located, being H from heavy and L from light. This CDRs may be CDR H1 42TVSGFSLTVYGV53, CDR H2 ₆9MIWGDGSTDY₇₈, CDR H3 115ARDRSYGGSSAWFGY129, CDR L1 43RASHDISNYL52, CDR L2 68YYTSRLHS75, and/or CDR L3 108QQGNYLPYT116. As this antibody recognizes a quaternary structure, some interactions between amino acids are key for the specific detection of the C9 neoantigen. Among them, the amino acids of aE11 L113 (CDR L3), V50 (CDR H1), Y120 (CDRH3), and Y112 (CDR L3) bind to with V68, L423, L423 and P72 of the C9. On top of this, the CDR H3 is relatively long and possesses two buried arginine residues that both contribute salt bridges (R116-E71 and R118- D78) with the predominantly negative C9 TSP-LDLRA loop. In addition, CDR H2 D73 contributes a third salt bridge to R65 positioned on the TSP domain of the lagging C9. The CDR L1 loop does not largely contribute to the aE11-Fab/C9 complex, and , CDR L2 only contributes minor polar contacts with the leading C9 interface. In this case, the detection of the neoepitope is not defined by a conformational change but by distinct interfaces that are discontinuous in the monomeric state of C9. This is merely one example of possible antibodies that may be used for detection. However, it is important to recognise that present invention enables detection of any protein in the complement system as will be detailed in the description.

As mentioned herein, present invention relates to a method for predicting an adverse response to gene therapy treatment in a patient and consequently predicting a complement activation potential in a subject. Put in different wording, present invention relates to a method for predicting the risk or potential of a subject responding adversely to a certain AAV-based therapy. It is considered that if the presence of complement activity is detected, there is a risk that a subject undergoing any AAV-based treatment of experiencing an adverse effect or unwanted side-effect.

In another aspect, the method is capable of measuring the AAV-mediated complement activation in a patient sample in a dose-dependent manner. Specifically, this may be either by way of the classical pathway (CP), lectin pathway (MP) and/or the alternative pathway (AP).

Consequently, in one aspect, present invention relates to a method comprising the steps of: a) providing a biological sample, b) contacting the sample with AAV or AAV-VLP particles of any serotype, wherein the complement activation is uninhibited or fully functional, c) further contacting the resulting mixture of b) with a conjugated detector antibody capable of specifically binding any neoepitope generated due to the complement system activation, such as e.g. one present in the complement membrane attack complex (C5b-9).

In another aspect, present invention relates to a method comprising the steps of: i) providing a biological sample, diluted in a sample dilution buffer, ii) contacting the diluted sample with AAV or AAV-VLP particles of any serotype wherein the AAV or AAV-particles are immobilised on a surface, iii) removing unbound sample from step ii), and thereafter adding to the well, the biological sample i) in a buffer and a sample dilution wherein the complement cascade and its activation pathway is not inhibited, iv) further contacting the resulting mixture of iii) with a conjugated detector antibody capable of specifically binding any protein generated due to the complement system activation, such as e.g. one present in the complement membrane attack complex (C5b-9), or detection of any protein in the complement system by any suitable means such as aptamers, affibodies, nanobodies, affimers, designed ankyrin repeat proteins (DARPins), molecularly imprinted polymers (MIPs), peptide ligands, lectins, synthetic small molecule ligands, fusion tags, or any other suitable binding molecules, thereby detecting the presence or absence of any complement activity in the sample.

According to the invention, and as is illustrated by step i) the sample is diluted in a buffer which inhibits the complement activation.

Suitable buffers for inhibiting complement activation is well known in the art and may e.g. be a buffer comprising any agent capable of chelating or binding to a bivalent ion, such as e.g. Ca2⁺ or Mg2⁺. Specific non-limiting examples are buffers comprising e.g. Ethylene Diamine Tetraacetic Acid (EDTA), Ethylene Glycol Tetraacetic Acid (EGTA), Sodium Citrate or the likes.

Moreover, the degree of dilution of the sample in i) may be in the range of about 1/2 to about 1/100, such as e.g. about 1/5 or more, such as e.g. about 1/10 or more, such as e.g. about 1/15 or more, such as e.g. about 1/20 or more, such as e.g. about 1/30 or more, such as e.g. about 1/50 or more, such as e.g. about 1/75 or more, such as e.g. about 1/100. The degree of dilution is intended to mean that if e.g. the dilution is 1/2, one part of sample is diluted with 2 parts of buffer etc.

The time for incubation employed in step ii) may be in during any period of from about 10 min to about 90 min, such as e.g. about 20 min, or about 30 min, or about 40 min, or about 50 min, or about 60 min.

The temperature of the sample added to the immobilised AAV or AAV-VLP may be any temperature of from about 20°C to about 40°C, such as e.g. about 37°C.

In another aspect, and as illustrated in step iii), once the diluted biological sample has been diluted in a buffer inhibiting the complement activity and added to a well comprising AAV of any serotype or AAV-VLP of any serotype, and after which an incubation period has passed, the well is emptied. As is illustrated in step iii) the same biological sample is added to the well. However, in this step the biological sample is diluted in a buffer not inhibiting the complement activity and i.e. supports the complement activity.

Thus, in one aspect, the buffer which may be employed in step iii) is different from the buffer employed in step i), such that the buffer in step i) inhibits complement activity, whereas the buffer in step iii) supports or does not inhibit complement activity.

Suitable exemplary buffers that may be employed in step iii) may be e.g. a buffer having an appropriate ionic strength of e.g. approximately 150 mM NaCI and may further be supplemented with calcium and magnesium ions, which are necessary for complement function.

The pH of these buffers supporting the complement activity may be close to physiological levels such as e.g. about 7.4.

Non-limiting examples of buffers supporting the complement activity may be e.g. Gelatin Veronal Buffer (GVB++). Other buffers used for this purpose may be e.g. Veronal Buffered Saline (VBS), Hank’s Balanced Salt Solution (HBSS), Phosphate-Buffered Saline (PBS) (which may be supplemented with magnesium and calcium).

The time for incubation employed in step iii) may be in during any period of from about 10 min to about 90 min, such as e.g. about 20 min, or about 30 min, or about 40 min, or about 50 min, or about 60 min, or about 90 min.

The temperature of the sample added to the immobilised AAV or AAV-VLP may be any temperature of from about 20°C to about 40°C, such as e.g. about 37°C.

In general, these buffers supporting the complement activity may exhibit similar ionic strength (approximately 150 mM NaCI) and maintain a pH close to physiological levels. Buffers mentioned above already contain magnesium or calcium ions, PBS requires supplementation with these cations.

In another aspect, a buffer supporting the complement activity may comprise one or more of the following;

NaCI: 120-180 mM pH: 7.2-7.6

CaCI₂: 0.1-0.3 mM

MgCI₂: 0.5-5 mM

According to the invention the sample dilution of step iii), i.e. the biological sample which is diluted in the buffer supporting the complement activity, may have a degree of dilution in the order of about 1/15 to about 1/150, such as e.g. about 1/18 or more, such as e.g. about 1/20 or more, such as e.g. about 1/30 or more, such as e.g. about 1/40 or more, such as e.g. about 1/50 or more, such as e.g. about 1/75 or more, such as e.g. about 1/100 or more, such as e.g. about 1/120 or more, such as e.g. about 1/150 or more. Present invention also relates to a product. The product comprises a surface element onto which a plurality of AAV-particles are attached or otherwise immobilised. Present invention also relates to a kit comprising a surface element onto which a plurality of AAV-particles are attached or otherwise immobilised. The surface element may be a well, or a plurality of wells in e.g. a microtiter plate.

The AAV-particle may be e.g. an AAV or an AAV-VLP (Virus Like Particle) particle.

Present invention also relets to a method of preparing e.g. a microtiter plate wherein the wells comprise a plurality of AAV or AAV-VLP attached to or otherwise immobilised onto the surface of the wells of the microliter plate.

In one aspect, the method comprises the steps of;

I) providing AAV or AAV-VLP in a suspended in a fluid, such as e.g. a buffering solution,

II) contacting the AAV or AAV-VLP suspended in the fluid with a surface of a container or well,

III) incubating the container or well with the AAV or AAV-VLP suspension for a period of time,

IV) removing of the suspension from each well or container and rinsing the wells or containers with the buffering solution,

V) adding a buffering solution comprising a blocking solution to each well,

VI) incubating the container or well with the buffering solution comprising a serum for a period of time,

VII) removal of all liquids from the container or wells,

VIII) allowing the container or wells to dry.

In one aspect, the suspension of AAV or AAV-VLP may have any density or concentration of about 2.5x10⁸ to about 45x10⁸ particles/ml of the suspension in step I).

In a further aspect, the incubation time illustrated in step III) may be at least about 16h, such as e.g. at least about 20h, such as e.g. at least about 24h.

The incubation illustrated in step III) may take place at any temperature of about 4°C to about 8°C.

The rinsing of each well may be at least once, such as at least two times or at least three times. As is illustrated in step VI), the incubation may take place during any period of about 2h to about 4h.

As is illustrated in step V), the incubation may take place at any temperature of about 15°C to about 25°C, such as e.g. about 20°C.

According to the invention, the drying step illustrated by step VIII), may take place at a temperature of about 25°C to about 35°C, such as e.g. about 30°C. The drying may be continued until the wells or container are considered sufficiently dry, such as e.g. until the relative humidity in the drying device is less than about 19%.

In another aspect, present invention relates to a product obtainable by the method comprising the steps of:

I) providing AAV or AAV-VLP in a suspended in a fluid, such as e.g. a buffering solution,

II) contacting the AAV or AAV-VLP suspended in the fluid with a surface of a container or well,

III) incubating the container or well with the AAV or AAV-VLP suspension for a period of time,

IV) removing of the suspension from each well or container and rinsing the wells or containers with the buffering solution,

V) adding a buffering solution comprising a blocking solution to each well,

VI) incubating the container or well with the buffering solution comprising a serum for a period of time,

VII) removal of all liquids from the container or wells,

VIII) allowing the container or wells to dry.

The product may in principle be any type of container such as e.g. a tube, flask or a container comprising a plurality of wells, such as e.g. a microtiter plate of any format.

The product may be made of any material such as e.g. a polymeric material and preferably a plastic material.

In one aspect, the blocking solution may be any such solution well-known in the art and such as e.g. solutions to block wells in ELISA format and which may be selected from:

• Bovine Serum Albumin (BSA): Used at concentrations of 0.1 -5% in buffer solutions to reduce non-specific binding.

• Non-fat Dry Milk (NFDM): Contains milk proteins, typically used at around 5% concentration in buffer solutions.

• Gelatin: Employed at concentrations of 0.1-5% to minimize background noise. • Normal Serum: Serum from non-immunized animals (e.g., goat or horse) used to block non-specific sites.

• Synthetic Polymers: Agents like polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP) that effectively block surfaces.

• Commercial Blocking Buffers: Proprietary formulations such as SuperBlock™ or Blocker™ Casein designed for efficient blocking, or the likes.

The product may thus comprise one or more containers wherein AAV or AAV-VLP have been attached or otherwise immobilised onto the surface of the one or more containers and wherein the surface has been blocked with respect to remaining binding sites and thus preventing nonspecific binding sites of the surface of the one or more containers.

According to present invention, the inventors thereof have surprisingly found that it is possible to design a method specific for a serotype AAV serotype, such as e.g. for AAV9 without using secondary anti-IgG antibodies. Moreover, and importantly, the inventors of present invention have designed methods enabling detection of complement activation not only through the classical pathway, but also the alternative pathway and the lectin pathway.

In one aspect, and importantly, present invention enables specific detection of the Membrane attack complex (Terminal complement complex C5b-9) without other interfering factors and consequently securing that result of the assay is a result of only activation of the host’s complement system without other interfering factors.

It is also important to understand the present invention does not rely on any specific detection antibodies. A person skilled in the art is well aware of the design of any antibody for specific detection of a protein or peptide. Detection may also be made with or without antibodies and a person skilled in the art is well aware of the design of an antibody for a specific purpose/detection.

Thus present invention relates to a method comprising the steps of: i) providing a biological sample, ii) contacting the sample with AAV or AAV-VLP particles of any serotype wherein the AAV or AAV-particles are immobilised on a surface, and diluting the sample using a buffer and a sample dilution inhibiting complement activation, iii) removing unbound sample and diluent from step ii), and thereafter contacting the resulting mixture with the biological sample i) in a buffer and a sample dilution where the complement cascade and its activation pathways of such a biological specimen typically are fully functional, iv) further contacting the resulting mixture of iii) with a conjugated detector antibody capable of specifically binding any neoepitope generated due to the complement system activation, such as e.g. one present in the complement membrane attack complex (C5b-9), or detection of any protein in the complement system by any suitable means such as aptamers, nanobodies, affimers, designed ankyrin repeat proteins (DARPins), molecularly imprinted polymers (MIPs), peptide ligands, lectins, synthetic small molecule ligands, fusion tags, or any other suitable binding molecules..

As is implied herein, present invention is capable of detecting key complement proteins involved in the classical, lectin, and alternative pathways of complement activation. These proteins may be e.g.;

• C1 complex: C1q, C1r, C1s

. C1 inhibitor (C1-INH)

• Mannose-Binding Lectin (MBL)

• MBL-associated serine proteases (MASP-1, MASP-2, MASP-3)

• C4: C4a, C4b, C4d

• C2: C2a, C2b

• C3: C3a, C3b, iC3b, C3c, C3dg, C3d, C3g

• Factor B: Bb, Ba

• Factor D

• C5: C5a, C5b

• Membrane Attack Complex (MAC) or Terminal Complement Complex (TCC): C5b, C6, C7, C8, C9

• Properdin

• Regulatory Proteins: Factor H, Factor I, C4-binding protein (C4BP)

These proteins and/or fragments thereof may be detectable via Antibody-Based Assays for Complement Activation.

In another aspect, the method may detect Surface-Bound Complement Components such as e.g.;

• C1 complex: C1q, C1r, C1s

. C1-INH

. MBL

. MASP-1, MASP-2, MASP-3

• C4 fragments: C4b, C4d

. C2b

• C3 fragments: C3b, iC3b, C3dg, C3d

• Factor Bb

. C5b

. MAC/TCC: C5b, C6, C7, C8, C9

• Properdin Specifically, present method also relates to detection of key Surface-Bound Components that can be used to monitoring the cascade’ progression during complement activation. This may include the detection of elements relates to the formation of C3 and C5 convertases and the initiation of the terminal complement pathway such as e.g.;

. C4b, C4d

• C3 fragments: C3b, iC3b, C3dg, C3d

. C5b

. MAC/TCC: C5b, C6, C7, C8, C9

Moreover, present invention may also relate to detecting the TCC. Thus, in one aspect, this may entail detection of e.g.; C5b, C6, C7, C8, and C9.

Embodiments of Antibody-Conjugation Methods:

Direct Antibody Conjugation:

In one embodiment, the detector antibody is directly conjugated to the enzyme label, Alkaline Phosphatase (AP) or Horseradish Peroxidase (HRP). This direct conjugation ensures a robust linkage between the antibody and the enzyme, facilitating accurate detection of complement activation products.

Biotin-Streptavidin Conjugation:

An alternative embodiment involves the use of biotinylated antibodies. In this method, antibodies specific to the complement activation products are biotinylated, and subsequently, the biotinylated antibodies are conjugated to streptavidin-linked AP or HRP. The biotinstreptavidin interaction offers strong binding affinity, enhancing the stability of the conjugates.

Hinge Region Conjugation:

The Hinge Region Conjugation embodiment entails the use of hinge-region-specific antibodies for conjugation. Antibodies are modified or engineered to expose their hinge regions, and the activated hinge regions are conjugated to AP or HRP. This conjugation method provides a stable linkage, preserving the antigen-binding sites and maintaining antibody specificity.

Enzyme-Tagged Antibodies:

In another approach, antibodies themselves are engineered to carry the enzymatic label. Antibodies are fused or genetically modified to include AP or HRP, resulting in enzyme-tagged antibodies. These engineered antibodies retain their antigen specificity while enabling direct detection of complement activation products without the need for additional enzyme conjugates. Secondary Antibody Conjugation:

In the Secondary Antibody Conjugation embodiment, a two-step detection process is employed. Primary antibodies specific to complement activation products are used to capture the analyte, and then a secondary antibody, specific to the primary antibody, is conjugated to AP or HRP. This approach allows for signal amplification and increased sensitivity in the ELISA.

Dual Enzyme Conjugation:

In certain scenarios, a combination of Alkaline Phosphatase (AP) and Horseradish Peroxidase (HRP) can be used to achieve higher sensitivity or multiplexing capabilities. Different antibodies specific to various complement activation products can be individually conjugated to AP and HRP, enabling the simultaneous detection of multiple targets in the same ELISA assay.

It is understood that the embodiments listed above are non-limiting examples, and other antibody-conjugation methods may also be utilized to detect signals for predicting AAV- mediated complement activation. The disclosed method's versatility and flexibility allow for seamless adaptation to specific experimental requirements and research objectives.

Embodiments of Potential Substrates for ELISA Detection:

Colorimetric Substrates for Alkaline Phosphatase (AP):

One embodiment involves the use of colorimetric substrates suitable for Alkaline Phosphatase (AP). These substrates react with AP in the presence of the enzyme-antibody complex and produce a colored product. Common colorimetric substrates include p-nitrophenyl phosphate (pNPP) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT). The development of a colored reaction product indicates complement activation, and the absorbance can be measured spectrophotometrically.

Chemiluminescent Substrates for Horseradish Peroxidase (HRP):

For Horseradish Peroxidase (HRP) enzyme label, chemiluminescent substrates can be utilized. These substrates produce light signals upon enzymatic reaction, which can be detected by a luminometer or other sensitive imaging systems. Examples of chemiluminescent substrates include luminol or acridan-based substrates, providing enhanced sensitivity and allowing for quantitative analysis of complement activation.

Fluorogenic Substrates for Alkaline Phosphatase (AP) or Horseradish Peroxidase (HRP): Fluorogenic substrates offer another approach for ELISA detection. They are non-fluorescent until acted upon by the enzyme label, resulting in the generation of a fluorescent product. Fluorogenic substrates can be used with either AP or HRP, and the fluorescence intensity correlates with the extent of complement activation. Common fluorogenic substrates include 4- methylumbelliferyl phosphate (4-MUP) for AP and 4-methylumbelliferone (4-Mll) for HRP.

Chromogenic-HRP Substrates:

Horseradish Peroxidase (HRP) can also be used in conjunction with chromogenic substrates. These substrates produce colored products upon reaction with the enzyme label, which can be quantified spectrophotometrically. Common chromogenic substrates for HRP include 3, 3', 5,5'- tetramethylbenzidine (TMB) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS).

Combined Use of Substrates:

In some embodiments, a combination of different substrates may be employed for multiplexing or to enhance the detection of complement activation products. For instance, both colorimetric and fluorogenic substrates can be used simultaneously, enabling the detection of two distinct signals from AP and HRP-conjugated antibodies.

Development of Novel Substrates:

The invention also encompasses the use of newly developed or custom-designed substrates suitable for ELISA detection. Novel substrates can be tailored to meet specific assay requirements, such as enhanced sensitivity, stability, or the ability to detect multiple targets.

It is important to note that the substrates mentioned above are examples of potential options that can be used for predicting AAV-mediated complement activation. The choice of substrate(s) can be tailored based on the specific needs of the assay, including sensitivity, instrumentation availability, and the desired level of multiplexing.

The present invention encompasses a kit or a set of components. This kit might include one or more antibodies, as detailed herein, which can optionally be conjugated to an appropriate enzyme, examples of which include Horse Radish Peroxidase (HRP), alkaline phosphatase (ALP), urease, or other relevant enzymes recognized in the field.

The antibodies contained within may act as primary or detection antibodies, as elaborated in this document, and may also be optionally linked to an enzyme.

Additionally, the kit can incorporate a secondary antibody, as specified herein, which might optionally be conjugated to enzymes like HRP, ALP, urease, or other enzymes familiar to those skilled in the art. For detection and subsequent quantification, a substrate that the conjugated enzyme can metabolize is introduced. This substrate can be any fitting compound digestible by the attached enzyme. Examples of these substrates, though not exhaustive, include tetramethyl benzidine (TMB), 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), p- Nitrophenyl Phosphate, Disodium Salt (PNPP), and o-phenylenediamine dihydrochloride (OPD), among others.

In some aspects, the enzyme used for antibody conjugation may be Alkaline Phosphatase (AP), or Horseradish Peroxidase (HRP) and the likes. The substrates that are suitable for these enzymes in the assay are para-Nitrophenyl Phosphate (pNPP) for AP and 3,3’,5,5’-Tetramethyl Benzidine (TMB) for HRP.

In particular, present invention relates to detection of response to all serotypes of AAV. In one aspect, the invention relates to detection of AAV2, i.e. detection of complement activation mediated by AAV2.

In another aspect, present invention relates to detection of AAV8, i.e. detection of complement activation mediated by AAV8.

In a further aspect, present invention relates to detection of AAV9, i.e. detection of complement activation mediated by AAV9.

In one aspect, present invention relates to predicting if a patient that is to receive gene therapy based on AAV is at risk of experiencing an adverse effect to the therapy owing to complement activation in the immune system.

In specific embodiments, present invention also relates to the following items;

1. A method of predicting a complement activation potential in a subject, the method comprising the steps of; a) providing a biological sample, b) contacting the sample with AAV or AAV-VLP particles of any serotype, wherein the complement activation is uninhibited or fully functional, c) further contacting the resulting mixture of b) with a conjugated detector antibody capable of specifically binding any neoepitope generated due to the complement system activation, such as e.g. one present in the complement membrane attack complex (C5b-9), thereby detecting the presence or absence of any complement activity in the sample. 2. The method according to item 1 , wherein step b) also comprises diluting the sample using a buffer and a sample dilution inhibiting complement activation.

3. The method according to items 1 and 2, wherein the method comprises a step x) comprising removing unbound sample and diluent from step b), and thereafter contacting the resulting mixture with the biological sample a) in a buffer and a sample dilution where the complement cascade and its activation pathways of such a biological specimen typically are fully functional, and wherein step x) takes place after step b) but before step c).

4. The method according to any one of the preceding items, wherein the AAV or AAV-VLP particles are bound to the walls of a well in a microtiter plate.

5. The method according to any one of the preceding items, wherein the conjugated antibody is conjugated with an enzyme such as e.g. Alkaline Phosphatase (AP), Horseradish Peroxidase (HRP) etc.

6. The method according to any one of the preceding items, wherein substrate is e.g. OPD (o- phenylenediamine dihydrochloride), TMB (3,3',5,5'-tetramethylbenzidine), ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), PNPP (p-Nitrophenyl Phosphate, disodium salt) etc.

7. The method according to any one of the preceding items, wherein the capsids is a full or an empty AAV-capsid.

8. The method according to any one of the preceding items, wherein the antibodies are different and binding to any neoepitope generated due to the complement system activation, such as e.g. one present in the complement membrane attack complex (C5b-9). 9. The method according to any one of the preceding items, wherein the antibodies are different and conjugates with different conjugates.

10. The method according to any one of the preceding items, wherein the complement activation potential in a subject is considered low if the optical density (OD) is below 0.6, and wherein the activation potential is considered at medium risk if the optical density is above 0.6 but below 1.5, and wherein the activation potential is considered at high risk if the optical density is 1.5 or above.

11. The method according to any one of the preceding items, wherein the detector antibodies are capable of recognizing a quaternary structure of the C9 neoantigen selected among the amino acids of aE11 L113 (CDR L3), V50 (CDR H1), Y120 (CDRH3), and Y112 (CDR L3) binding to V68, L423, L423 and P72 of the C9.

12. The method according to any one of the preceding items, wherein the detector antibodies comprises one or more of the CDRs set forth in;

H1 : 42TVSGFSLTVYGV53,

H2: 69MIWGDGSTDY78,

H3: 115ARDRSYGGSSAWFGY129,

L1 : 43RASHDISNYL52,

L2: 68YYTSRLHS75,

L3: 108QQGNYLPYT116, wherein H stand for heavy chain and L stands for light chain.

13. The method according to any one of the preceding items, wherein the method comprises including positive and negative controls. The invention is further illustrated in the below non-limiting examples. The examples merely serve to illustrate the invention but is not to be construed as limiting the overall scope of the invention.

Examples

Example 1

Tests were performed to predict the complement activation potential of different sera to the presence of AAV vectors. Used sera samples presented different amounts of NAb, and 2 of the samples had a high amount of TAb (Figure 3). Assay results indicate that the complement activation potential does not depend only on the amount of TAb, but also on the intrinsic capacities of the sera to be activated by AAV vectors (Figure 4). Samples with low TAb such as sample 4 presented an increased complement activation potential in comparison with samples with a similar amount of anti-AAV antibodies (Figure 4). The test was performed according to the procedure described in the section below, Material & Methods.

Material and methods

Reagents

Serum samples were bought from Discovery Life Science and stored at -80°C until use. Serum samples with complement system compounds depleted were bought from Complement Technologies and stored at -80°C until use.

Enzyme-Linked Immunosorbent Assay for predicting complement activation potential

AAV-VLP (Catalog # 5EAAV2-b; VectorBuilder), or full AAV capsids (Catalog # AAV2SP(VB210527-1364ygg); VectorBuilder) were diluted in PBS (Medicago, REF 09-9400- 100, working solution) containing MgCh, to a density between 2.5x10⁸ to 45x10⁸ particles/mL. The suspension of virus particles was applied to 96-well microtiter plates (Nunc Maxisorp Lockwell C8), 100 pL/well. The plates were sealed with plastic film and incubated at 4-8°C overnight i.e. >16 h to allow for virus particle adherence to wells’ plastic surface. After incubation, the plates were washed 3x with 300pL of PBS buffer with Tween 20 in each wash. Then, in order to block any remaining binding sites in the microtiter plate wells, PBS buffer with BSA and sucrose (200 pL/well) was added, and the plates incubated for 2-4 h at 21°C. After incubation, all liquid was removed from the wells and the plates dried in a plate drier at 30°C until the relative humidity in the cupboard was <19 %. Plates were subsequently stored at 4- 8°C in sealed aluminium foil bags with desiccant until use. On the day of assay, plasma and/or serum samples were diluted 1/10 in PBS buffer with BSA, EDTA and Tween 20. The diluted samples were applied in duplicates to AAV-coated microtiter plate wells, 100 pL/well, and an IgG preparation with known AAV-ADA content (Kiovig;

Takeda) was used as calibrator. The plates were subsequently incubated at 37°C for 60 min. Then, plates were washed with 3x300 pL PBS with Tween 20 as above. Plasma/serum samples were again diluted, this time 1/101 in TBS-based buffer with CaCh, MgCh. The serum dilutions were added to the washed plates, 100 pL/well. The plates were subsequently incubated at 37°C for 90 min. After incubation, plates were washed with 3x300 pL PBS with Tween 20 as above. To all wells, the HRP -conjugated antibody aE11 (Diatec Monoclonals), or anti-C3b, or anti-C4dwas added, 100 pL/well, followed by incubation of the plates at 37°C for 30 min to allow the conjugated antibody to bind to any TCC (Terminal Complement Complex)/MAC (Membrane Attack Complex), C3b, or C4d adhering to the wells of the plate. Experiments with anti-C5b were using a secondary antibody. After incubation with conjugate the plates were washed with 3x300 pL PBS with Tween 20 as above, and colorimetric ELISA substrate for HRP - TMB - was added, 100 pL/well. The plates were then incubated at 21 °C for 30 min to let the colour signal develop. The colour signal in each well was subsequently assessed by reading the plates in a Tecan Sunrise instrument at 450 and 620 nm.

Enzyme-Linked Immunosorbent Assay for evaluating the presence of anti-AAV antibodies

The ELISA AAV TAb assay (Catalog # AAV2TAbRUO, AAV8TAbRUO and AAV9TAbRUO; Svar Life Science) was used to evaluate the presence of anti-AAV neutralizing antibodies. The assay was performed as indicated in the application note from the manufacturer. Briefly, samples were diluted 1/30 - 1/100. The diluted samples were applied in duplicates to AAV- coated microtiter plate wells. The plates were subsequently incubated and then washed with washing buffer (PBS with Tween 20). After washing, HRP-conjugated anti-human IgG (or antihuman IgM) antibody was added to all wells, followed by incubation of the plates at 21 °C for 30-60 min to allow the conjugated antibody to bind to any IgG (or IgM) molecules adhering to the wells of the plate. After incubation with conjugate the plates were washed with washing buffer, and colorimetric ELISA substrate for HRP - TMB - was added. The plates were then incubated at 21°C for 30 min to let the colour signal develop. The colour signal in each well was subsequently assessed by reading the plates in a Tecan Sunrise instrument at 450 and 620 nm.

Neutralizing Antibody Assay

The iLite® AAV2 NAb Platform (Catalog # BM6100, BM6002; Svar Life Science) was used to evaluate the presence of anti-AAV neutralizing antibodies. The assay was performed as indicated in the application note from the manufacturer. Briefly, human serum samples were diluted 1:5 (in assay concentration 1:10), and a serial dilution of IVIg (ranging from 10000 to 0 pg/ml) in the same diluent with the same serum concentration as the diluted samples, using NAb negative serum was performed. Then 40pl of IVIg references, controls, and diluted test samples were added to assigned wells of a 96-well plate (Catalog # 6055680, PerkinElmer). BM6100 and BM6002 vials were thawed and mixed carefully. After that, BM6100 and BM6002 cells were diluted in 5.5ml of diluent, and 40pl of the diluted cells were added to each well. The plate was then mixed and incubated for 18 hours at 37 °C with 5% CO2. Next day, firefly luciferase substrate (Catalog # E2920, Promega) was prepared according to supplier instructions. Cells were then lysed by adding 80pl of firefly substrate to each well followed by 10 minutes in incubation. The luminescence signal was measured using a plate reader (GloMax® Explorer Multimode Microplate Reader, Promega). The neutralizing activity of the serum samples is reported as a percentage of the most negative sample.

Graphing and statistical analysis

All graphs were generated using the GraphPad Prism v10.3.0 software.

Discussion

By employing the above protocol the assay is demonstrated to be applicable on any AAV serotype. These results are highlighted in Fig. 5

The results showcased in Fig. 6 indicates a correlation between complement activation and presence of IgG. The same samples as seen in Fig. 5 was used. Notably, complement activation does not always correlate with the quantity of anti-AAV IgG antibodies, reflecting the known influence of antibody isotype and other antibody-dependent characteristics on complement activation.

The corresponding situation for IgM is illustrated in Fig. 7. The results illustrated in Figure 7 shows the presence of IgM for the same samples used in Figures 5C and 6C. These results had been obtained by using a newly developed AAV-IgM-Tab ELISA which is highly sensitive in detecting IgM and all tested samples exhibited signal levels near the background, indicating an absence of samples with markedly elevated anti-AAV IgM concentrations.

Comparing results from Figure 5, 6 and 7 we can identify that sample 12, which is anti-AAV9 IgG negative (Figure 6C) but exhibits high complement activation (Figure 5C), shows the highest levels of anti-AAV9-lgM antibodies (Figure 7) compared to other samples. This indicates that our assay has proven to be highly sensitive to even small amounts of anti-AAV IgM-driven complement activation. This highlights the increased sensitivity by the method of present invention.

Again, the assay is illustrated in Fig. 6 and 7 by employing various AAV serotypes. Figs. 6 and 7 also highlights the assays for detection of the classical pathway.

With respect to the alternative pathway, the role of the alternative pathway in complement activation by AAV is via an antibody-independent pathway (non-classical pathway). In present assay design/method, which involves well-exposed coated AAV particles, buffer composition and sample dilution, allows capturing activation of the alternative pathway, which can be further accelerated by the presence of anti-AAV antibodies and as illustrated in Fig. 8 and 9.

In the assay, the signal observed with factor H (FH)-depleted serum (representing uncontrolled alternative pathway) without I Vlg indicates that the assay may measure a higher predisposition to complement activation, even in the absence of antibodies. Moreover, other potential genetic variations could also be identified. C3-depleted serum was used (Fig. 9), a hub protein of the complement cascade necessary for complement activation, as another potential genetic variations. This variation results in the inefficacy of activation complement system. Overall, the results showcase specificity and the possibility to measure individual variations in complement activity.

With present assay, the use of an antibody targeting the downstream protein or protein complexes within all complement pathways are to ensure that complement activation is the actually measured parameter. However, any complement antigen, protein or protein complex would suffice as is seen in Fig. 9 wherein the detection antibody is changed to detect any deposited complement proteins, or exchanged with an antibody detecting other parts: i.e. constituting proteins of the MAC complex .

AAV Complement Activation ELISA Assay is a solid-phase ELISA assay designed to determine e.g . AAV2-triggered complement activation in vitro. In such a scenario, AAV2 particles are immobilized on a microtiter plate, allowing for the comprehensive capture of the entire AAV2-mediated complement activation process. This includes interactions between AAV2 and anti-AAV2 binders, as well as the complement system, all within a single well from initiation to readout.

This approach differs from other methods, available in the art, which prescreen AAV gene therapy patient candidates for a higher risk of complement. Those methods involve reactions occurring entirely in the liquid phase (addition of AAV particles to serum/blood) before being measured by an ELISA specific to complement-activated component. Unlike 'in-solution' ELISA, present invention’s assay measures de novo complement activation, meaning that the complement activation measured is not influenced by the initial status of the individual's sample's complement activity. In the present invention, the complement cascade is built ab initio on AAV serving as an anchor for complement cascade initiation, thus ensuring that the signal measured is only due to the AAV-mediated complement activation (and not sporadic activation of the complement system).

Sera from four donors, all of which tested negative for anti-AAV2 antibodies (data not shown), were either activated by incubation at 37°C for 3 hours or kept on ice as non-activated controls.

As showed in Figure 10, serum incubation at 37°C results in spontaneous activation of the complement system, as indicated by increased TCC (measured using a TCC ELISA). Moreover, a natural variability in complement activity can be observed between individuals (non-activated sera). It's important to note that this step is essential for ‘in-solution’ ELISA to confirm AAV-specific complement activation.

To determine whether the complement activation status of the samples could lead to erroneous readouts with our solid-phase ELISA assay, the sera were tested both in the presence and absence of intravenous immunoglobulin (I Vlg) . The addition of I Vlg served as an additional control to verify that, even with higher concentrations of anti-AAV2 antibodies, the complement activity status of the samples does not affect the outcome.

According to the invention, AAV Complement Activation ELISA Assay (Fig. 11), both activated and non-activated samples, representing different levels of complement activity (Fig. 10), show similar responses both with and without AAV particles. This indicates that the assay reliably measures AAV-specific complement activation regardless of the initial complement status of the sample. Statistical analysis using repeated measures two-way ANOVA reveals no significant differences between activated and non-activated sera. This confirms that TCC or other activated complement components already present in the sample do not non-specifically nucleate the complement system within the plate, thereby eliminating the need for control samples without AAV particle addition to measure specific signals. Additionally, this underscores the superiority of the method of present invention over 'in-solution' ELISAs. List of references:

Gao, G., Vandenberghe, L. H., Alvira, M. R., Lu, Y., Calcedo, R., Zhou, X., & Wilson, J. M. (2004). Clades of Adeno-Associated Viruses Are Widely Disseminated in Human Tissues. Journal of Virology, 78(12), 6381-6388. https://doi.Org/10.1128/jvi.78.12.6381 -6388.2004

Lek, A., Wong, B., Keeler, A., Blackwood, M., Ma, K., Huang, S., Sylvia, K., Batista, A. R., Artinian, R., Kokoski, D., Parajuli, S., Putra, J., Carreon, C. K., Lidov, H., Woodman, K., Pajusalu, S., Spinazzola, J. M., Gallagher, T., LaRovere, J., ... Flotte, T. (2023). Death after High-Dose rAAV9 Gene Therapy in a Patient with Duchenne’s Muscular Dystrophy. New England Journal of Medicine, 389(13), 1203-1210. https://d0i.0rg/l 0.1056/nejmoa2307798

Mietzsch, M., Jose, A., Chipman, P., Bhattacharya, N., Daneshparvar, N., McKenna, R., &

Agbandje-McKenna, M. (2021). Completion of the AAV Structural Atlas: Serotype Capsid

Structures Reveals Clade-Specific Features. Viruses, 13(1), 101. https://doi.org/10.3390/v130101Q1

Claims

Claims

1. A method of predicting a complement activation potential in a subject, the method comprising the steps of; i) providing a biological sample, diluted in a sample dilution buffer which inhibits the complement activity, ii) contacting the diluted sample with AAV or AAV-VLP particles of any serotype wherein the AAV or AAV-particles are immobilised on a surface, iii) removing unbound sample from step ii), and thereafter the well is contacted with a mixture comprising the biological sample i) in a buffer sample dilution wherein the complement cascade and its activation pathway is not inhibited, iv) further contacting the resulting mixture of iii) with a conjugated detector antibody capable of specifically binding any protein generated due to the complement system activation, such as e.g. one present in the complement membrane attack complex (C5b-9), or detection of any protein in the complement system by any suitable means such as aptamers, affibodies, nanobodies, affimers, designed ankyrin repeat proteins (DARPins), molecularly imprinted polymers (MIPs), peptide ligands, lectins, synthetic small molecule ligands, fusion tags, or any other suitable binding molecules, thereby detecting the presence or absence of any complement activity in the sample.

2. The method according to any one of the preceding claims, wherein the AAV or AAV-VLP particles are bound to the walls of a well in a microtiter plate or flask.

3. The method according to any one of the preceding claims, wherein the buffers for inhibiting complement activation in i) is e.g. a buffer comprising any agent capable of chelating or binding to a bivalent ion, such as e.g. Ca2⁺ or Mg2⁺. Specific non-limiting examples are buffers comprising e.g. Ethylene Diamine Tetraacetic Acid (EDTA), Ethylene Glycol Tetraacetic Acid (EGTA), Sodium Citrate or the likes.

4. The method according to any one of the preceding claims, wherein the sample dilution in i) is in the range of about 1/2 to about 1/100, such as e.g. about 1/5 or more, such as e.g. about 1/10 or more, such as e.g. about 1/15 or more, such as e.g. about 1/20 or more, such as e.g. about 1/30 or more, such as e.g. about 1/50 or more, such as e.g. about 1/75 or more, such as e.g. about 1/100.

5. The method according to any one of the preceding claims, wherein the buffers supporting or not inhibiting the complement activation in iii) is e.g. a buffer comprising may be e.g. a buffer having an appropriate ionic strength of e.g. approximately 150 mM NaCI and may further be supplemented with calcium and magnesium ions, which are necessary for complement function and which has a pH close to physiological levels such as e.g. about 7.4.

6. The method according to any one of the preceding claims, wherein the buffers supporting or not inhibiting the complement activation in iii) is e.g. Gelatin Veronal Buffer (GVB++). Other buffers used for this purpose may be e.g. Veronal Buffered Saline (VBS), Hank’s Balanced Salt Solution (HBSS), Phosphate-Buffered Saline (PBS) (which may be supplemented with magnesium and calcium).

7. The method according to any one of the preceding claims, wherein the buffers supporting or not inhibiting the complement activation in iii) is a buffer comprising one or more of the following or having one or more of the following attributes;

NaCI: 120-180 mM, pH: 7.2-7.6,

CaCI₂: 0.1-0.3 mM,

MgCI₂: 0.5-5 mM.

8. The method according to any one of the preceding claims, wherein the sample dilution in iii) is in the order of about 1/15 to about 1/150, such as e.g. about 1/18 or more, such as e.g. about 1/20 or more, such as e.g. about 1/30 or more, such as e.g. about 1/40 or more, such as e.g. about 1/50 or more, such as e.g. about 1/75 or more, such as e.g. about 1/100 or more, such as e.g. about 1/120 or more, such as e.g. about 1/150.

9. The method according to any one of the preceding claims, wherein the neoepitope or protein in the complement cascade , e.g membrane attack complex (C5b-9), is detected by the aid of the detection antibody, optionally conjugated antibody with an enzyme or by other suitable means such as any fluorescent technique, luminescent or chemiluminescent technique, electrochemical detection method, mass spectrometric technique, or by utilizing binding agents selected from aptamers, nanobodies, affimers, designed ankyrin repeat proteins (DARPins), molecularly imprinted polymers (MIPs), peptide ligands, lectins, synthetic small molecule ligands, fusion tags, or any other suitable detection method.

10. The method according to any one of the preceding claims, wherein the conjugated antibody is conjugated with an enzyme such as e.g. Alkaline Phosphatase (AP), Horseradish Peroxidase (HRP) etc.

11. The method according to any one of the preceding claims, wherein a substrate is employed and wherein the substrate is specific for the enzyme conjugated to the detection antibody and selected from e.g. OPD (o-phenylenediamine dihydrochloride), TMB (3, 3', 5,5'- tetramethylbenzidine), ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]- diammonium salt), PNPP (p-Nitrophenyl Phosphate, disodium salt) etc.

12. The method according to any one of the preceding claims, wherein the AAV or AAV-VLP are capsid and which is a full or an empty AAV-capsid.

13. The method according to any one of the preceding claims, wherein the antibodies are different and binding to any proteins generated due to the complement system activation, such as e.g. one present in the complement membrane attack complex (C5b-9), C3b or C4d.

14. The method according to any one of the preceding claims, wherein the antibodies are different and conjugated with different conjugates.

15. The method according to any one of the preceding claims, wherein the detector antibodies are capable of recognizing a quaternary structure of a product resulting from the complement system activation, such as C5b, C3b, C4d or the C9 neoantigen generated in C5bC9.

16. The method according to any one of the preceding claims, wherein the detector antibodies are capable of detecting epitopes or structural conformations of complement proteins and their fragments, thereby measuring complement activity selected from proteins and fragments of:

• C1 complex: C1q, C1 r, C1 s

• C1 inhibitor (C1-INH)

• Mannose-Binding Lectin (MBL)

• MBL-associated serine proteases (MASP-1 , MASP-2, MASP-3)

• C4: C4a, C4b, C4d

• C2: C2a, C2b

• C3: C3a, C3b, iC3b, C3c, C3dg, C3d, C3g

• Factor B: Bb, Ba

• Factor D

• C5: C5a, C5b

• Membrane Attack Complex (MAC) or Terminal Complement Complex (TCC): C5b, C6, C7, C8, C9

• Properdin

• C3(H2O)

• Regulatory Proteins: Factor H, Factor I, C4-binding protein (C4BP).

17. The method according to any one of the preceding claims, wherein the detection is made with respect to surface-bound complement components and selected from, but not limited to;

• C1 complex: C1q, C1 r, C1 s

• C1 -INH

• MBL

• MASP-1 , MASP-2, MASP-3

• C4 fragments: C4b, C4d

• C2a

• C3 fragments: C3b, iC3b, C3dg, C3d

• Factor B

• C5b

• MAC/(TCC: C5b, C6, C7, C8, C9

• Properdin

18. The method according to any one of the preceding claims, wherein the detection is made with respect to the formation of C3 and C5 convertases and the initiation of the terminal complement pathway selected from:

• C2a

• C4b, C4d

• C3 fragments: C3b, iC3b, C3dg, C3d

• C5b

• MAC/TCC: C5b, C6, C7, C8, C9

19. The method according to any one of the preceding claims, wherein the detection is made with respect to TCC selected from C5b, C6, C7, C8, and C9.

20. The method according to any one of the preceding claims, wherein the method comprises including positive and negative controls.

21. A method comprises for preparing AAV or AAV-VLP immobilised on a surface, the method comprising the steps of;

I) providing AAV or AAV-VLP in a suspended in a fluid, such as e.g. a buffering solution,

II) contacting the AAV or AAV-VLP suspended in the fluid with a surface of a container or well,

III) incubating the container or well with the AAV or AAV-VLP suspension for a period of time,

IV) removing of the suspension from each well or container and rinsing the wells or containers with the buffering solution,

V) adding a blocking solution to each well,

VI) incubating the container or well with the blocking solution for a period of time,

VII) removal of all liquids from the container or wells,

VIII) allowing the container or wells to dry.

22. The method according to claim 21, wherein the suspension of AAV or AAV-VLP has any density or concentration of about 2.5x10⁸ to about 45x10⁸ particles/ml of the suspension in step I).

23. The method according to claims 21-22, wherein the incubation time in step III) may be at least about 16h, such as e.g. at least about 20h, such as e.g. at least about 24h.

24. The method according to claims 21-23, wherein the incubation in step III) may take place at any temperature of about 4°C to about room temperature of about 20°C .

25. The method according to claims 21-24, wherein the incubation in step VI) takes place during any period of about 2h to about 4h.

26. The method according to claims 21-25, wherein the incubation in step VI) takes place at any temperature of about 15°C to about 25°C, such as e.g. about 20°C.

27. The method according to claim 21-26, wherein the drying step VIII), takes place at a temperature of about 25°C to about 35°C, such as e.g. about 30°C.

28. The method according to claim 21-27, wherein the drying step VIII) is conducted until the wells or container are considered sufficiently dry, such as e.g. until the relative humidity in the drying device is less than about 19%.

29. A product obtainable by the method according to any one of claims 21-28.

30. The product obtainable according to claim 29, wherein the product is tube, flask or a container comprising a plurality of wells such as e.g. a microtiter plate of any format or dimension.

31. A product comprising one or more containers wherein AAV or AAV-VLP have been attached or otherwise immobilised onto the surface of the one or more containers and wherein the surface has been blocked with respect to unspecific activation of the complement cascade.

32. The product according to claim 31, wherein the product is a flask or a tube or a microtiter plate of any format or dimension such as e.g. a 96 well plate.

33. A kit comprising a product according to any of claims 31-32.

34. The kit according to claim 33, further comprising one or more of buffering solutions, substrates, positive and negative controls etc.