EA041088B1 - AAV-MEDIATED EXPRESSION OF ANTIBODIES AGAINST INFLUENZA AND METHODS FOR THEIR USE - Google Patents

Description

Electronically filed materials that are incorporated by link

Applicant hereby incorporates by reference the sequence listing material filed in electronic form with the present disclosure. This file is named UPN_15-7484PCT_ST25.txt.

Statement of federally funded research

This invention is made in part with government support under W911NF-13-2-0036 issued by the Defense Advanced Research Projects Agency (DARPA). The government has certain rights to the invention.

Background of the invention

Influenza infection is the seventh leading cause of death in the US and accounts for up to 49,000 deaths per year, a significant proportion of nearly 500,000 deaths worldwide [Prevention, C.f.D.C.a. Estimates of deaths associated with seasonal influenza: United States, 1976-2007. 2010; available in MMWR Morb. Mortal. Wkly. Rep. 59:1057-1062]. The economic burden of annual influenza epidemics is estimated at about US$87 billion. More than half of this amount is hospital care required for almost 1 million patients, of which 70% are elderly patients (aged >65 years) [Molinari, N.A., et al., Vaccine, 2007. 25(27), p. 5086-96]. In addition, immunocompromised individuals, such as HIV/AIDS patients, organ transplant recipients, or those suffering from autoimmune diseases, are at high risk for influenza and have an increased susceptibility to infection and its complications, which include fatal pneumonia and acute respiratory distress syndrome [Oliveira, E.C., et al., J. Intensive Care Med, 2003. 18(2), p. 80-91]. Influenza is an RNA virus that belongs to the Orthomyxoviridae family. There are three genera of influenza viruses: A [Medina, R.A. and A. Garcia-Sastre, Nat. Rev. Microbiol., 2011. 9(8), p. 590-603.], In [Paul Glezen, W., et al., Am. J. Public. Health, 2013. 103(3), p. e43-51] and C. These types of influenza are divided on the basis of antigenic differences between matrix and nuclear proteins. In the US, influenza A and B cause seasonal epidemics during the winter months. Influenza C is not known to cause epidemics or be associated with mild respiratory illnesses.

Person-to-person transmission of influenza A or B usually occurs through the spread of an aerosol or droplets through the sneezing or coughing of an infected subject. The influenza virus usually enters the nasal respiratory tract. There, hemagglutinin (HA) binds to sialic acid receptors present on airway epithelial cells and the virus envelope fuses with the host cell membrane. Subsequently, the viral RNA enters the cytosol and eventually the nucleus of the host cell, where it replicates. Following viral replication, the host cell is lysed and several thousand viruses are released. This cycle continues, the virus replicates and eventually disseminates to the underlying respiratory tract, where it causes severe disease that can be fatal in certain high-risk groups of subjects [Medina, cited above].

Although influenza vaccine coverage in the United States has increased in the last decade, studies have shown a low effect of seasonal influenza vaccines in elderly and immunocompromised patients [Ljungman, P., Clin Microbiol Infect, 2012. 18 Suppl 5, p. 93-9]. Several aspects of the influenza virus and the human host's immune response to influenza infection stand in the way of a simple preventive drug. Key targets of the adaptive immune response, such as the HA protein of the virus, change rapidly, making the immunological memory response only partially protective against new infections [Medina, cited above]. A person's response to a natural infection or influenza vaccine is usually limited in scope, providing protection only against closely related influenza subtypes. This has resulted in the annual vaccination of subjects 6 months of age and older against seasonal strains of the influenza virus that are predicted to emerge during the coming season.

Influenza A viruses can infect humans and various other mammals, including pigs, horses, dogs, and birds [Medina, cited above]. These viruses are divided into two subtypes based on the structure of the two surface proteins hemagglutinin (HA) and neuraminidase (NA). There are 18 known subtypes of HA and 11 subtypes of NA [CDC. Types of Influenza Viruses. 2014; available at: http://www.cdc.gov/flu/approx/viruses/-types.htm]. Influenza A is associated with major epidemics and pandemics, especially the 1918 pandemic, which is associated with significant disease prevalence and death. Epidemic influenza, also known as seasonal influenza, is usually uncomplicated and remains confined to the upper respiratory tract of the individual only [Molinari, cited above; Glezen, cited above]. Viral pneumonia is rare, but susceptible populations, including pregnant women, the elderly, patients with pre-existing cardiovascular and pulmonary disease, are considered to be at high risk for influenza B complications. Seasonal influenza is generally considered to lead to mild illness.

The emergence of a new influenza pandemic remains a threat that could lead to substantial deaths and economic losses around the world. It is believed that the immune memory repertoire arising from previous influenza infections and vaccinations helps blunt the complications of a new infection and enhances the effect of the vaccine. This does not apply when an influenza virus in animal reservoirs acquires a tropism for the human respiratory tract and is transmitted to humans.

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[Juno, J., et al., Clin. dev. Immunol., 2012. 2012, p. 797180]. These zoonotic strains are quite different from those normally circulating in the human population and can lead to fatal pandemics because they are not effectively controlled by vaccines designed for human virus strains [Juno, cited above]. As established during the 2009 H1N1 pandemic [Shapshak, P., et al., Diagn. Ther., 2011. 15(2), p. 63-81], the vaccine development time is not short enough to ensure that the population is vaccinated in response to an emerging pandemic. Unlike an epidemic, an influenza pandemic does not occur often, but can result in significant mortality. Pandemic influenza viruses arise from a genetic mix between non-human (ie avian) and human viruses. The recombination of virus segments is a key mechanism for the rapid creation of a new virus. These events of antigenic variation introduce into the human population an immunologically novel influenza virus that lacks pre-existing immunity. Two human influenza pandemics in the last century have been associated with genetic lineages that arose from the recombination of segments of the genome with a genome of non-human origin. During the most recent pandemic (2009), younger people were disproportionately affected by lower respiratory diseases that required hospitalization relative to years between pandemics [Dawood, F.S., et al., Lancet Infect. Dis., 2012. 12(9), p. 687-95].

Influenza vaccines are not always effective in protecting against influenza. In early 2013, an influenza outbreak was reported in the vaccinated population on US Navy minesweepers of 102 young healthy males (ages 21 to 44). Nearly 25% of these vaccinated subjects had flu-like symptoms that required medical attention. PCR analysis indicated an H3N2 influenza strain that shares 99% homology with strains circulating in the 2013-2014 influenza season, which are similar to the H3N2 antigenic component of the 2013-2014 influenza vaccine [T.L. Aquino, et al., Influenza Outbreak in a Vaccinated Population - USS Ardent, mmwr 63(42); 947-949 2014].

Two monoclonal antibodies have emerged as candidates for influenza prophylaxis. For influenza A, the FI6 antibody [Oliveira, et al., cited above], which was developed by the Lanzavecchia group [US 2010/0080813], and for influenza B, the CR8033 antibody [Dreyfus, C., et al., Science, 2012. 337(6100 ), p. 1343-8; US patent 8852595], developed by the Wilson (The Scripps Research Institute) and Friesen (Crucell) groups. FI6 has been reported to be highly effective against several strains of influenza A (including pandemic strains A/H1N1/1918 and A/H1N1/2009) and has a broad strong reactivity and neutralizing profile for all group 1 and group 2 HAs [Corti, D., et al ., Science, 2011. 333(6044), p. 850-6]. Importantly, these antibodies have been shown not to create escape mutants, even after several successive passages [Oliveira, cited above; Paul Glezen, cited above]. When delivered via a vector, this antibody has been reported to protect mice and ferrets from lethal infection with several influenza A strains [Limberis, M.P., et al., Sci. Transl. Med., 2013. 5(187), p. 187ra72; Limberis, M.P., et al., Clin. Vaccine Immunol., 2013. 20(12), p. 1836-7; Adam et al. Clin. Vaccine Immunol. Nov 2014; 21(11):1528-33].

There is one universal antibody, CR9114 [Dreyfus., C. et al, Science, 2012, 337(6100), p. 1343-8], which binds to both influenza A group 1 and group 2 viruses and influenza B virus. However, the usefulness of this antibody is limited by the relatively high concentrations of antibody required to neutralize one of the various strains of influenza A and B. IC ₅₀ for viruses group 1 ranges from 0.1 to 100 µg/ml, with only two strains falling between 0.1 and 1 µg/ml and the remaining between 1 and 100 µg/ml. The IC ₅₀ for group 2 viruses is even less favorable, with only one strain falling below 1 μg/ml [Dreyfus, cited above]. Relatively high concentrations of CR8071 are required for in vivo protection against influenza B, and suboptimal doses resulted in more pronounced weight loss compared to unimmunized mice (Fig. S2 and escape mutants after only 15 passages [Dreyfus, cited above]. Another CR8020 antibody to influenza B led to the emergence of escape mutants after only four passages [Dreyfus, cited above].

There is currently one approved therapy (FluMist Quadrivalent) that is delivered intranasally. However, it is not suitable for subjects who are severely allergic to eggs and subjects who are 2 to 17 years old and who are taking aspirin or medicines containing aspirin [Shapiro R.J., S.K., et al. The potential American market for generic biological treatments and the associated cost savings. 2008; available at: http://www.sonecon.com/docs/studies-/0208_GenericBiologicsStudy.pdf].

Adeno-associated viruses (AAVs) are members of the Parvoviridae family. It has been shown that these small DNA viruses have significant prospects as vectors for achieving stable transgene expression after in vivo delivery. AAVs were initially found as contaminants in adenovirus laboratory preparations [Melnick, J.L., et al., Association of 20-Millimicron Particles with Adenoviruses. J. Bacteriol., 1965. 90(1), p. 271-4]. Immunological characterization of these isolates indicates the existence of six AAV serotypes. Seroepidemiological studies indicate broad human exposure to various AAV serotypes, with more than 60% of the population demonstrating Nab to most of the six AAV serotypes by age 10 years [Calcedo, R., et al.,

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J. Infect. Dis., 2009. 199(3), p. 381-90]. At the beginning of 2000 The repertoire of AAV vectors has been expanded with the isolation of several hundred new AAV viruses in humans and non-human primates

[Gao, G., et al., J. Virol., 2004. 78(12), p. 6381-8]. More than 120 AAV vector genotypes have been identified, including the original six serotypes from human and NHP tissue sources, and have been characterized phylogenetically and organized into six distinct clades [Gao, G., et al., J. Virol., 2004. 78(12), p. 6381-8].

There remains a need in the art for an anti-influenza therapy that is effective in therapeutic and/or prophylactic use.

The essence of the invention

In one aspect, the invention relates to a composition that can be used for passive immunization against influenza infection. The composition contains an anti-influenza A antibody expressed from an AAV vector and an anti-influenza B antibody expressed from a second AAV vector. In one embodiment, the first non-replicating recombinant AAV has an AAV9 capsid (rAAV9) and a vector genome that contains nucleic acid sequences encoding (a) an AAV inverted terminal repeat (ITR) (b) an enhancer; (b) chicken beta actin promoter; (c) intron; (d) 5'-UTR; (e) a leader peptide operably linked to the F16v3 heavy chain; (f) FI6v3 heavy variable chain; (g) Fc chain of human IgG1 (CH2-3); (h) a leader peptide operably linked to an immunoglobulin kappa light variable chain; (i) a constant immunoglobulin light chain; (j) furin recognition site; (k) F2A linker; (l) polyadenylation signal and (m) AAV inverted terminal repeat.

In certain embodiments, the composition comprises a second non-replicating rAAV9 that has a vector genome that contains nucleic acid sequences encoding (a) an AAV inverted terminal repeat (ITR) (b) an enhancer; (b) chicken beta actin promoter; (c) intron; (d) 5'-UTR; (e) a leader peptide operably linked to the CR8033 heavy chain; (f) heavy variable chain CR8033; (g) Fc chain of human IgG1 (CH2-3); (h) a leader peptide operably linked to an immunoglobulin kappa light variable chain; (i) constant light chain cDNA; (j) furin recognition site; (k) F2A linker; (l) polyadenylation signal; and (m) AAV inverted terminal repeat and aqueous liquid suspension base.

In another aspect, a method is provided for protecting a human patient from influenza, which comprises administering an effective amount of an anti-influenza composition as described herein. Suitably, the patient is dosed in an amount of from about 1 x 10 ¹⁰ to about 3 x 10 ¹³ copies of the genome. In certain embodiments, the composition is administered intranasally. In other embodiments, the composition is administered intramuscularly or intravenously.

In an additional aspect, a product is provided that contains a container containing an anti-influenza composition as described herein, an optional diluent, and instructions for administration.

In another additional aspect, the invention relates to a method for preventing influenza, which includes the introduction of a vector expressing a synthetic antibody against influenza, as disclosed in the present description. Such therapy may be in combination with other vectors expressing various antibodies or other antiviral compositions.

Optionally, the method can be used as a vaccine, i. before exposure to the flu.

Other aspects and advantages of the invention are readily apparent from the following detailed description of the invention.

Brief description of the figures

In FIG. 1A, 1B are schematic representations of the components of the GTP101 vector.

In FIG. 1A is a schematic representation of mAb AAV-FI6.

In FIG. 1B is a representation of mAb AAV-CR8033.

In FIG. 2 is a flow diagram with an overview of the manufacturing process.

In FIG. 3A and 3B depict the determination of the minimum effective dose (MED) of AAV2/9.CB7.FI6 required to protect against challenge using PR8.

Fig. 3A shows the masses of mice treated intranasally (IN or in) with varying doses of AAV2/9.CB7.FI6 after challenge with 5 LD ₅₀ PR8.

Fig. 3B shows the survival of infected mice. Mice were sacrificed when they appeared to be in distress or their body weight decreased >30%.

In FIG. 4A and 4B show determination of FI6 expression in the lung (BALF) and nasal (NLF) surfaces. Mice were given IN various doses of AAV2/9.CB7.FI6 and the level of expressed antibody in the superficial fluid lining the lungs and nose, which was collected at necropsy, was assessed.

In FIG. 4A shows the results for FI6 expression in BALF.

In FIG. 4B shows the results for FI6 expression on NLF surfaces.

In FIG. 5 shows the quantitative determination of viral load in the lungs of naive mice and mice vaccinated with AAV2/9.CB7.FI6. Three mice from the unvaccinated (naive) and vaccinated AAV2/9.CB7.FI6 groups were dissected on day 6 in order to quantify

- 3 041088 determine the viral load in the lungs.

In FIG. 6A and 6B show the rapid onset of protection against influenza infection. 1x1011 k.g. (genome copies) AAV2/9.CB7.FI6 were delivered IN to BALB/c mice and 8 h later mice were challenged using

LD50PR8.

In FIG. 6A shows the weights of mice after infection on day 0.

In FIG. 6B shows the survival of infected mice. Mice were sacrificed when they appeared to be in distress or their body weight decreased >30%.

In FIG. 7A and 7B show the determination of the MED for AAV2/9.CB7.CR8O33 that is required for protection against challenge using B/Lee/40.

In FIG. 7A shows the masses of mice pre-treated with IN varying doses of AAV2/9.CB7.CR8O33 after challenge with 5 LD ₅₀ B/Lee/40.

In FIG. 7B shows the survival of infected mice. Mice were sacrificed when they appeared to be in distress or their body weight decreased >30%.

In FIG. 8 shows the effect of mixing two AAV vectors on protection against infection with influenza A (PR8). Mice were dosed or lx 109 kg. AAV2/9.CB7.FI6 (triangles) or mix 1x109 k.g. AAV2/9.CB7.FI6 and 1x109 kg AAV2/9. CB7.CR8O33 (squares). Mice were challenged 14 days later with 5 LD ₅₀ PR8 (influenza A). Mice were sacrificed when they appeared to be in distress or their body weight decreased >30%.

In FIG. 9 shows the effect of mixing two AAV vectors on protection against influenza B infection. Mice were dosed with or 1x109 kg. AAV2/9.CB7.CR8033 (triangles) or mix 1x109 kg. AAV2/9.CB7.CR8033 and 1x109 k.g. AAV2/9.CB7.FI6 (squares). Mice were challenged 14 days later with 5 LD ₅₀ B/Lee/40 (influenza B). Mice were sacrificed when they appeared to be in distress or their body weight decreased >30%.

In FIG. 10 shows the levels of circulating serum AAV2/9-specific NAbs. Mice were intranasally (i.n.) injected with the AAV2/9 vector at doses ranging from 1x109 to 1x1011 kg/mouse and analyzed for serum AAV2/9 neutralizing antibody on day 28 post AAV2/9 delivery.

In FIG. 11A-11G show vector-mediated prevention of influenza A (PR8) infection. Female BALB/c mice at 6 weeks of age (n=5/group) were given an in mixture of 1x109 kg. AAV2/9.CB7.CR8033 and 1x109 k.g. AAV2/9.CB7.FI6 in a total volume of 50 µl PBS. The vector was prepared in three different solutions: PBS-pH 6.8, PBS-pH 7.2 or PBS-pH 7.4. Mice were challenged with 5 LD ₅₀ PR8 7 days after vector injection. Animal weights were recorded daily and mice were sacrificed when they appeared to be in distress or when they lost <30% of their pre-challenge body weight. Mice were given in 1x109 kg. AAV2/9.CB7.CR8033 and 1x109 k.g. AAV2/9. CB7.FI6 in PBS-pH 6.8 (FIG. 11A), PBS-pH 7.2 (FIG. 11D or PBS-pH 7.4 (FIG. 11G), and challenged with PR8 after 7 days.

In FIG. 11A, 11D and 11G show mouse weights over time; black circles represent mice that received vector via pipette (liquid delivery), dark gray squares represent mice that received vector that was processed via IMAD, and light gray triangles represent mice that did not receive vector (naive mice) and served in as controls for PR8 infection. Naive mice were sacrificed on day 8 due to severe weight loss. At the end of the experiment (day 21), survivors were sacrificed, BALF was harvested to assess antibody expression, and lungs were processed to quantify AAV9 genomes by Taqman PCR.

In FIG. 11B, 11E and 11H show protein A ELISA antibody expression as quantified in BALF mice treated with a mixture of 1x109 k.g. each of AAV2/9.CB7.CR8O33 and AAV2/9.CB7.FI6 prepared in PBS-pH 6.8, PBS-pH 7.2 and PBS-pH 7.4, respectively. Results are shown in ng/mL for device (IMAD) and pipette control (liquid).

In FIG. 11C, 11F and 11-I are AAV9 genomes that were quantified by Taqman PCR in the lungs of mice that received a mixture of 1x109 k.g. each of AAV2/9.CB7.CR8O33 and AAV2/9.CB7.FI6 prepared in PBS-pH 6.8, PBS-pH 7.2 and PBS-pH 7.4, respectively.

In FIG. 12A-12G show vector-mediated infection prevention using PR8 and influenza B virus (B/Lee/40). Female BALB/c mice at 6 weeks of age (n=5/group) were given an in mixture of 1x109 kg. AAV2/9.CB7.CR8O33 and 1x109 k.g. AAV2/9.CB7.FI6 in a total volume of 50 µl PBS. The vector was prepared in PBS-pH 7.4. Mice were challenged with 5 LD ₅₀ PR8 (FIG. 12A) or B/Lee/40 virus (FIG. 12D) after 7 days. Animal weights were recorded daily and mice were sacrificed when they appeared to be in distress or when they had lost <30% of their pre-challenge body weight. Shown are the weights of mice challenged with PR8 (FIG. 12A) or B/Lee/40 (FIG. 12D) over time; black circles represent mice that received vector via pipette (liquid delivery), dark gray squares represent mice that received vector that was processed via IMAD, and light gray triangles represent mice that did not receive vector (naive mice) and served in as controls for PR8 infection. With the exception of one survivor,

- 4,041,088 naïve mice 4 out of 5 were sacrificed on day 8. At the end of the experiment (day 21), survivors were sacrificed, BALF was collected to assess antibody expression, and lungs were processed for quantitation

AAV9 genomes by Taqman PCR.

In FIG. 12B shows protein A ELISA antibody expression as quantified in BALF.

In FIG. 12C shows the AAV9 genomes present in the lungs, which were quantified by Taqman PCR in mice treated with a mixture of 109 k.g. each of AAV2/9.CB7.CR8033 and AAV2/9.CB7.FI6 prepared in PBS-pH 7.4 and infected with PR8.

In FIG. 12E shows BALF antibody expression as quantified by Protein A ELISA, and FIG. 12F shows the AAV9 genomes present in the lungs, which were quantified by Taqman PCR in mice treated with a mixture of 1x109 k.g. each of AAV2/9.CB7.CR8033 and AAV2/9.CB7.FI6 prepared in PBS-pH 7.4 and infected with B/Lee/40.

In FIG. 12G represents the amount of the vector suspension (0.5 ml) passed through the IMAD in order to assess the physical loss of the vector solution. As shown, a physical fluid loss of 4-6.6% of the initial volume was observed when the vector solution was passed through the IMAD.

Detailed description of the invention

Provided herein is a composition that is a two-part drug product consisting of two non-replicating recombinant serotype 9 adeno-associated viral (AAV) vectors that express recombinant antibodies that provide passive immunization against influenza A and influenza B, respectively.

The composition provided herein has several advantages over currently available influenza vaccines. Influenza A and influenza B antibody constructs co-expressed in vivo provide passive immunization against influenza A and influenza B infection. In other words, unlike a traditional vaccine that delivers influenza antigen and relies on the patient's immune system to to induce an immune response and generate antibodies, the composition provided herein delivers anti-influenza antibodies to a patient. Thus, the vaccine can be used in patients in whom the immune system is unable to generate a satisfactory protective immune response after immunization with influenza antigen. In addition, the composition provided herein may provide a more rapid onset of protection after administration than an antigen-based vaccine approach.

Other advantages include the fact that the composition can be delivered intranasally; thus, the approach is minimally invasive and is not associated with the risk of mild infection or other side effects associated with the delivery of existing attenuated virus-containing intranasal vaccines. Another advantage is that the composition can be used for patients who are allergic to eggs.

Also, the composition is not contraindicated in patients taking aspirin or medicines containing aspirin. In addition, the rAAV compositions provided herein substantially reduce the number of repeated parenteral administrations required for protein therapeutics to be effective. In certain embodiments, re-administration is only needed about 1 time per year and is limited to delivery to the epithelial cells of the nose. In accordance with yet another advantage, the compositions presented herein can be obtained in a narrower time frame than many traditional virus-based influenza vaccines. Thus, the composition according to the invention relates to a more practical way to protect the population at risk.

In certain embodiments, the vectors provided herein express effective levels of functional antibody when delivered intranasally at a dose of about 3 ml or less, 2 ml or less, or 1 ml or less, 0.5 ml, or less, such as in the range of about from 100 to 250 µl. In general, the compositions are prepared at a pH of about 5.5 to about 8.5, 6 to 8, or 6.5 to 7.5, or 6.8, 7.2, or 7.4. Thus, the vectors provided herein are highly effective in presenting therapeutic levels of antibody at doses that are convenient for metered doses, or in products or kits containing pre-metered doses. In certain embodiments, the compositions are formulated for intranasal delivery such that the volume and size of the spray droplet is preferably directed to intranasal epithelial cells. Expression in the nasal epithelium has been shown to confer protective passive immunity. The compositions can also be formulated for intranasal delivery that is directed to the lungs. Targeting to the lungs can be in addition to, or preferable to, the nasal epithelium, for example, by adjusting the volume and/or size of the intranasally delivered droplet. Compared to the standard influenza vaccine delivered intramuscularly, AAV delivered intranasally leads to a faster onset of Ab expression (within hours or a couple of days). However, in certain embodiments, the compositions described herein may be formulated for delivery via other routes, such as intramuscular injection, intravenous injection, or other suitable routes. In such cases, suitable formulations and volumes can be determined by one skilled in the art.

Antibody constructs.

In certain embodiments, a composition provided herein delivers two influenza antibodies that are expressed in vivo in a cell through a non-replicating viral vector and secreted from it to confer protective immunity. As illustrated in the examples herein, the viral vector is an rAAV and the composition contains two different rAAV stock solutions, each containing a different vector genome. In the first stock solution, the vector genome contains the coding sequences for the influenza A antibody. In the second stock solution, the vector genome contains the coding sequences for the influenza B antibody.

The influenza A antibody is a synthetic FI6 construct. See, for example, US Patent Publication No. 2010/0080813 for the amino acid sequences of the heavy chain, light chain variable chain, variable regions, and complementarity-determining regions of FI6v3. See also U.S. Patent 8,124,092. In certain embodiments, an F16v3 expression cassette is constructed that encodes F16 FI6 light variable regions (Genbank PDB accession numbers: AEL31310.1, GI: 342674599 and 342674581) linked to constants (CH1, CH2, and CH3) human IgG1 domains, and germline kappa light chain sequences linked to the human CL domain, creating a monoclonal antibody architecture. In another embodiment, the FI6 expression cassette contains a full-length FI6v3 heavy chain (see n. 2440-2760 (CH1), n. 27613426 (CH2-CH3) in SEQ ID NO: 1). cDNA encoding an exemplary light chain is provided in n. 3511-3909 in SEQ ID NO: 1. (leader n. 3511-3510, kappa chain n. 3571-3909) In another embodiment, the FI6 synthetic construct comprises at least the FI6v3 heavy chain variable region, CH1 heavy chain constant regions, CH2 and CH3) and an immunoglobulin light chain.

In certain embodiments, a nucleic acid sequence encoding the immunoglobulin domains that make up an influenza A antibody is designed for expression in human cells. Such sequences may include, for example, the FI6v3 heavy chain variable region (n. 2053-2439 in SEQ ID NO: 1) and CH2 and CH3 constant regions (n. 2761-3426 in SEQ ID NO: 1). However, other coding sequences for the F16v3 amino acid sequences provided herein can be designed and are within the scope of this invention. See, for example, other heavy chain variable region encoding sequences (e.g., amino acids from SEQ ID NO: 2), CH1 (e.g., amino acids from SEQ ID NO: 3), and/or CH2-CH3 (e.g., amino acids from SEQ ID NO: 3). NO: 4). See also SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23.

In certain embodiments, the variable light chain is a kappa chain derived from a germline source. For example, in the examples provided herein, the lcl|IGKV4-1*01 [Homo sapiens] germline gene is used (see, for example, N. 35713909 in SEQ ID NO: 1 or N. 3265-3606 in SEQ ID NO : 8). Optionally, another nucleic acid sequence may be selected encoding the kappa light chain amino acid sequence (SEQ ID NO: 5) and/or the light chain constant (SEQ ID NO: 7). In another alternative, another suitable germline sequence may be selected. In another embodiment, the light chain is a lambda chain. Preferably, a germline sequence that does not alter the antigenic specificity of the heavy chain partner is selected as the source of the light chain. Sources of such germline immunoglobulin sequences are provided, for example, in the Kabat database, www.ncbi.nlm.nih.gov/ and http://www.imgt.org/genedb/table?mode=3d&selectGenes=IGKV4-l&selectSpecies=Homo% 20sapiens.

The influenza B antibody is a synthetic antibody CR8033. See, for example, US Pat. No. 8,852,595 for the amino acid sequences of the CR8033 heavy chain, light chain, variable regions, and complementarity determining regions. The antibody has a heavy chain which is an engineered sequence derived from the influenza antibody CR8033. In one embodiment, the engineered antibody provided herein comprises a codon optimized variable heavy chain (e.g., n. 1753-2133 in SEQ ID NO: 8), CH1 (e.g., n. 2134-2454 in SEQ ID NO: 8) and CH2-CH3 (eg n. 2455-3120 in SEQ ID NO: 8). In certain embodiments, the CH1 region may not be used. In other embodiments, different nucleic acid sequences can be selected encoding a heavy chain variable region (e.g., SEQ ID NO: 9), CH1 (e.g., SEQ ID NO: 10) and/or CH2-3 (e.g., SEQ ID NO: 11 ).

In other embodiments, light chains can be derived from an F16 antibody. See, for example, Corti et al., Science, 2011 Aug 12; 333(6044):860-6, Epub 2011 Jul 28.

In certain embodiments, the synthetic F16 antibody construct provided herein can be expressed in vitro and used, for example, in a protein

- 6 041088 therapy or to create anti-idiotypic antibodies. This antibody construct consists of at least a combination of an F16 heavy chain with a light chain from a germline source.

In other embodiments, the CR8033 synthetic antibody construct provided herein can be expressed in vitro and used, for example, in protein therapy or to generate anti-idiotypic antibodies. This antibody construct consists of at least the CR8033 heavy chain in combination with a light chain from a germline source.

Such expression methods and uses are known in the art.

Vector genomes.

In order to express a selected immunoglobulin domain, a nucleic acid molecule can be designed that contains codons that are selected for optimal expression of immunoglobulin polypeptides in a selected mammalian species, such as a human. In addition, the nucleic acid molecule may contain a heterologous leader sequence for each heavy chain and light chain of the selected antibody, which encodes an IL-2 signal leader peptide fused upstream from heavy and chain polypeptides consisting of variable and constant regions. However, another heterologous leader sequence may be used in place of one or both of the IL-2 signal/leader peptides. The signal/leader peptides may be the same or different for each heavy chain and light chain of an immunoglobulin construct. They may be signal sequences that occur natively in an immunoglobulin (eg, IgG) or may be from a heterologous source. Such heterologous sources may be secretory signal peptides of a cytokine (e.g., IL-2, IL-12, IL-18, or the like), insulin, albumin, β-glucuronidase, alkaline protease, or fibronectin, or sequences from tissue-specific secreted proteins, among other things.

As used herein, an expression cassette refers to a nucleic acid sequence that contains at least a first open reading frame (ORF) and optionally a second ORF. The ORF may contain two, three or four antibody domains. For example, an ORF may contain a full length heavy chain. Alternatively, the ORF may contain one or two antibody domains. For example, an ORF may comprise a heavy chain variable domain and one heavy chain constant domain. In another example, the ORF may comprise a light chain variable region and a light chain constant region. Thus, the expression cassette can be designed as a bicistronic, i.e. containing regulatory sequences that control the expression of open reading frames in it from common regulatory sequences. In this case, the two ORFs are usually separated by a linker. Suitable linkers, such as an internal ribozyme binding site (IRES) and/or a furin-2a (F2a) self-cleaving peptide linker [see e.g. Radcliffe and Mitrophanous, Gene Therapy (2004), 11, 1673-1674], are known in the art. areas. Suitably, the ORF is operably linked to regulatory control sequences that control expression in the target cell. Such regulatory control sequences may include poly-A, a promoter and an enhancer. In order to promote co-expression with the AAV vector, the first and second expression cassettes may share at least one enhancer and/or poly-A sequence.

In one embodiment, the rAAV is packaged in a selected AAV capsid, the nucleic acid molecule contains an expression cassette containing: an AAV 5' inverted terminal repeat (ITR) sequence, a promoter, a 5'-UTR an optional Kozak sequence, a first signal peptide operably linked to the first an immunoglobulin chain containing a heavy chain, a linker sequence, a second signal peptide operably linked to a second immunoglobulin chain, and a 3' AAV ITR, where one of and the second immunoglobulin are an immunoglobulin light chain, where said expression cassette co-expresses immunoglobulin chains in a cell- host under conditions that allow the chains to assemble into a functional antibody construct having antibody specificity, given the heavy chain. In one embodiment, the heavy chain is a synthetic influenza immunoglobulin FI6v3 heavy chain and the light chain is a germline kappa light chain.

In one embodiment, the expression cassette contains an AAV ITR from a source other than the AAV capsid to form a pseudotyped AAV. In one embodiment, the expression cassette further comprises a constitutive promoter and RGB poly-A. Other suitable vector elements may be selected, such as promoters and poly-A sequences. For example, a minimal promoter and/or a minimal poly-A can be chosen to save space. Typically, in this embodiment, each promoter is adjacent (left or right (or 5' or 3')) to the enhancer sequence and the poly-A sequences are adjacent to the ITR, with an ORF in between. While it is preferred to express the heavy chain sequences first, the order of the ORFs can be varied as the immunoglobulin domains encoded by them allow. For example, light chain constant and variable sequences

- 7 041088 can be located to the left of the enhancer, and the heavy chain can be encoded by ORFs located to the right of the enhancer. Alternatively, the heavy chain can be placed to the left of the enhancer and the ORF to the right of the enhancer to encode the light chain. Alternatively, the opposite configuration is possible.

In another embodiment, the rAAV is packaged in a selected AAV capsid, the nucleic acid molecule contains an AAV 5' inverted terminal repeat (ITR) sequence, a promoter, a 5'-UTR, an optional Kozak sequence, an IL-2 signal peptide operably linked to an FI6 immunoglobulin heavy chain , F2a, an IL-2 signal peptide operably linked to the germline kappa light chain, and 3' AAV ITR. In one embodiment, the AAV capsid is AAV9 or AAV8. In a further embodiment, the ITR is from AAV2 or another source that differs from the source of the AAV capsid.

Suitable regulatory escape sequences can be selected and obtained from various sources. In one embodiment, minimal promoter and/or minimal poly-A can be used for size retention.

As used herein, the term minimal promoter refers to a short DNA sequence consisting of a TATA box and other sequences that serve to accurately indicate the site of transcription initiation to which regulatory elements are added to control expression. In one embodiment, a promoter refers to a nucleotide sequence that contains a minimal promoter plus regulatory elements that are capable of directing the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal elements upstream, the latter elements often referred to as enhancers. In one embodiment, the minimal promoter is the cytomegalovirus (CMV) minimal promoter. In another embodiment, the minimal promoter is derived from human CMV (hCMV), e.g., the minimal promoter derived from the hCMV immediate early promoter (see US 20140127749 and Gossen and Bujard (Proc. Natl. Acad. Sci. USA, 1992, 89:5547- 5551), which are incorporated herein by reference). In another embodiment, the minimal promoter is derived from a viral source, such as, for example, SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoters, or Rous sarcoma virus (RSV) early promoters; or from eukaryotic cell promoters, e.g. beta-actin promoter (Ng, Nuc. Acid Res. 17:601-615, 1989; Quitsche et al., J. Biol. Chem. 264:9539-9545, 1989), GADPH promoter ( Alexander, M. C. et al., Proc. Nat. Acad. Sci. USA, 85:5092-5096, 1988, Ercolani, L. et al., J. Biol. Chem. 263:15335-15341, 1988), TK promoter -1 (thymidine kinase), HSP (heat shock protein) promoters, UbB or UbC promoter, PGK, Ef1-a promoter or any eukaryotic promoter containing a TATA box (US Published Application No. 2014/0094392). In another embodiment, the minimal promoter contains a mini-promoter, such as the CLDN5 mini-promoter described in US Published Application No. 2014/0065666. In another embodiment, the minimal promoter is a thymidine kinase (TK) promoter. In one embodiment, the minimal promoter is tissue-specific, such as one of the muscle cell specific promoters, the TnISlow minimal promoter, the TnIFast minimal promoter, or the muscle creatine kinase promoter (US Published Application No. 2012/0282695). Each of these documents is incorporated herein by reference.

A recombinant AAV vector (AAV virus particle) may contain an AAV capsid-packaged nucleic acid molecule expressing a functional antibody as described herein. The expression cassette may contain regulatory elements for the open reading frame(s) in each expression cassette, and the nucleic acid molecule may optionally contain additional regulatory elements.

The AAV vector may contain a full length 5' inverted terminal repeat (ITR) and a full length 3'-ITR AAV. A truncated version of the 5'-ITR, referred to as AITR, is described in which the D-sequence and the terminal clearance site (trs) are deleted. The abbreviation sc refers to self-complementary. Self-complementary AAV refers to a construct in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, instead of waiting for cell-mediated second-strand synthesis, the two complementary halves of scAAV combine to form a single double-stranded DNA (dsDNA) block that is ready for immediate replication and transcription. See, for example, D.M. McCarty et al., Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis, Gene Therapy, (August 2001), vol. Nov. 8, 16, p. 1248-1254. Self-complementary AAVs are described, for example, in US patent No. 6596535; 7125717 and 7456683, each of which is incorporated into the present description by reference in its entirety.

When a pseudotyped AAV is to be obtained, the ITR is selected from a source that is different from that of the AAV capsid. For example, an AAV2 ITR can be selected for use with an AAV capsid having a specific potency for a selected cellular receptor target tissue.

- 8 041088 or a viral target. In one embodiment, the ITR sequences from AAV2, or a deletion version thereof (AITR), are used for convenience and expedite regulatory approval.

However, you can select ITR from other AAV sources. When the source of the ITR is AAV2 and the source of the AAV capsid is another AAV, the resulting vector can be called pseudotyped. However, other ITR AAV sources may be used.

Various AAV capsids have been described. Methods for creating AAV vectors are extensively described in the literature and patent documents, including, for example, WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2. AAV capsid sources can be selected from AAVs that target the desired tissue. For example, suitable AAVs may include, for example, AAV9 [US 7906111; US 2011|0236353A1], rh10 [WO 2003/042397] and/or hu37 [see, for example, US 7906111; US 2011|0236353-A1]. However, other AAVs include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 [US Pat. No. 7,790,449; 7282199] and others. However, other sources of AAV capsids and other viral elements can be selected, as other immunoglobulin constructs and other vector elements allow.

Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, for example, US Pat. Nos. 7,790,449; 7282199; W02003/042397; WO 2005/033321, WO 2006/110689 and US 7588772 B2]. In one system, a producer cell line is transiently transfected with a construct that encodes an ITR-flanked transgene and construct(s) that encodes rep and cap. In a second system, a packaging cell line that stably delivers rep and cap is transiently transfected with a construct encoding a transgene flanked by an ITR. In each of these AAV systems, virions are produced in response to infection with a helper adenovirus or herpesvirus, requiring separation of the rAAV from the contaminating virus. More recently, systems have been developed that do not require infection with a helper virus in order to extract the necessary AAV helper functions (i.e., El, E2a, VA, and E4 adenovirus or UL5, UL8, UL52, and UL29 herpesvirus and herpesvirus polymerases), which the system also supplies, in trance. In these newer systems, helper functions can be provided by transient transfection of cells with constructs that encode the desired helper functions, or cells can be engineered to stably contain genes encoding helper functions whose expression can be directed at the transcriptional or post-transcriptional level. In yet another system, the ITR-flanked transgene and the rep/cap genes are introduced into insect cells via infection with baculovirus vectors. For reviews of these production systems, see, in general, for example, Zhang et al., 2009, Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production, Human Gene Therapy, 20:922-929, which included in the present description by reference in its entirety. Methods for making and using these and other AAV production systems are also described in the following US patents, the contents of which are incorporated herein by reference in their entirety: 5,139,941; 5741683; 6057152; 6204059; 6268213; 6491907; 6660514; 6951753; 7094604; 7172893;7201898;7229823 and 7439065.

In one embodiment, the polyadenylation signal (poly(A)) is the minimum poly(A) signal, i. the minimum sequence required for efficient polyadenylation. In one embodiment, the minimal poly(A) is a synthetic poly(A), such as that described in Levitt et al, Genes Dev., 1989 Jul, 3(7):1019-25 and Xia et al., Nat. Biotechnol. 2002 Oct; 20(10):1006-10. Epub 2002 Sep 16. In another embodiment, the poly(A) is derived from rabbit poly(A) beta-globin. In one embodiment, poly-A acts bidirectionally (An et al., 2006, PNAS, 103(49):18662-18667. In one embodiment, poly(A) is derived from an early SV40 poly-A signal sequence. Each of of these documents is incorporated into the present description by reference.

Optionally, a single enhancer or the same enhancer may regulate the transcription of multiple heterologous genes in a plasmid construct. Various enhancers suitable for use in the invention are known in the art and include, for example, the early CMV enhancer, the Hoxc8 enhancer, nPE1 and nPE2. Additional enhancers that can be used in the present description are described in Andersson et al., Nature, March 2014, 507(7493):455-61, incorporated herein by reference. Other enhancer elements may include, for example, an apolipoprotein enhancer, a zebrafish enhancer, a GFAP enhancer element, and tissue-specific enhancers such as described in WO 2013/1555222, the woodchuck post-hepatitis post-transcriptional regulatory element. Additionally or alternatively, another, eg, human cytomegalovirus (HCMV) hybrid immediate early (IE)-PDGR promoter, or other promoter-enhancer elements can be selected. To enhance expression, other elements may be introns (such as the promega intron or a chimeric intron from chicken globin and human immunoglobulin). Other enhancers that can be used in the present description can be found in the Mammalian Promoter/Enhancer Database, which is located at http://promoter.cdb.riken.jp/.

The constructs described herein may additionally contain other expression control or regulatory sequences, for example including suitable transcription initiation, termination, promoter and enhancer sequences; signals for efficient RNA processing, such as splicing and polyadenylation (poly-A) signals; sequences that stabilize cytoplasmic mRNA; sequences that increase the efficiency of translation (i.e., the Kozak consensus sequence; sequences that increase protein stability; and, when desired, sequences that enhance the secretion of the encoded product. In the demonstration examples below, the Kozak sequence used is CCACCATG (n (1988), (1992) in SEQ ID NO: 1); however, other suitable sequences may be selected.The promoter may be selected from a constitutive promoter, a tissue-specific promoter, a specific cell-specific promoter, a promoter that responds to physiological signals, or a regulated promoter [see ., for example, WO 2011/126868 and WO 2013/049492].

These control sequences are operably linked to the gene sequences of the immunoglobulin constructs. As used herein, the term operably linked refers to both expression control sequences that run contiguous with the gene of interest and expression control sequences that act in trans or at a distance to drive the gene of interest.

Examples of constitutive promoters suitable for driving the expression of antibody domains include, but are not limited to, promoters of beta-actin of chicken (CB) or beta-actin of other species, the human cytomegalovirus (CMV) promoter, early and late promoters of simian virus 40 (SV40) , U6 promoter, metallothionein promoters, EFla promoter, ubiquitin promoter, hypoxanthine phosphoribosyltransferase (HPRT) promoter, dihydrofolate reductase (DHFR) promoter (Scharfmann et al., Proc. Natl. Acad. Sci. USA, 88:4626-4630 (1991), promoter adenosine deaminase, phosphoglycerine kinase (PGK) promoter, pyruvate kinase promoter, phosphoglycerol mutase promoter, β-actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA, 86:10006-10010 (1989), UbB, UbC, long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, herpes simplex virus thymidine kinase promoter, and other constitutive promoters known to those skilled in the art. ok suitable for use in the present invention include, but are not limited to, endothelin-I (ET-I) and Flt-I, which are specific to endothelial cells, FoxJ1 (which targets ciliated cells).

Although less desirable, inducible promoters suitable for directing the expression of antibody domains can be used, which include promoters that are sensitive to exogenous agents (eg, pharmacological agents) or physiological signals. These sensing elements include, but are not limited to, a hypoxia sensing element (HRE) that binds HIF-Ia and β, a metal ion sensing element such as described in Mayo et al. (1982 Cell 29:99-108); Brinster et al. (1982, Nature, 296:39-42) and Searle et al. (1985, Mol. Cell. Biol. 5:1480-1489); or a thermal shock sensitive element such as described in Nouer et al. (in Heat Shock Response, eds. Nouer, L., CRC, Boca Raton, Fla., p. 167-220, 1991).

In one embodiment, open reading frame expression is driven by a regulatable promoter that provides tight control of ORF (gene) transcription, such as a pharmacological agent or transcription factors activated by a pharmacological agent or, in alternative embodiments, physiological signals. Examples of regulatable promoters that are ligand-dependent transcription factor complexes that can be used include, but are not limited to, members of the nuclear receptor family activated by their respective ligands (e.g., glucocorticoid, estrogen, progestin, retinoid, ecdysone, and their analogs and mimetics) , and rTTA activated by tetracycline. Examples of such systems include, without limitation, ARGENT™ Transcriptional Technology (ARIAD Pharmaceuticals, Cambridge, Mass.). Examples of such promoter systems are described, for example, in WO 2012/145572, incorporated herein by reference. In other embodiments, RNA-based small switches are described at http://www.ncbi.nlm.nih.gov/pubmed/25605380.

Other promoters may include, for example, human cytomegalovirus (CMV) immediate early enhancer/promoter, SV40 early enhancer/promoter, polymovirus JC promoter, myelin basic protein (MBP) or gliofibrillary acidic protein (GFAP) promoters, latency associated promoter (LAP) of the virus herpes simplex (HSV-1), Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, specific neuron promoter (NSE), platelet-derived growth factor (PDGF) promoter, hSYN, melanin-concentrating hormone (MCH) promoter, CBA, gliofibrillary acidic protein (GFAP) promoter, matrix metalloprotein (MPP) promoter, and chicken beta-actin promoter. The promoters may be the same or different for each expression cassette.

In certain embodiments, each of the vector genomes comprises an immunoglobulin expression cassette flanked by inverted terminal repeats (ITRs). The expression cassettes contain coding sequences driven by the CB7 promoter, a fusion between the cytomegalovirus (CMV) immediate early enhancer (C4) and the chicken beta actin promoter. Transcription from this promoter is enhanced by the presence of the chicken beta-actin intron (CI).

- 10 041088

The 5' untranslated region (UTR) of the human c-myc gene is located upstream of the mAb coding region as a translation enhancer. The polyadenylation signal for the expression cassette is poly-A rabbit beta globin (RBG). Leader (signal) peptides precede both the heavy and light chains of the antibody and mediate the secretion of the antibody from the transduced cell. The heavy and light chains are separated by a linker containing a furin cleavage site and the FMDV 2A sequence. This linker mediates post-translational cleavage of the heavy and light chains and, with the exception of one amino acid change (the terminal lysine in the heavy chain is replaced by an arginine residue), is completely removed from the final mAb product.

In one embodiment, the vector contains a constitutive promoter. In another embodiment, the 5'-UTR is a truncated UTR from the human c-myc gene. The linker sequence may be F2A or IRES. The heavy signal peptide and the light signal peptide may be the same or different. In one embodiment, the leader sequence is an IL-2 interleukin (IL) leader, which may be wild-type IL-2 (MYRMQLLSCIALSLALVTNS, SEQ ID NO: 24) as used in the FI6v3 heavy chain, or a synthetic leader sequence , such as the mutated IL-2 used in the germline kappa light chain (MYRMQLLLIALSLALVTNS, SEQ ID NO: 5), or the mutated IL-2 leader used in the CR8033 heavy chain (MRMQLLLIALSLALVTNS, SEQ ID NO: 25). Other leader sequences may be used, including innate antibody signal peptides or other heterologous leader sequences. In one embodiment, wild-type poly-A or synthetic poly-A can be selected. As used herein, a vector can be any suitable genetic element that transfects, transduces, or infects a host cell and expresses immunoglobulins that assemble into a functional antibody. Such vectors may be selected from a lentiviral vector, a baculovirus vector, a parvovirus vector, a plasmid, a modified RNA, and a DNA molecule, where the mRNA and DNA may be in the form of nanoparticles.

In one embodiment, the vector genome for an AAV vector carrying an influenza A antibody contains the following.

Inverted Terminal Repeats (ITRs): AAV2 ITRs (GenBank No. NC001401) represent only the cis sequences required for vector genome replication and packaging. An AAV that has an ITR from a source other than its capsid is called pseudotyped. In certain embodiments, an ITR from a source other than AAV2 may be selected for this construct to generate another pseudo-typed AAV. Alternatively, you can choose the ITR of the same source as the capsid.

Cytomegalovirus (CMV) immediate early enhancer (260 bp, C4; GenBank No. K03104.1). In another embodiment, you can choose a different enhancer. See discussion of enhancers below.

Chicken beta actin promoter (281 bp; CB; GenBank No. X00182.1). In certain embodiments, a different promoter may be selected. See discussion of promoters below. In other embodiments, a plurality of enhancers and/or promoters may be included.

Chicken beta-actin intron: intron 875 b.p. from the chicken beta actin gene (GenBank No. X00182.1) is present in the expression cassette of the vector.

c-myc 5'-UTR: nucleotides 381-428 from mRNA homologue of avian myelocytomatosis viral oncogene vmyc Homo sapiens (MYC) GenBank accession number NM002467).

cDNA leader (signal) peptide: cDNA sequence with codon optimization (http://www.uniprot.org/uniprot/P60568) preceding and in-frame with the FI6 heavy chain. FI6v3 heavy variable chain cDNA: human codon optimized cDNA sequence encoding the influenza human heavy chain variable region (GenBank accession number AEL31303.1).

Constant Heavy 1 (CH1) Chain: A codon-optimized cDNA coding for the human immunoglobulin heavy chain protein sequence, GenBank accession number BAF64540.1, was synthesized.

FI6 Fc chain (CH2-3): codon-optimized cDNA encoding part of human immunoglobulin GenBank accession number 2J6E_A, except for the C-terminal lysine, which was substituted for arginine, and position 57, where aspartic acid was substituted for glutamic acid).

cDNA leader (signal) peptide: synthesized cDNA sequence with optimization of codons, which precedes the universal light chain (ILhy1 described in [Zhang et al, cited above).

Universal Light Chain cDNA: A human cDNA sequence was synthesized with codon optimization (GeBank accession number ACJ71709.1).

constant light chain cDNA: synthesized human codon sequence GenBank accession number AGH70219.1).

Furin recognition site: arginine-lysine-arginine-arginine and cDNA were synthesized with codon optimization.

F2A linker: a 24 amino acid peptide derived from FMDV (GenBank No.

CAA2436.1), and cDNA with codon optimization.

Polyadenylation signal: rabbit beta-globin polyadenylation signal 127 bp. (GenBank

no. V00882.1) provides cis sequences for efficient polyadenylation of the antibody mRNA.

In certain embodiments, the vector genome for an AAV vector carrying an influenza B antibody contains inverted terminal repeats (ITRs): AAV2 ITR; immediate early enhancer of cytomegalovirus (CMV); chicken beta actin promoter; chicken beta-actin intron; truncated 5' UTR from cmyc; cDNA of the leader (signal) peptide; heavy variable chain cDNA CR8033; constant heavy 1 (CH1) chain: cDNA with codon optimization; Fc chain CR8033 (CH2-3); cDNA of the leader (signal) peptide; cDNA universal light chain; constant light chain cDNA; furin recognition site; linker F2A; polyadenylation signal.

In certain embodiments, the influenza A and/or influenza B vector elements may vary. For example, a vector genome as illustrated above, but without the AAV ITR, can be used in another expression system (eg, baculovirus, lentiviral, plasmid, deproteinized DNA). Other suitable vector elements, including, for example, promoters, enhancers, linkers, poly-A, introns, for use in an AAV or non-AAV expression system are described herein.

The two or more ORFs carried by a nucleic acid molecule packaged in a vector can be expressed from two expression cassettes, one or both of which can be bicistronic.

Coding sequences for the selected immunoglobulin (eg, heavy and/or light chain(s)) or other elements (eg, leader sequences) can be generated and/or synthesized. Nucleic acid (eg, RNA and DNA) sequencing methods are known to those skilled in the art. Once a nucleic acid sequence is known, there are web-based and commercially available computer programs and service companies that reverse-translate amino acid sequences into nucleic acid coding sequences. See, for example, backtranseq from EMBOSS, http://www.ebi.ac.uk/Tools/st/; Gene Infinity (http://www.geneinfinity.org/sms/sms_backtranslation.html); ExPasy (http://www.expasy.org/tools/). In one embodiment, RNA and/or cDNA coding sequences are designed for optimal expression in human cells. Methods for synthesizing nucleic acids are known to those skilled in the art and can be used for whole or partial nucleic acid constructs described herein.

Codon optimization coding regions can be designed in many different ways. This optimization can be done using methods that are available online (eg GeneArt), published methods, or a company that provides codon optimization services such as DNA2.0 (Menlo Park, CA) for example. One codon optimization algorithm is described, for example, in US Patent Application No. WO 2015/012924, incorporated herein by reference. See also, for example, US Patent Publication No. 2014/0032186 and US Patent Publication No. 2006/0136184. The entire length of the open reading frame (ORF) is modified appropriately for the product. However, in some embodiments, only a fragment of the ORF may be modified. Using one of these methods, it is possible to apply frequencies to any given polypeptide sequence and obtain a nucleic acid fragment of a codon-optimized coding region that codes for the polypeptide.

For use in the production of an AAV viral vector (eg, recombinant (r) AAV), expression cassettes may carry any suitable vector, eg, a plasmid, that is delivered to a packaging host cell. Plasmids that can be used in this invention can be designed to be suitable for replication and packaging in prokaryotic cells, mammalian cells, or both. Suitable transfection methods and packaging host cells are known and/or can be easily developed by one of skill in the art.

Methods for generating and isolating AAVs suitable for use as vectors are known in the art. See, in general, for example, Grieger & Samulski, 2005, Adenoassociated virus as a gene therapy vector: Vector development, production and clinical applications, Adv. Biochem. Engin/Biotechnol. 99:119-145; Buning et al., 2008, Recent developments in adenoassociated virus vector technology, J. Gene Med. 10:717-733; and sources cited below, each of which is incorporated into the present description by reference in its entirety. For transgene packaging into virions, ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the expression cassettes. The cap and rep genes can be represented in trans.

In one embodiment, the expression cassettes described herein are constructed in a genetic element (eg, a shuttle plasmid) that carries the sequences of the immunoglobulin constructs it carries into a packaging host cell to produce a viral vector. In one embodiment, the selected genetic element can be delivered to an AAV packaging cell by any suitable method, including transfection,

- 12 041088 electroporation, liposome delivery, membrane fusion methods, high speed DNA coated beads, viral infection and protoplast fusion. It is also possible to obtain stable packaging AAV cells. Alternatively, expression cassettes can be used to generate a non-AAV viral vector or to generate in vitro antibody mixtures. Methods used to obtain such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic methods. See, for example, Molecular Cloning: Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

AAV vectors.

As used herein, AAV9 capsid refers to AAV9 having the amino acid sequence GenBank Accession Number AAS99264, incorporated herein by reference. Some variation in this encoded sequence is within the scope of this invention and may include sequences having approximately 99% identity with the specified amino acid sequence in GenBank accession number AAS99264 and in US7906111 (also WO 2005/033321) (i.e. less than about 1% variation regarding the mentioned sequence). However, in other embodiments, other AAV9 or AAV9 capsid variants having at least about 95% identity with the above sequences can be selected. Methods for creating a capsid, its coding sequences, and methods for obtaining rAAV viral vectors are described. See, for example, Gao, et al., Proc. Natl. Acad. sci. U.S.A. 100 (10), 6081-6086 (2003) and US 2013/0045186 A1.

The term AAV9 intermediate or AAV9 vector intermediate refers to an assembled rAAV capsid that does not have the desired genomic sequences packaged into it. It can also be called an empty capsid. Such a capsid may not contain detectable genomic sequences of the expression cassette, or contain only partially packaged genomic sequences that are insufficient to achieve expression of the gene product. These empty capsids are not functional to transfer the gene of interest into the host cell.

The recombinant adeno-associated virus (AAV) described herein can be created using methods that are known. See, for example, WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2. Such a method includes culturing a host cell that contains a nucleic acid sequence encoding an AAV capsid; functional rep gene; an expression cassette consisting of at least AAV inverted terminal repeats (ITR) and a transgene; and sufficient helper functions to allow packaging of the expression cassette into an AAV capsid protein.

Briefly, cells are obtained in suitable cell culture cells (eg, HEK293). Methods for making the gene therapy vectors described herein include methods well known in the art, such as generating a DNA plasmid used to generate gene therapy vectors, generating vectors, and purifying vectors. In some embodiments, the gene therapy vector is an AAV vector and the generated plasmids are an AAV cis plasmid encoding the AAV genome and a gene of interest, an AAV trans plasmid containing the AAV rep and cap genes, and an adenoviral helper plasmid. The vector generation process may include method steps such as initiating a cell culture, passaging the cells, seeding the cells, transfecting the cells with plasmid DNA, changing the medium after transfection to serum-free medium, and collecting the vector-containing cells and culture media. The harvested vector-containing cells and culture media are referred to herein as crude cell harvest.

The crude cell harvest can then be subjected to method steps such as vector harvest concentration, vector harvest diafiltration, vector harvest microfluidization, vector harvest nuclease digestion, microfluidized intermediate filtration, chromatography coarse purification, ultracentrifugation coarse purification, buffer exchange by tangential flow filtration and/or cooking and filtering in order to get a large amount of vector.

Two-step high salt affinity chromatography purification followed by anion exchange resin chromatography is used to purify the vector drug product and remove empty capsids. These methods are described in more detail in U.S. Patent Application No. 62/322,071, filed April 13, 2016, and U.S. Patent Application No. 62/226,357, filed December 11, 2015, entitled Scalable Purification Method for AAV9, which are incorporated herein by links. Purification methods for AAV8 are given in US Patent Application No. 62/322,098, filed April 13, 2016, and US Patent Application No. 62/266,341, filed December 11, 2015, and rh10, US Patent Application No. 62/322,055, filed April 13 2016, and US Patent Application No. 62/266,347 entitled Scalable Purification Method for AAVrh10, filed December 11, 2015, and for AAV1, US Patent Application No. 62/322,083, filed April 13, 2016, and 13 041088 U.S. Patent Application No. 62/26,351, Scalable Purification Method for AAV1, filed December 11, 2015, all of which are incorporated herein by reference.

In order to calculate the content of empty and full particles, plot the volumes of the VP3 bands for a selected sample (for example, in the examples in the present description of the drug purified in the iodixanol gradient, where the number of k.g. = the number of particles) from the loaded k.g. particles. The resulting linear equation (y=mx+c) is used to calculate the number of particles in the band volumes for the test product peaks. The number of particles (p.c.) loaded in 20 µl is then multiplied by 50 to obtain particles (p.c.)/ml. Ch-c/ml divided by kg/ml gives the ratio of particles and copies of the genome (p-c/kg). Ch-c./ml-k. g/ml gives empty p.c./ml. Empty p.c./ml divided by p.c./ml and multiplied by 100 gives the percentage of empty particles.

In general, methods for analyzing empty capsids and AAV vector particles with packaged genomes are known in the art. See, for example, Grimm et al., Gene Therapy (1999), 6:1322-1330; Sommer et al., Molec. Ther. (2003), 7:122-128. In order to test the denatured capsid, the methods include SDS electrophoresis of the treated AAV stock solution in a polyacrylamide gel, which consists of any gel capable of separating the three capsid proteins, for example, in a gradient gel containing 3-8% Tris-acetate in buffer where the gel is run to separate the sample material and transfer the gel to nylon or nitrocellulose membranes, preferably nylon. Anti-AAV capsid antibodies are then used as primary antibodies that bind to denatured capsid proteins, preferably an anti-AAV capsid monoclonal antibody, most preferably an anti-AAV-2 monoclonal antibody B1 (Wobus et al., J. Virol. (2000), 74: 9281-9293). A secondary antibody is then used that binds to the primary antibody and contains an agent for detecting binding to the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently linked to it, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A binding detection method is used to semi-quantify binding between primary and secondary antibodies, preferably the detection method is capable of detecting radioactive isotope radiation, electromagnetic radiation or colorimetric changes, most preferably a chemiluminescence detection kit. For example, for SDS-PAGE, samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing a reducing agent (eg, DTT) and capsid proteins are resolved on precast gradient polyacrylamide gels (eg, Novex). Silver staining can be done using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions or other suitable staining method, ie SYPRO Ruby or Coomassie staining. In one embodiment, the concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real-time PCR (Q-PCR). Samples are diluted and digested with DNase I (or other suitable nuclease) in order to remove exogenous DNA. After nuclease inactivation, samples are further diluted and amplified using primers and TaqMan™ fluorogenic probe with DNA sequence specificity between the primers. The number of cycles required to reach a certain level of fluorescence (cycle threshold, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing identical sequences to that contained in the AAV vector is used to create a calibration curve in the Q-PCR reaction. The cycle threshold (Ct) values obtained from the samples are used to determine the vector genome titer by normalizing them to the Ct value for the plasmid calibration curve. An endpoint analysis based on digital PCR can also be used.

In one aspect, an optimized Q-PCR method is used that uses a broad spectrum serine protease, eg proteinase K (eg, commercially available from Qiagen). More specifically, the optimized qPCR genome titer assay is similar to the standard assay, except that after digestion with DNase I, samples are diluted with proteinase K buffer and treated with proteinase K, followed by heat inactivation. Suitably, the samples are diluted with proteinase K buffer in an amount equal to the size of the sample. Proteinase K buffer can be concentrated up to 2 times or more.

Typically, proteinase K treatment is about 0.2 mg/ml, but may vary from 0.1 to about 1 mg/ml. The processing step is generally carried out at about 55°C for about 15 minutes, but it can be carried out at a lower temperature (for example, from about 37 to about 50°C) for a longer period of time (for example, from about 20 to about 50°C). 30 minutes) or at a higher temperature (eg, up to about 60° C.) for a shorter period of time (eg, about 5 to 10 minutes). Similarly, heat inactivation generally occurs at about 95°C for about 15 minutes, but the temperature may be lower (for example, from about 70 to about 90°C) and the time is increased (for example, from about 20 to about 30 min). Samples are then diluted (e.g. 1000-fold) and subjected to TaqMan assay as described in

- 14 041088 standard analysis.

Additionally or alternatively, droplet digital PCR (ddPCR) can be used. For example, methods for determining single-stranded and self-complementary genome titers of an AAV vector using ddPCR are described. See, for example, M. Lock et al., Hu Gene Therapy Methods, Hum. Gene Ther.

methods. 2014 Apr; 25(2):115-25, doi:10.1089/hgtb.2013,131. Epub 2014 Feb 14.

Briefly, a method for separating rAAV9 particles having packed genomic sequences from AAV9 intermediates without genomes involves performing fast resolution liquid chromatography on a suspension containing recombinant AAV9 virus particles and AAV9 intermediates capsid products, wherein the AAV9 virus particles and the AAV9 intermediates bind with strong an anion exchange resin equilibrated at pH 10.2 and exposed to a salt gradient while monitoring the eluate for UV absorption at about 260 and about 280. Although less optimal for rAAV9, the pH can range from about 10.0 to 10.4. In this method, complete AAV9 capsids are collected from a fraction that is eluted when the A260/A280 ratio reaches the inflection point. In one example for the affinity chromatography step, the diafiltered product can be applied to Capture Select™ Poros-AAV2/9 affinity resin (Life Technologies), which efficiently captures the AAV2/9 serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, while AAV particles are efficiently retained.

A suitable formulation is an aqueous suspension buffered at a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation.

A suitable surfactant or combination of surfactants can be selected from non-ionic surfactants that are non-toxic. In one embodiment, a bifunctional block copolymer surfactant terminated in primary hydroxyl groups is selected, such as Pluronic® F68 [BASF], also known as poloxamer 188, which has a neutral pH and an average molecular weight of 8400. Other surfactants can be selected. active agents and other poloxamers, ie. non-ionic tri-block copolymers consisting of a central hydrophobic polyoxypropylene (poly(propylene oxide)) chain flanked by two hydrophilic polyoxyethylene (poly(ethylene oxide)) chains, SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic glyceride), simple polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. The name of these copolymers usually contains the letter P (for poloxamer) followed by three digits: the first two digits multiplied by 100 give the approximate molecular weight of the polyoxypropylene core, and the last digit multiplied by 10 gives the percentage of polyoxyethylene content. In one embodiment, the poloxamer 188 is selected. The surfactant may be present in an amount of up to about 0.0005 to about 0.001% of the slurry.

Usages and schemes

In one embodiment, a method for treating influenza and/or preventing influenza virus infection comprises co-administration of a synthetic FI6v3 antibody and a synthetic CR8033 antibody provided herein. Optionally, such a passive immunization regimen may be combined with a conventional vaccine, with additional influenza antibody, or with other antiviral ingredients may be selected.

In certain embodiments, the two antibodies are co-expressed from different rAAV9 vectors. In another embodiment, rAAV-mediated delivery of an artificial antibody is combined with another antibody-expressing system.

The vectors are preferably suspended in a physiologically compatible carrier for administration to a human or non-human mammal patient. Suitable carriers can be easily selected by one of skill in the art, given the indication to which the virus being carried is directed. For example, one suitable carrier includes saline, which can be prepared with various buffer solutions (eg, phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, maltose, and water. The choice of carrier is not a limitation of the present invention. Optionally, compositions of the invention may contain, in addition to rAAV and carrier(s), other standard pharmaceutical ingredients such as preservatives or chemical stabilizers.

In certain embodiments, two stock solutions of different vectors (eg, rAAV9.FI6v3+rAAV9.CR8033) are prepared separately. In such an embodiment, they can be delivered separately, but substantially simultaneously with each other, for example, within minutes to about 1 hour. In certain embodiments, two stock solutions of different vectors are mixed and combined in one composition for administration. Whether they deliver

- 15 041088 alone or in a combined suspension, the two vector stocks are mixed in a ratio of approximately 1:1 based on genome copies. In other embodiments, this ratio can be changed, for example, from about 3:1, about 2:1 to about 1:2, based on complete copies of the genome.

In certain embodiments, the rAAV9 formulation is a suspension that contains an effective amount of the AAV vector suspended in phosphate buffered saline (PBS) at a total concentration of approximately 200 mM, 0.001% (w/v) Pluoronic F68 and 5% glycerol. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example in the range of pH 6 to 9 or pH 6.5 to 8, pH 7.0 to 7.7 or pH 7.2 to 7.8. However, other pH ranges within the broadest ranges and these sub-ranges may be chosen for a different delivery route.

In one embodiment, the composition may have, for example, a concentration of at least about 1x109 to 3x10 ¹³ kg/ml, as measured by oqPCR or digital droplet PCR (ddPCR), as described, for example, in M. Lock et al., Hu Gene Therapy Methods, Hum. Gene Ther. methods. 2014 Apr; 25(2):115-25, doi:10.1089/hgtb.2013,131. Epub 2014 Feb 14, incorporated herein by reference.

In certain embodiments, a composition as described herein is formulated for intranasal delivery. This invention relates to products containing kits that contain an intranasal delivery device and a container for a composition of the invention, optionally with other components such as diluents, etc.

In certain embodiments, the intranasal delivery device includes a jet nebulizer that delivers an aerosol of particles having an average size ranging from about 30 to about 100 microns in size. In certain embodiments, the average size range is from about 10 to about 50 microns. Suitable devices are described in the literature and some are commercially available, eg LMA MAD NASAL™ (Teleflex Medical; Ireland); Teleflex VaxINator™ (Teleflex Medical; Ireland); Controlled Particle Dispersion® (CPD) from Kurve Technologies. See also, PG Djupesland, Drug Deliv and Transl. Res (2013), 3:42-62. In certain embodiments, particle size and delivery volume are controlled to preferentially target nasal epithelial cells and minimize lung targeting. In other embodiments, the aerosol particles are from about 0.1 to about 20 microns or less in order to deliver to lung cells. Such finer particle sizes can minimize retention in the nasal epithelium.

Any suitable method or route can be used to administer an AAV-containing composition as disclosed herein, and optionally to co-administer other active drugs or therapies in combination with the AAV-mediated antibodies described herein. document. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration.

In one embodiment, a frozen composition is provided that contains rAAV in a buffer solution as disclosed herein in frozen form. Optionally, one or more surfactants (eg Pluronic F68), stabilizers or preservatives are present in the composition. Suitably, for use, the composition is thawed and titrated to the desired dose using a suitable diluent, such as sterile saline or buffered saline.

Optionally, the composition described herein can be used in combination with other antiviral drugs and/or vaccines against other viral targets, including other influenza vaccines, including influenza A, influenza B, and influenza C. Type A viruses are the most virulent pathogens. person. Influenza A serotypes that have been associated with pandemics include H1N1, which caused the Spanish flu in 1918 and swine flu in 2009; H2N2, which caused the Asian flu in 1957; H3N2, which caused the Hong Kong flu in 1968; H5N1, which caused bird flu in 2004; H7N7; H1N2; H9N2; H7N2; H7N3 and H10N7. Broad-spectrum neutralizing antibodies against influenza A are described. As used herein, a broad-spectrum neutralizing antibody refers to a neutralizing antibody that can neutralize multiple strains from multiple subtypes. For example, the monoclonal antibody CR6261 [The Scripps Institute/Crucell] has been described that binds to a wide range of influenza viruses, including the 1918 Spanish flu (SC1918/H1) and the H5N1 avian influenza virus that jumped from chickens to humans in Vietnam in 2004 ( Viet04/H5). CR6261 recognizes a highly conserved helical region in the membrane-proximal hemagglutinin stalk, the predominant protein on the surface of the influenza virus. This antibody is described in WO 2010/130636, which is incorporated by reference into the present specification. Another neutralizing antibody, F10 [HOMA Ltd], has been described that can be used against H1N1 and H5N1. [Sui et al., Nature Structural and Molecular Biology (Sui,

- 16 041088 et al. 2009, 16(3):265-73)] Other anti-influenza antibodies such as Fab28 and Fab49 can be selected. See, for example, WO 2010/140114 and WO 2009/115972, which are incorporated by reference. Other antibodies can be easily selected, such as those described in WO 2010/010466, published patent publication

USA US 2011/076265 and WO 2008/156763.

Methods for using these rAAVs, for example for passive immunization, are described, for example, in WO 2012/145572. Other modes of delivery and use will be apparent to those skilled in the art. For example, a regimen as disclosed herein may include, in addition to one or more of the combinations described herein, an additional combination with one or more of a biological drug, small molecule drug, chemotherapeutic agent, immunostimulants, radiation, surgical interference, etc. A biological drug as disclosed herein is based on a peptide, polypeptide, protein, enzyme, nucleic acid molecule, vector (including viral vectors), or the like.

In combination therapy, the AAV-deliverable immunoglobulin construct described herein is administered before, during, or after initiation of therapy with another agent such as antiviral therapy, antibiotics, or any combination thereof, i. before and during, before and after, during and after, or before, during and after the start of therapy. For example, AAV can be administered between days 1 and 30, preferably days 3 and 20, more preferably between days 5 and 12 prior to therapy.

As described above, the term approximately, when used to modify a numerical value, means ±10% variation, unless otherwise indicated.

As used throughout this specification and claims, the term comprise and variations thereof, including comprises and comprising, among other variations, include other components, elements, integers, steps, and the like. The term consists of or consisting of includes other components, elements, integers, steps, and the like.

The terms identical or percent identity, in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or share a precisely defined percentage of amino acid residues or nucleotides that are the same (i.e., approximately 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity) in a well defined region (e.g. any one of the modified ORFs presented herein description, when compared and aligned to the maximum match in the comparison window or designated area) as measured using the BLAST or BLAST 2.0 sequence comparison algorithms with the default parameters described below, or by manual alignment and visual inspection (see, for example, web - NCBI website, etc.). As another example, polynucleotide sequences can be compared using Fasta, a program in CG version 6.1. Fasta represents the alignments and percentage of region sequence identity for the best overlap between the query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta with its default parameters (word size 6 and NOPAM factor for substitution matrix) as presented in CG version 6.1, incorporated herein by reference. In general, these programs are used with default settings, although one skilled in the art can change these settings as needed. Alternatively, one of skill in the art may use another algorithm or computer program that represents at least a level of identity or alignment as such algorithms and programs represent. This definition also applies to, or may be applied to, a complementary sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, preferred algorithms may take into account gaps and the like. Preferably, identity exists over a region that is at least about 25, 50, 75, 100, 150, 200 amino acids or nucleotides in length, and often over a region that is 225, 250, 300, 350, 400, 450, 500 amino acids or nucleotides in length, or over the full length of an amino acid or nucleic acid sequence.

Typically, when an alignment is derived based on an amino acid sequence, the alignment contains insertions and deletions that are identified as such relative to the AAV reference sequence, and the amino acid residue numbering is based on the reference scale provided for the alignment. However, any given AAV sequence may have fewer amino acid residues than the reference scale. In the present invention, when discussing a parent sequence, the term same position or corresponding position refers to the amino acid located at the same residue number in each of the sequences relative to the reference scale for aligned sequences. However, out of alignment, each of the proteins can have these amino acids located at different residue numbers. Alignment is performed using any of the various publicly or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, such as Clustal X, MAP, PIMA, MSA, BLOCKMAKER, MEME, and Match-Box programs. In general, any of these programs are used with default settings, although one skilled in the art can change these settings as needed. Alternatively, a person skilled in the art may use another algorithm or computer program that represents at least the level of identity or alignment as these algorithms and programs represent. See, for example, J.D. Thomson et al., Nucl. Acids. Res., A comprehensive comparison of multiple sequence alignments, 27(13):2682-2690 (1999).

As used herein, an effective amount refers to an amount of a rAAV9 composition that delivers and expresses in target cells an amount of anti-influenza antibodies sufficient to reduce or prevent influenza infection. An effective amount can be determined based on an animal model instead of a human patient. Examples of a suitable mouse model are described herein.

As used herein, the term patient generally refers to a person diagnosed with or at risk of infection with influenza. Such a patient may also be referred to herein as a subject. The term subject can also refer to non-human animals, such as cats, dogs, and non-human primates.

A functional antibody may be an antibody or immunoglobulin that binds to a selected target (e.g., an antigen on a cancer cell or pathogen such as a virus, bacterium, or parasite) with sufficient binding affinity to elicit a desired physiological outcome, which may be protective (e.g., , passive immunization) or therapeutic.

As used herein, an immunoglobulin domain refers to the heavy chain or light chain domain of an antibody, as defined with reference to a standard full length antibody. More specifically, a full-length antibody contains a heavy (H) chain polypeptide that contains four domains: one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2 and CH3) regions, and a light (L) chain polypeptide, which contains two domains: one N-terminal variable (VL) region and one C-terminal constant (CL) region. The Fc region may contain two domains (CH2-CH3). The Fab region may contain one constant and one variable domain for each of the heavy and light chains. Constant domain allotypes suitable for these constructs may include, for example, G1m17.1 and G1m3.

As used herein, different specificities indicate that said immunoglobulin constructs (eg, full length antibody, heavy chain, or other construct capable of binding to a particular target) bind to different target sites. This may refer to different targets on the same antigen, different strains of the same pathogen (eg different viral strains), or different antigens.

Equal specificity refers to the ability of an immunoglobulin to bind to a particular target site, which may be present on multiple strains of a pathogen (eg, influenza virus) or for one or a subset of strains of a virus or other pathogen. Suitably, these specificities are such that there is no significant or measurable binding to non-target sites.

The term heterologous, when used in relation to a protein or nucleic acid, indicates that the protein or nucleic acid contains two or more sequences or subsequences that do not occur in the same relationship with each other in nature. For example, a nucleic acid is typically produced recombinantly, having two or more sequences from unrelated genes arranged to produce a new functional nucleic acid. For example, in one embodiment, the nucleic acid has a promoter from one gene located to direct expression of a coding sequence from another gene. Thus, with respect to the coding sequence, the promoter is heterologous. The term heterologous light chain is a light chain containing a variable domain and/or a constant domain from an antibody that has a target specificity different from that of the heavy chain.

As used throughout the description, the singular form refers to one or more. As such, the singular form, one or more, and at least one are used interchangeably herein.

Unless otherwise defined in this specification, the technical and scientific terms used in this specification have the same meaning as generally understood by one of ordinary skill in the art and by reference to published texts which provide one of ordinary skill in the art with general guidance regarding many terms used in the present description.

The following examples illustrate how to obtain and use a mixture of AAV2/9 vectors,

- 18 041088 in which one expresses a broad-spectrum neutralizing antibody to influenza A, called FI6; the other expresses a broad-spectrum influenza B neutralizing antibody called CR8033. In one embodiment, the composition, herein referred to as GTP101, is prepared in phosphate buffered saline (PBS) with total salt adjusted to 200 mM, 0.001% (w/v) Pluronic F68 and 5% glycerol. AAV2/9 is administered intranasally (IN) after being diluted in sterile buffer at 4 dose levels. Starting dose levels are selected based on pre-clinical effect data and adjusted based on non-clinical safety assessment, at the highest concentration-limited dose that can be achieved without aggregation of the AAV vector particles. Dose range from 3x10 ¹² to 2.4x10 ¹³ copies of the genome (k.g.) was administered as 4x0.2 ml per nostril via intra-nasal mucosal atomizing device (IMAD). It is understood that this invention covers variations of these illustrative studies. For example, other intranasal agents, doses, volumes, and schedules may be used. Additionally, the composition can be adapted for other routes of administration.

The following examples are illustrative only and do not limit the invention described herein.

Example 1. AAV vectors.

AAV9 vectors expressing the heavy and light chains of the monoclonal antibodies FI6v3 and CR8 033 under the control of a cytomegalovirus enhancer hybrid of the chicken β-actin promoter were designed, cloned, and packaged. The cis plasmids used to construct each vector used in further studies are shown in SEQ ID NO: 1 (FI6v3) and SEQ ID NO: 8 (CR8033), which are incorporated by reference together with their features. AAV9 vectors were generated using triple transfection of human HEK293 MCB cells using methods as previously described, for example, US Published Application No. US 2009/0275107; see also M. Lock et al., Hum. Gene Ther., 2010; 21(10):1259-1271.

Human HEK293 MCB cells are transfected using:

(i) plasmids with vector genomes, (ii) an AAV helper plasmid called pAAV2/9.KanR, which contains the wild-type AAV rep2 and cap9 genes, and (iii) an adenoviral helper plasmid called pAdAF6(Kan).

The genome size of the packaged GTP101 vectors is 1) AAV-FI6: 4650 bp. and 2) AAV-CR8033: 4585 N. You can also use other methods using producer cell lines of baculovirus systems. Alternative cis plasmids and vectors can be generated using other coding sequences provided herein.

A. Plasmid 1 with the AAV vector GTP101 genome: pAAV.CB7.CI.FI6.RBG.

Plasmid pAAV.CB7.CI.FI6.RBG with the genome of the vector mAb AAV-FI6 (p3193) is 7471 bp. in size (Fig. 7B). The vector genome derived from this plasmid is a single stranded DNA genome with AAV2 derived ITRs that flank the mAb expression cassette. The FI6 coding sequences encode an influenza A-specific mAb. Expression from the transgene cassettes is driven by the CB7 promoter, a fusion between the cytomegalovirus (CMV) immediate early enhancer (C4) and the chicken beta-actin promoter, while transcription from this promoter is enhanced by the presence of the chicken beta-actin intron. (CI). The 5' untranslated region (UTR) of the human c-myc gene is located upstream of the mAb coding region as a translation enhancer. The poly-A signal for the expression cassette is rabbit beta-globin (RBG) poly-A. Leader (signal) peptides precede both the heavy and light chains of the antibody and mediate the secretion of the antibody from the transduced cell. The heavy and light chains are separated by a linker containing a furin cleavage site and the FMDV 2A sequence. This linker mediates post-translational cleavage of the heavy and light chains and, with the exception of one amino acid change (the terminal lysine in the heavy chain is replaced by an arginine residue), is completely removed from the final mAb product. An antibody translation cassette containing the 5'UTR and the mAb coding sequence with codon optimization was synthesized de novo and subcloned into pZac2.1 to generate p3191. Subsequently, the gene cassette was shuttled into the CB7 backbone, pN406 using NheI/NotI enzymes to change the polylinker to the antibody translation cassette to generate pAAV.CB7.CI.FI6.RBG (p3193). All constituent parts of the plasmid were verified by direct sequencing using Qiagen Genomic Services.

C. Plasmid 2 with the genome of the AAV vector GTP101: pAAV.CB7.CI.CR8 033.RBG.

Plasmid pAAV.CB7.CI.CR8033.RBG with the genome of the vector mAb AAV-CR8033 (p3523) is 7462 bp. at the rate of. The vector genome derived from this plasmid is a single stranded DNA genome with AAV2 derived inverted terminal repeats (ITRs) that flank the mAb expression cassette. The CR8033 coding sequences encode an influenza B specific mAb. Expression from the transgene cassettes is driven by the CB7 promoter, a fusion between the cytomegalovirus (CMV) immediate early enhancer (C4) and a modified beta-actin promoter.

- 19 041088 chicken, while transcription from this promoter is enhanced by the presence of the chicken beta-actin intron (CI). The truncated 5' UTR of the human c-myc gene is positioned upstream of the mAb coding region as a translation enhancer. The poly-A signal for the expression cassette is rabbit beta-globin (RBG) poly-A. Leader (signal) peptides precede both the heavy and light chains of the antibody and mediate the secretion of the antibody from the transduced cell. The heavy and light chains are separated by a linker containing a furin cleavage site and the FMDV 2A sequence. This linker mediates post-translational cleavage of the heavy and light chains and, with the exception of one amino acid change (the terminal lysine in the heavy chain is replaced by an arginine residue), is completely removed from the final mAb product. An antibody translation cassette containing a 5' untranslated region and a codon optimized mAb coding sequence encoding mAb TCN032 was synthesized de novo and subcloned into pZac2.1 to generate p3187. The TCN032 light chain was replaced with a universal light chain taken from pAAV.CB7.CI.FI6.RBG (p3193) described above using the EcoRV/BsiWI enzyme sites. The CR8033 heavy variable chain coding sequence was codon optimized, synthesized de novo, and used to replace the TCN032 heavy variable chain through SphI/SalI restriction sites. The entire translation cassette was shuttled into a CB7 backbone plasmid, pN437, using SphI/NotI enzymes to change the TCN032 translation cassette to the CR8033 translation cassette and create the pAAV.CB7.CI.CR8033.RBG (p3523) plasmid. All constituent parts of the plasmid were verified by direct sequencing using Qiagen Genomic Services.

C. Cloning of the kanamycin backbone.

The transgene cassettes from (pAAV.CB7.CI.FI6.RBG (p3193) and pAAV.CB7.CI.CR8033.RBG (p3523) were cloned into an AAV2 ITR flanked construct. The backbone of the plasmid in this construct was derived from pZac2.1, a plasmid based on pKSS and it contains the kanamycin resistance gene.The final plasmids with vector genomes are pAAV.CB7.CI.FI6.RGB.KanR and pAAV.CB7.CI.CR8033.RBG.Kan.All components of the final plasmids with vector genomes are verified through sequencing and validate sequences as part of the GMP manufacturing process.

D. Description of sequence elements.

1. Inverted Terminal Repeats (ITRs): AAV ITRs (GenBank No. NC001401) are sequences that are identical at both ends but in opposite orientation. The AAV2 ITR sequences function as both an origin of vector DNA replication and a vector genome packaging signal when AAV and adenovirus helper functions are presented in trans. As such, the ITR sequences represent only the cis sequences required for vector genome replication and packaging.

2. Cytomegalovirus (CMV) immediate early enhancer (260 bp, C4; GenBank No. K03104.1). This element is present in both plasmids with vector genomes.

3. Chicken beta actin promoter (281 bp; CB; GenBank No. X00182.1) promoter and is used to drive high level antibody expression. This element is present in both plasmids with vector genomes.

4. Chicken beta-actin intron: intron 875 b.p. from the chicken beta actin gene (GenBank No. X00182.1) is present in the expression cassette of the vector. The intron participates in transcription but is removed from the mature mRNA by splicing, bringing together the sequences on both sides of it. The presence of the intron in the expression cassette has been shown to facilitate the transport of mRNA from the nucleus to the cytoplasm, thus enhancing the accumulation of a constant level of mRNA for translation. This is a common feature in vectors with genes that are designed for increased levels of gene expression. This element is present in both plasmids with vector genomes.

5. c-myc 5'-UTR: nucleotides 381-428 of v-myc Homo sapiens (MYC) viral oncogene mRNA homologue (GenBank accession number NM002467). This element is present in both plasmids with vector genomes.

6. cDNA of the leader (signal) peptide: a codon-optimized cDNA sequence (GenBank accession number AAB8686.1) preceding and in-frame of the FI6 heavy chain was synthesized.

7. FI6v3 heavy variable chain cDNA: cDNA sequence was synthesized with human codon optimization (GenBank accession number AEL31303.1).

8. cDNA of leader (signal) peptide: cDNA sequence was synthesized with codon optimization (ILco2, described in [Zhang, L., Q. Leng, and A.J. Mixson, J. Gene Med., 2005. 7(3), p. 354 -6]) preceding the CR8033 heavy chain and in frame with it.

9. CR8033 heavy variable chain cDNA: The human cDNA sequence was synthesized with codon optimization (GenBank accession number AFP87542.1).

10. Constant heavy 1 (CH1) chain: cDNA was synthesized with codon optimization (access number

- 20 041088 tupa GenBank BAF64540.1). This element is present in both plasmids with vector genomes.

11. Fc chain of CR8033 (CH2-3): cDNA was synthesized with codon optimization (accession no.

GenBank 226787), except for the C-terminal lysine, which was replaced by arginine).

12. FI6 Fc chain (CH2-3): GeneArt synthesized cDNA with codon optimization (GenBank accession number 2J6E_A), except for the C-terminal lysine, which was substituted for arginine, and position 57, where aspartic acid was substituted for glutamic acid.

13. Leader (signal) peptide cDNA: A codon-optimized cDNA sequence that precedes the universal light chain (ILhy1, which is described in [Zhang et al., cited above)] was synthesized.

14. Universal light chain cDNA: The human cDNA sequence was synthesized with codon optimization (GeBank accession number ACJ71709.1). This element is present in both plasmids with vector genomes.

15. Light chain constant cDNA: human codon sequence was synthesized (GenBank accession number AGH7 0219.1). This element is present in both plasmids with vector genomes.

16. Furin recognition site: Arginine-lysine-arginine-arginine and cDNA were synthesized with codon optimization. This element is present in both plasmids with vector genomes.

17. F2A Linker: A 24 amino acid peptide derived from FMDV (GenBank No. CAA2436.1) and codon optimized cDNA were synthesized. This element is present in both plasmids with vector genomes.

18. Polyadenylation signal: 127 bp polyadenylation signal. rabbit beta globin (GenBank No. V00882.1) provides cis sequences for efficient polyadenylation of antibody mRNA. This element functions as a transcriptional termination signal, a specific cleavage event at the 3' end of the nascent transcript, and the addition of a long polyadenyl tail. This element is present in both plasmids with vector genomes.

E. AAV2/9 helper plasmid pAAV2.9.KanR.

This AAV2/9 helper plasmid p5E18.AAV2/9n (p0061; 7330 bp) is an AAV helper plasmid that encodes four wild-type AAV2 rep proteins and three wild-type AAV VP capsid proteins from AAV serotype 9 [J . Virol. 2004Ju; 78(12):6381-8].

In order to create a chimeric packaging construct, the AAV2 cap gene from plasmid p5E18 containing the wild-type AAV2 rep and cap genes was deleted and replaced with a PCR fragment from the AAV2/9 cap gene amplified from liver DNA to obtain plasmid p5E18VD2/AAV2/ 9. Of note, the p5 AAV promoter, which normally drives rep expression, is moved in this construct from the 5' end of rep to the 3' end of cap. This arrangement serves to introduce a spacer between the promoter and the rep gene (ie the backbone of the plasmid), downregulate rep expression and increase the ability to maintain vector production. The backbone of the plasmid in p5E18 is derived from pBluescript KS. All constituent parts of the plasmid were verified by direct sequencing. The ampicillin resistance gene will be replaced with a kanamycin resistance gene to generate pAAV2/9n.KanR.

F. Adenovirus helper plasmid pAdDeltaF6(Kan).

Plasmid pAdDelta (A)F6(Kan) has a size of 15774 bp.

The plasmid contains regions of the adenoviral genome that are important for AAV replication, namely the E2A, E4 and VA RNA (adenovirus E1 functions are provided by 293 cells), but does not contain other adenoviral replication or structural genes. The plasmid does not contain cis elements critical for replication, such as adenoviral inverted terminal repeats, and therefore does not expect to generate an infectious adenovirus. It was obtained from an Ad5 molecular clone with E1 and E3 removed (pBHG10, pBR322 based plasmid). Deletions were introduced into Ad5 DNA in order to eliminate the expression of unnecessary adenoviral genes and reduce the amount of adenoviral DNA from 32 kb to 12 kb). Finally, the ampicillin resistance gene was replaced with a kanamycin resistance gene to give pAdAF6(Kan). The adenoviral genes E2, E4 and VAI that remain on this plasmid, along with E1 that is present in HEK293 cells, are required for the production of the AAV vector.

Example 2. Manufacturing process.

Cells are cultured on 10-layer Corning CellStack (CS-10) and HS-36 and all open manipulations are performed in biosafety class II rooms in an ISO class 5 environment. The purification process is carried out in a closed system where possible, however, column chromatography manipulations are not considered as a completely closed system. To minimize this risk, disposable flow paths are used as part of the GE ReadyMate production mobile platform for column chromatography. After purification by column chromatography, the product is subjected to diafiltration using the final composition buffer (1x PBS (200 mm NaCl) 0.001% Pluronic F68, 5% glycerol) and sterile filtration to obtain BDS, which is frozen at <-60°C. After testing the BDS, the BDS is thawed, pooled and diluted as needed with sterile final formulation buffer and filtered for SAFC in its Fill Suite. In the case where the two components of the GTP101 vector are produced separately, the components are mixed in

- 21 041088 1:1 ratio before filling.

An overview of the manufacturing process is shown in FIG. 2.

A. Cell seeding: HEK293 cells express the E1a and E1b gene products required for the production of high titer rAAV. HEK293 cells are adherent and highly susceptible to transfection, resulting in high titers of rAAV when transfected with DNA with the plasmid. Cells were expanded to 5x109-5x101° cells using Corning T-flask and CS-10, which allowed sufficient cell mass to be generated to seed 2° HS-36 for vector production per BDS batch. The cells are cultured in a medium consisting of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% gamma irradiated fetal bovine serum (FBS) from New Zealand. The cells are substrate-dependent and cell disassociation is performed using TrypLE Select, a non-animal cell disassociation reagent. Cell seeding is performed using sterile disposable bioprocessing bag and tube sets. Cells contain at 37°C (±1°C), in an atmosphere of 5% (±0.5%) CO ₂ .

B. Transient transfection: After 3 days of growth (DMEM + 10% FBS), HS-36 cell culture media are replaced with fresh serum-free DMEM media and transfected with three production plasmids using an optimized PEI-based transfection method. All plasmids used in the preparation process are manufactured by Aldevron Inc. using their GMP-Source™ quality system and infrastructure using the most salient features of cGMP production; traceability, document control and material segregation.

Sufficient DNA transfection complex with plasmid is prepared in BSC in order to transfect 20 HS-36 (per BDS batch). Initially, a DNA/PEI mixture is prepared containing 3.0 mg of the plasmid with the genome of the vector pAAV.CB7.CI.FI6.RBG.KanR or pAAV.CB7.CI.CR8033.RBG.KanR, 60 mg of pAdDeltaF6(Kan), 30 mg of AAV helper plasmid pAAV2.rh10.KanR and GMP grade PEI (PEIPro, PolyPlus Transfection SA). Alternatively, when mixed plasmid transfections with the vector genome are used, 1.5 mg of each of the two cis plasmids is used. After good mixing, the solution is allowed to stand at room temperature for 25 min, and then added to serum-free media in order to quench the reaction, and then added to HS-36. The transfection mixture is leveled between all 36 layers in HS-36 and the cells are incubated at 37°C (±2°C) in 5% (±0.5%) CO ₂ for 5 days.

C. Collection of Cell Media: Transfected cells and media are harvested from each HS-36 using disposable bioprocessing bags by aseptic draining of media from blocks. After collecting the medium, MgCl ₂ is added to a volume of ~80 L to a final concentration of 2 mM (benzonase cofactor) and nuclease benzonase (Catalog No: 1.016797.0001, Merck Group) is added to a final concentration of 25 U/ml. The product (in a disposable bioprocessing bag) is incubated at 37° C. for 2 hours in an incubator to allow sufficient time for enzymatic digestion of the residual cellular and plasmid DNA present in the crop from the transfection procedure. This step is carried out in order to minimize the amount of residual DNA in the final vector FDP. After an incubation period, NaCl is added to a final concentration of 500 mM to assist in product recovery during filtration and subsequent tangential flow filtration (see steps 4 and 5 below).

D. Clarification: Cells and cell debris are removed from the product using a volumetric filter capsule (1.2 µm/0.22 µm) connected in series as a sterile sealed tube and bag set driven by a peristaltic pump. The clarification ensures that downstream filters and chromatography columns are protected from clogging, and the reduction of filtration bioburden ensures that at the end of the filter run, any bioburden potentially introduced during the upstream production process is removed prior to downstream purification. Media are passed through a Sartorius Sartoguard PES (1.2/0.22 µm) capsule filter (Sartorius Stedim Biotech Inc.).

E. Large-Scale Tangential Flow Filtration: Volume reduction (10-fold) of clarified product is achieved using Tangential Flow Filtration (TFF) using a special sterile closed bioprocessing tubing, bag, and membrane kit manufactured by Spectrum Labs. The principle of TFF is to flow a pressurized solution parallel to a membrane of suitable porosity (100 kDa). The pressure difference moves smaller molecules through the membrane and efficiently into the waste stream, while retaining molecules larger than the pores of the membrane. By recirculating the solution, a parallel flow sweeps across the membrane surface, preventing clogging of the membrane pores. By selecting the appropriate membrane pore size and surface area, the volume of a liquid sample can be rapidly reduced while retaining and concentrating the desired molecule. Diafiltration in TFF applications involves adding fresh buffer to the recirculating sample at the same rate that the liquid passes through the membrane and into the waste stream. Increasing amounts of low molecular weight compounds are removed from the recirculating sample by increasing the volume of diafiltration. This leads to moderate cleaning of the clarified product, but also allows up to

- 22 041088 to be replaced with a buffer compatible with the subsequent affinity column chromatography step. Accordingly, a 100 kDa PES membrane (Spectrum Labs) was used for concentration followed by diafiltration using 4 volumes of buffer consisting of: 20 mM Tris pH 7.5 and 400 mM NaCl. The diafiltered product was stored overnight at 4° C. and then further clarified using a 1.2 μm/0.22 μm volumetric filter capsule in order to remove any precipitated material.

F. Affinity Chromatography: The diafiltered product is applied to Capture Select™ Poros-AAV2/9 affinity resin (Life Technologies), which effectively captures the AAV2/9 serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, and efficient capture of the AAV particles occurs. After loading, the column is washed to remove additional added impurities, followed by a stepwise elution at low pH (400 mM NaCl, 20 mM sodium citrate; pH 2.5), which is immediately neutralized by collection in 1/10 volume of neutralizing buffer ( bis-tris propane, 200 mM, pH 10.2).

G. Anion Exchange Chromatography: To achieve further reduction of process impurities including blank AAV particles, the pooled Poros-AAV2/9 eluate is diluted 50-fold (20 mM bis-tris propane, 0.001% Pluronic F68; pH 10.2) to reduce ionic strength to allow binding to the CIMultus Q monolithic matrix (BIA Separations). After a low salt wash, the vector product was eluted using a 60 CV linear NaCl salt gradient, 10-180 mM NaCl). This gentle salt gradient effectively separates capsid particles without the vector genome (empty particles) from particles containing the vector genome (full particles) and leads to a preparation that is substantially free of empty capsids. Fractions were collected in tubes containing 1/100 volume of 0.1% Pluronic F68 and 1/27 volume of Bis-Tris pH 6.3 to minimize non-specific binding to the tubes and duration of exposure to high pH, respectively. The purity of the peak and eluted crop fractions were identified by qPCR, pooled and stored at 4° C. until final preparation using TFF. qPCR data on the AEX titer of the combined product is used to estimate the target final volume to achieve a titer of 2x10 ¹³ kg/ml for the final preparation (stage immediately following).

H. Final preparation and sterilizing filtration to obtain BDS: TFF was used to obtain the final composition from the pooled AEX fractions using a 100 kDa membrane (Spectrum Labs, Inc.). This is achieved by diafiltration with 4 volumes of preparation buffer (1 x PBS (200 mM NaCl) 0.001% Pluronic F68, 5% glycerol) and concentrated to the calculated volume to give the target concentration. After diafiltration, the final product is filtered through a 0.22 µm filter (Millipore, Billerica, MA) to obtain BDS. Samples are withdrawn for BDS testing (described in the section below). The filtered clarified batch is stored in sterile polypropylene tubes and frozen at <-60°C in a quarantined area until released for final filling.

I. Final Fill: Frozen BDS is thawed (and pooled if necessary) and filled into West Pharmaceutical 2 ml Ready-to-Use (pre-sterilized) glass vials with 13 mm stoppers and sealant at fill volumes >0.6 to <2 .0 ml per vial. Filling operations occur in accordance with standardized filling operations provided by the technician and process qualification testing at SAFC. Upon completion of the final filling, all operators working in the filling unit are monitored using RODAC cups (to detect viable solids) on their sterile gloves as they leave the BSC. The surface of the BSC is also monitored for viable organisms upon completion of filling. SAFC QC inspects vials for appearance, particulate matter, volume and integrity. Individually labeled vials are labeled to include protocol number, product name, lot number, location number, and stored in labeled boxes. Labeled vials are quarantined <-60°C until release.

J. ddPCR titer qg: The accuracy and reliability of quantitative PCR (qPCR) titration of genome copies (qg) is excellent for AAV quality control and vector dosing. Genome titers are measured using a new qPCR method based on droplet digital PCR [Lock, M., et al., Absolute determination of single-stranded and self-complementary adenoassociated viral vector genome titers by droplet digital PCR. Hum Gene Ther Methods, 2014. 25(2), p. 115-25], which gives unprecedented accuracy and reproducibility. DNA detection is performed using sequence-specific primers and fluorescently labeled probes targeting the FI6 and CR8033 coding regions to enable the quantification of component genomes in FDP. In addition, primers and probes targeting the poly-A region of the RBG are used to determine the total content of the FDP genome. Many standards, validating samples and controls (for background and DNA contamination) were included in the analysis. An assay is qualified by establishing and defining assay parameters, including sensitivity, limit of detection, skill range, and inter-assay and within-assay reproducibility. Internal reference lot AAV2/9, semi

- 23 041088 obtained using the newly developed production process, installed and used in order to carry out qualifying studies. It should be noted that the inventors' previous experience suggests that the titer obtained by spot digital PCR is typically 2-fold higher than that obtained by the inventors' standard pPCR method. Once a new titration method has been qualified, all previous test items used in the pilot studies described in Sections 6 and 8 should be re-analyzed and dosing adjusted. This may affect the actual dosing used in non-clinical and clinical studies, although the expectation is that the difference will not be more than 2-fold.

L. Infection titer: infectious unit (IU) assay is used to determine productive uptake and replication of the GTP101 vector in RC32 cells (rep2-expressing HeLa cells). A 96-well end point format was used similar to that published previously. Briefly, RC32 cells are co-infected with serial dilutions of GTP101 BDS and a uniform dilution of Ad5 with 12 repetitions at each dilution of rAAV. Cells were lysed 72 hours after infection and qPCR was performed to detect amplification of the rAAV vector via injection. The calculation of TCID ₅₀ at endpoint dilution (Spearman-Karber) is performed in order to determine the replication titer, expressed as IU/ml. Because infectivity values depend on particles coming into contact with cells, receptor binding, internalization, nuclear transport, and genome replication, they are affected by the geometry of the assay and the presence of suitable receptors and post-binding pathways in the cell line used. Receptors and pathways after binding are generally not retained in immortalized cell lines, and thus infectivity assay titers are not an absolute measure of the number of infectious particles present. However, the ratio of encapsulated k.g. and infectious units (described as the kg/IU ratio) can be used as a measure of lot-to-lot conformity of a product.

M. host cell (human) DNA qPCR analysis was used to detect residual human 293 DNA in the BDS. After the introduction of irrelevant DNA, total DNA (irrelevant, vector and residual genomic) is extracted from ~1 ml of the product. Host cell DNA is quantified using qPCR targeting the 18S rDNA gene. The amounts of detectable DNA are normalized based on the recovery of non-relevant DNA added.

N. Host cell (human) protein.

An ELISA is performed on a BDS sample in order to measure the levels of HEK 293 host cell contaminating proteins. A Cygnus Technologies HEK293 Host Cell Proteins 2nd Generation ELISA kit is used. Samples and pre-diluted HEK293 HCP standards are added to microtiter wells pre-coated with affinity-purified capture anti-HEK293 HCP antibody, along with peroxidase-conjugated polyclonal detection antibody against HEK293 HCP. After incubation, the wells are washed to remove unbound reagents and TMB, a peroxidase substrate, is added. After development, the reaction is stopped using a sulfuric acid solution. The absorbance of the resulting colored product is measured using a microplate reader and the amount of HEK293 HCP in each sample is calculated from the calibration curve.

A. AAV competent for replication (rcAAV).

As part of the BDS test plan, a GTP101 FDP sample is analyzed for the presence of replication-competent AAV2/9 (rcAAV), which could potentially occur during the production process. A three-passage assay has been developed that consists of cell-based amplification and passaging, followed by detection of rcAAV DNA by real-time qPCR (Cap9 target). The cell-based component consists of inoculating monolayers of HEK293 (P1) cells with dilutions of the test sample and wild-type human adenovirus 5 (Ad5). 10 ¹⁰ DRP of the vector product is the maximum amount of product tested. Due to the presence of an adenovirus competent for replication, AAV will be amplified in cell culture. After two days, a cell lysate is obtained and Ad5 is inactivated with heat. The clarified lysate is then passaged in a second round of cells (P2) in order to maximize sensitivity (again the presence of Ad5). After 2 days, a cell lysate is obtained and Ad5 is inactivated with heat. The clarified lysate is then passaged in the third round of cells (P3) in order to maximize sensitivity (again the presence of Ad5). After 2 days, cells are lysed to release DNA, which is then subjected to qPCR in order to detect AAV2/9 cap sequences. Amplification of the AAV2/9 cap sequences in an Ad5 dependent manner indicates the presence of rcAAV. The use of an AAV2/9 surrogate positive control containing the AAV2 rep and AAV2/9 cap genes allows determination of the limit of detection (LOD) of the assay (0.1, 1 10 and 100 IU), and using a serial dilution of the GTP101 vector (1 x 10 ¹⁰ , 1x109, 1x10 ⁸ , 1x10 ⁷ DRP) the approximate level of rcAAV present in the test sample can be quantified.

R. Biological activity in vitro.

To link qPCR titer to.g. with gene expression, an in vitro biological assay is performed by transducing HEK293 cells with a known large amount of k.g. per cell and measurements of secreted expression of FI6 and CR8033 72 hours after transduction using an anti-idiotypic ELISA with specificity for two mAbs. Comparison with highly active pre-clinical and tox vector preparations allows interpretation of product activity.

Q. Total protein, capsid protein and determination of protein purity.

In vector samples, total protein is first quantified by a protein calibration curve for bovine serum albumin (BSA) using a bicinchoninic acid assay. The determination is performed by mixing equal parts of the sample with the Micro-BCA reagent provided in the kit. The same procedure is applied to BSA Standard dilutions. The mixtures are incubated at 60° C. and the absorbance is measured at 562 nm. A calibration curve is generated from the absorbance of standards of known concentrations using a four-parameter approximation. Unknown samples are quantified according to four parameter regression.

To ensure semi-quantitative determination of AAV purity, samples are then normalized for genome titer and 5x109 k.g. separated on SDS-polyacrylamide (SDS-PAGE) gel. The gel is then stained with SYPRO Ruby. Bands of any impurities are quantified by comparison with co-electrophoresed BSA standards of 25, 50 and 100 ng of protein per lane. These amounts represent 1, 2 and 4% of the total AAV protein sample. Colored bands that appear in addition to the three specific AAV proteins VP1, VP2 and VP3 are considered protein contaminants. All contaminant bands are compared to reference proteins and the percent contaminant by weight is reported as well as the approximate molecular weight. SDS-PAGE gels are also used to quantify VP1, VP2 and VP3 proteins and determine their ratio.

R. BSA protein.

This assay is performed for pre-release testing using the Bovine Albumin ELISA kit obtained from Bethyl Laboratories. This kit is an ELISA sandwich. The BSA present in the test sample is captured with an anti-bovine albumin antibody that has been pre-adsorbed onto the surface of the microtiter wells. After sample binding, unbound proteins and molecules are washed away and a biotinylated identification antibody is added to the wells to bind to the captured albumin. Streptavidin-conjugated horseradish peroxidase (SA-HRP) is then added to catalyze the colorimetric reaction with the chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine). The colorimetric reaction gives a blue product which turns yellow when the reaction is stopped by the addition of dilute sulfuric acid. The absorbance of the yellow product at 450 nm is proportional to the amount of albumin analyte present in the sample and a four parameter calibration curve can be generated. The albumin concentrations in the test samples can then be quantified by interpolating their absorbance from a calibration curve created in parallel with the samples. After factoring the sample dilutions, albumin concentrations in the original sample can finally be calculated.

S. Endonuclease benzonase.

Benzonase is used in the production process to degrade nucleic acids to help purify the vector and is essentially a process impurity. For pre-release testing, a commercial ELISA is used to measure the concentration of residual benzonase. Since the amount of benzonase is likely to be a trace amount, if at all, it is necessary to run the ELISA using a spectrum of standards that includes <1 ng/mL concentrations.

T. Ratio to.g. and I.U.

The kg/IU ratio is a measure of the conformity of a product. The qPCR titer (kg/mL) is divided by the analysis of infectious units (IU/mL) to obtain the calculated k.r./IU ratio.

U. Osmolality, pH and appearance testing.

These assays are performed for pre-release testing by BREL using osmolality testing protocols to lower freezing point. The appearance of the product is determined by visual inspection for clarity, color and absence/presence of foreign particles. The product is inspected against white and black backgrounds. The pH in the FDP is determined using a calibrated temperature compensated pH microelectrode.

Example 3 Evaluation of individual components of GTP101.

A. Determination of the Minimum Effective Dose (MED) of AAV2/9.CB7.FI6 required to protect against lethal Influenza A challenge AAV2/9.CB7.FI6 was administered intranasally (IN) to 6-week-old female BALB/c mice at doses ranging from 1x107 to 1x1010 kg/mouse and challenged with IN using 5 LD ₅₀ mouse-adapted H1N1 (PR8) after 14 days. As shown in FIG. 3A, 3B, MED (which was defined as the dose required for 100% of infected mice to survive challenge using 5 LD ₅₀ PR8) for AAV2/9.CB7.FI6 in mice is 1x109 k.g./mouse, with partial protection , which gives a dose reduced by half the logarithm (3x108 kg/mouse).

- 25 041088

On day 14, three mice per treatment group that were not challenged were sacrificed in order to assess the expression level of FI6 on airway surfaces, namely bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF). As shown in FIG. 4A and 4B, the amount of Ab FI6 measured at the surface of the lung (BALF) or nose (NLF) correlated with the dose of the AAV2/9 vector. The amount of antibody secreted in NLF is difficult to accurately quantify due to the small volume of mouse airway surface fluid (ASL) estimated at 1 µl.

Viral load (plaque forming units; PFU) was assessed 6 days after challenge with PR8 (FIG. 5) and found that mice treated with 1x10 ¹⁰ k.g. AAV2/9.CB7.FI6, their lungs were clear of PR8, and a significant reduction in viral load was observed in mice treated with 3x10 ⁹ kg. and also 1 x 10 ⁹ k.g. AAV2/9.CB7.FI6. Both of these doses have been found to be protective against influenza infection.

B. Initiation of AAV2/9 prophylaxis for inhaled influenza.

The rapid onset of effect is useful when dealing with an influenza outbreak that requires a short response time. As such, the interval between IN delivery of AAV2/9.CB7.FI6 and challenge with 5 LD5o PR8 was varied to determine how quickly protection could be achieved. The shortest interval between vector treatment and challenge was 8 hours. Mice pre-vaccinated and challenged 8 hours later lost some weight, but recovered and survived the challenge (FIGS. 6A and 6B).

C. Determination of the MED for AAV2/9.CB7.CR8O33, which is required in order to protect against lethal influenza infection.

In parallel studies, AAV2/9 expressing anti-influenza Ab CR8033 was evaluated for potential protection against influenza B challenge. Female BALB/c mice were given AAV2/9.CB7 at 6 weeks of age. CR8033 at doses ranging from 1 x 10 7 to 1 x 10 ¹⁰ kg/mouse and challenged them with IN using 5 LD ₅₀ mouse-adapted B/Lee/40. After 14 days, the inventors found that, similarly, the MED was 1x10 ⁹ kg/mouse (Fig. 7A and 7B). Mice did not survive challenge with the B/Lee/40 virus when they were given IN at a half log dose of 3 x 10 ⁸ kg/mouse.

D. Dissemination of the AAV2/9 vector after IN delivery to mice.

An important safety aspect of the AAV-mediated prevention strategy described herein is the targeting of vector-mediated transduction to terminally differentiated cells on respiratory surfaces. The dissemination potential of the AAV2/9 vector throughout the body was determined in mice that survived PR8 challenge. Mice were given AAV2/9.CB7.FI6 at varying doses and mice were dissected after infection and tissues (lung, liver, spleen, ovary, brain, heart) were harvested in order to assess the biodistribution of the vector. At vector doses of 1 x 109-1 x 10 ¹⁰ kg/mouse, all mice (5 per group) survived challenge. At all doses, the largest numbers of vector genomes were found in the lungs (eg, at the highest dose of vector, the ratio of AAV2/9 genomes in lung/liver was 1783). For the higher dose of 1 x 10 ¹⁰ kg/mouse, no GG/diploid genome was observed in spleen, brain and heart. Very low levels of the AAV2/9 vector were found in the liver and ovary and, as expected, most copies of the AAV2/9 genome were observed in the liver. In mice treated with 3x10 ⁹ kg, AAV2/9 was not detected in the liver, ovaries and spleen, and low capsid levels were observed in the brain and heart. As expected, there was a decrease in vector genomes in the lungs. In mice treated with 1 x 10 ⁹ kg, the vector genome, despite a significant decrease, was observed only in the lungs. For survivors in the 3×10 ⁸ kg/mouse treatment group, the copy level of the AAV2/9 genome was undetectable (below the limit of detection, LD).

Presence of vector/diploid genomes

Lungs Liver Spleen ovaries Brain Heart 1> <10 ¹⁰ r . G. 0.1783 0.0001 <LD 0.0005 <LD <LD 3> <10 ⁹ k . G. 0.0178 <LD <LD <LD 0.0043 0.0003 1> <10 ⁹ k . G. 0.0011 <LD <LD <LD <LD <LD 3> <10 ⁸ k . G. <LD <LD <LD <LD <LD <LD

Example 4 Effect of mixing two AAVs on the protective effect against influenza A and influenza B.

The effect on the prevention of influenza A and influenza B was evaluated by mixing two different AAV preparations. Mice were dosed with an IN mixture of 1 x 10 ⁹ kg. with AAV2/9.CB7.FI6 and 1x10 ⁹ kg. AAV2/9.CB7.CR8O33 and infected with 5 LD ₅₀ of PR8 virus or B/Lee/40 virus. If there is mutual interference or competition for receptor binding, then the protective effect will be significantly lower than the protection conferred by the introduction of a single protective AAV2/9 vector.

- 26 041088

In the first case, a mixture of 1x10 ⁹ kg. AAV2/9.CB7.FI6 and 1x10 ⁹ kg. AAV2/9.CB7.CR8033 was evaluated for protection against challenge using PR8 and compared to the protection against PR8 provided when only 1×10 ⁹ kg was delivered. AAV2/9.CB7.FI6. As shown in FIG. 8, no mutual interference was observed.

Then a mixture of 1x10 ⁹ k.g. with AAV2/9.CB7.FI6 and 1x10 ⁹ kg. AAV2/9.CB7.CR8O33 was evaluated for protection against challenge using B/Lee/40 (Influenza B) and compared to the protection against Influenza B provided when only 1x10 ⁹ kg was delivered. AAV2/9.CB7.CR8O33. As shown in FIG. 9, there was no mutual interference.

Example 5 Efficient Vector Re-Introduction and Protection Against Influenza A Infection

An important feature of the AAV2/9 vector delivery system is that it can be administered more than once. To mimic the situation of already existing immunity against the AAV2/9 capsid, the inventors delivered the IN AAV2/9 vector expressing the influenza-irrelevant transgene product at doses ranging from 1 x 10 ⁹ to 1 x 10 ¹¹ kg/mouse. After 28 days, the level of circulating serum AAV2/9-specific neutralizing antibodies (NAb) was assessed (Fig. 10). As expected, the level of AAV2/9 NAb in serum was directly proportional to the dose of AAV2/9 delivered by IN.

Then pre-immunized AAV2/9 mice were given 1x10 ¹¹ kg. AAV2/9.CB7.FI6. If the anti-AAV2/9 capsid NAb prevented effective re-introduction of AAV2/9, pre-immunized mice would not survive PR8 challenge. Despite high levels of circulating serum AAV2/9 Nab, which reached a serum dilution of 1:5120,

AAV2/9.CB7.FI6 was efficiently re-introduced, resulting in complete protection against 5 LD ₅₀ PR8 challenge.

Example 6 Effect of transient infection on the efficacy of AAV2/9-mediated FI6 prevention of PR8.

Following IN administration of AAV2/9.CB7.FI6, the possibility exists that the patient may be transiently infected with an airborne infection other than influenza, such as rhinovirus or RSV. As a result of infection, a subset of the respiratory cells that line the surface of the nasal and pulmonary airways will be damaged and lost. The effect of positively transduced cells damaged by viral infection on the effect of AAV2/9.CB7.FI6-mediated influenza prophylaxis was assessed. Briefly, 1x10 ¹⁰ k.g. AAV2/9.CB7.FI6 was delivered IN to female BALB/c mice at 6 weeks of age. To induce epithelial damage, two weeks later, one group was challenged with 1×10 ⁶ PFU of RSV and the other group was treated with IN using PBS. Four weeks later, all mice were challenged with IN using 5 LD ₅₀ PR8 and weight loss and survival were monitored. In the case of transient infection, there was no effect on the effect of AAV2/9-mediated prophylaxis.

Example 7 Pilot Toxicity and Expression Studies.

A. An interim non-GLP study is performed in immunocompetent CD-I mice in order to assess safety, kinetics, and immune response. Outbred CD-I mice were expected to respond to GTP101 (vectors AAV2/9.CB7.FI6 and AAV2/9.CB7.CR8033; investigational product (IP) GTP101) in a human-like manner due to their genetic heterogeneity. This transitional study is necessary since most of the previous work has been done using different inbred strains. Toxicity caused directly by the transgene product cannot be assessed in CD-1 mice, and instead can be assessed in immunocompromised inbred mice, as described elsewhere in this document.

To modulate mouse IP deposition via IMAD in the human nasal airways, we reduced the volume of fluid in which the vector is delivered to target the mouse nasal airways. Previously, the inventors conducted studies in which reducing the volume to 20 μl limits the expression of the nasal airways [Limberis, M.P., et al., Sci. Transl. Med., 2013. 5(187), p. 187ra72]. In mice, this can additionally be limited to 5 µl/nostril. This small volume still allows testing in mice of the maximum dose of GTP101 proposed in non-clinical studies.

dose groups, 10 animals per dose at one time (5 males and 5 females), control (PBS), 3.2 x 10 ¹⁰ , 3.2 x 10 ¹¹ and 3.2 x 10 ¹² k.g./kg IP (see next table). IP was administered on day 0, peak expression was observed on day 10, and a recovery period occurred on day 60. Safety assessments include body weight, clinical observations, serum chemistry and hematology at sacrifice, total necropsy, organ masses and tissue preservation at each sacrifice, histopathology of selected tissues only - lung, brain, liver, kidney, heart at high dose and controls . Histopathology in the medium and low dose group is performed only if abnormalities are observed at the highest dose. Expression of the two transgenes was assessed by sandwich ELISA at the time of sacrifice in nasal lavage fluid (NLF), BALF and serum. In immunological

- 27 041088 studies assess the levels of anti-drug antibodies (ADA) by ELISA (transgenes and capsid), and the levels of T-cell responses are assessed in the spleen.

Non-GLP Pilot Toxicology Study in Immunocompetent CD 1 Mice

Dose (kg/kg) Moment necropsy (day) Number of male CD1 mice per group Number of female CD1 mice per group 3.2x1O ¹⁰ 10 5 5 60 5 5 3.2x10 ^s 10 5 5 60 5 5 3.2x10 ¹² 10 5 5 60 5 5 PBS 10 5 5 60 5 5

B. Toxicological study in immunodeficient mice.

This toxicological study was carried out as the maximum dose suggested for humans, which is approximately 3.2x1011 kg/kg, in RAG-1 KO mice, and the middle dose group used for previous studies. Group size is 10 animals per time point, per sex, with sacrifice on days 10 and 60. IP is administered in a volume up to 10 μl/nostril.

Non-clinical toxicology study in immunodeficient mice

Dose Moment necropsy Number of male Rag-Ι mice per group Number of female Rag-Ι mice per group 3.2x1ο ¹¹ kg/kg 10 10 10 60 10 10 PBS 10 10 10 60 10 10

One dose group, 20 animals per time point dose (10 males and 10 females), dose IP 3.2x1ο ¹¹ kg/mouse. IP was introduced on day 0, peak expression on day 10, and recovery period on day 60. Safety evaluations include body weight, clinical observations, serum chemistry and hematology at sacrifice, total necropsy, organ masses and tissue preservation at each sacrifice, histopathology of selected tissues only (listed in Tables 8.8), low-dose group histopathology performed, only if abnormalities are observed at the highest dose. Expression of the two transgenes is assessed by ELISA in both NLF and BALF.

C. Toxicity and biodistribution studies in non-human primates.

This study is performed at a dose that is 10 times higher than the highest dose delivered in human studies using the same device and volumes used in the human study when normalized according to total body weight. In the human study, the highest dose is 3.2x1011 kg/kg (total 2.4x10 ¹³ kg for a 75 kg human), administered as 2x0.2 ml/nostril. NHP receive 3.2 x 10 ¹² k.g./kg (1.28 x 10 ¹³ k.g. for a 4 kg macaque), which is the highest dose that can be administered using the IMAD device in volumes intended for humans, which is 2. 4x10 ¹³ kg administered as 2x0.2 ml/nostril.

Toxicity and biodistribution studies are performed in rhesus monkeys. This study uses 6 males and 6 females for a total of 12 rhesus monkeys. Three time points are assessed, naive animals are sacrificed on day 0 prior to IP administration, day 10 corresponds to peak expression, and day 60 corresponds to the long-term recovery period.

At each point in time, two males and two females are killed. Toxicity is assessed by evaluation of SHS/chemical panels.

Example 8 Evaluation of the intranasal delivery device.

The next group of studies assesses the ability of AAV9 expressing antibodies against influenza A and B (FI6 and CR8033, respectively) in conjunction with the LMA® MAD Nasal™ atomizing device for intranasal mucosa (IMAD) to effectively protect mice from pandemic or seasonal influenza infection. First, it was found that an equimolar combination of AAV9.FI6 and AAV9.CR8033 vectors delivered intranasally (in) at a dose of 5x10 ¹⁰ k.g./KG (or

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1x10 ⁹ kg) per mouse effectively protected mice from lethal infection with influenza A (adapted to H1N1, PR8 mice) or influenza B (adapted to B/Lee/40 mice), respectively (FIGS. 11A, 11D, 11G and 12A , 12D). For the purpose of clinical advancement of AAV9, the compatibility of the vector with IMAD was assessed by the loss of volume and activity of the vector. For these studies, 0.5 ml of the vector suspension was passed through the IMAD in order to evaluate the physical loss of the vector solution, and also to evaluate the effect of the device on vector activity. As indicated in FIG. 12G, a physical fluid loss of 4-6.6% of the initial volume was observed when the vector solution was treated with IMAD. The inventors also evaluated three different vector formulations: PBS-pH 6.8, PBS-pH 7.2 or PBS-pH 7.4 for compatibility with the vector. Groups of mice were injected with a mixture of 5x101° k.g./KG (or 1x10 ⁹ k.g.) of each of AAV9.FI6 and AAV9.CR8033, as well as a mixture of 5x101° k.g./KG (or 1x1° ⁹ k.g.). g.) each of AAV9.FI6 and AAV9.CR8033, which was processed using IMAD. After 7 days, mice were challenged with 5 LD ₅₀ PR8 or B/Lee/40 (FIGS. 11A, 11D, 11G and 12A, 12D). No difference in protection against influenza A or B was observed when the mixture of vectors was treated with IMAD (dark gray squares). Additionally, the use of IMAD is compatible with the three different formulations, and no decrease in efficiency was observed when the vector that was prepared with each of the three formulations was treated with IMAD. Only mice injected with the vector survived infection. At the end of the experiment (day 21), mice were sacrificed and bronchoalveolar lavage fluid (BALF) was collected in order to evaluate antibody expression. Lungs were also taken in order to estimate the amount of vector genome present at the time of necropsy. No differences were observed in antibody expression on airway surfaces when the vector was delivered as a liquid or as a liquid after treatment with IMAD (FIGS. 11B, 11E, 11H and 12B, 12E). In addition, no significant differences were observed in the number of AAV9 vector genomes in the lungs.

This approach was then transferred to the monkey nose, a model that is more closely related to the human nose, which is the target tissue. This model assessed the kinetics of onset of transgene expression in nasal lavage fluid (NLF) and the stability of expression when the AAV9 vector was delivered via IMAD compared to traditional direct fluid delivery. Immune responses against the human antibody FI6 or CR8033 distort the experiment and the interpretation of the results. As such, self antigen (rhesus α-fetoprotein, rhAFP) was used as the reporter transgene in these studies. The expression of rhesus α-fetoprotein (rhAFP) is labeled here with the presence of a plus (+) sign, since accurate quantification of the antibody on mucosal surfaces is limited by the accuracy of NLF sampling (FIG. 13). At the end of the study, the biodistribution of the vector was also studied. Overall, no significant differences were observed between the two delivery methods in the kinetics and expression profile of the gene or the biodistribution of the AAV9 vector.

In conclusion, AAV-mediated prophylaxis of influenza A and B is safe and effective when vectors are delivered via an easy-to-use IMAD device that localizes transduction to the site of influenza infection, the nasal mucosa.

Example 9 Initial phase 1 study design.

The aim of the phase 1 study is to assess preliminary safety and tolerability, to understand the pharmacokinetics of IP in healthy volunteers, and to select a dose level for further development. The study is an open dose escalation study at dose levels based on preclinical data. Up to five cohorts are enrolled in the study, with four subjects per cohort. Subjects are screened for 4-12 weeks prior to treatment and observed for 6 months.

Each volunteer receives one dose of 0.8 ml IP IN through two consecutive 0.2 ml/nostril applications in each nostril using an approved IMAD. Up to twenty-eight (24) volunteers are included in five cohorts. Cohorts 1-4 receive increasing dose levels of IP, and the final cohort (cohort 5) is expanded to 8 volunteers at the optimal dose level (as determined by safety and pharmacokinetic parameters in cohorts 1-4).

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Initial Phase 1 Study Design in Healthy Adult Volunteers

Cohort N active Dose level of AAV2/9 transgene product (k.g.) Total volume to be injected (ml) Route of administration 1 4 3, OhYu ¹² 0.2 ml in each nostril; two application series, only 0.8 ml IN 2 4 6, wow ¹² IN 3 4 1.2x10 ¹³ IN 4 4 2.4x10 ¹³ IN 5 8 Optimal dose 0.2 ml in each nostril; IN two application series, only 0.8 ml Total 24

A. Inclusion/exclusion criteria for the target population.

Healthy adults aged 18-45 years of either sex with a body mass index of 19-30 kg/m ² and a body weight of 50-100 kg are entered into the study in order to evaluate the safety, immunological and pharmacokinetic parameters of GTP101.

Inclusion Criteria

Men or women aged 18-45 inclusive.

Able to give written informed consent to participate.

Healthy as determined by history, physical examination, vital signs, and clinical laboratory safety studies at baseline.

Non-Tobacco (Tobacco addicts are people who smoke more than 4 cigarettes or other tobacco products per week) and agree not to use tobacco products while participating.

Women must meet one of the following criteria:

at least one year after menopause;

surgically sterile;

will use oral, implantable, transdermal or injectable contraceptives within 30 days before IP administration and within 60 days after vaccination;

wish to abstain from sexual intercourse or use another reliable form of contraception approved by the investigator (eg, intrauterine device (IUD), female condom, spermicidal diaphragm, cervical cap, sexual partner use of a condom, or sterile sexual partner) from screening to study day 28.

Women of childbearing potential should have a negative urine pregnancy test within 24 hours before IP administration.

Understanding the requirements of the study, expressed availability during the required study period.

Exclusion Criteria

Significant history of seasonal hay fever or seasonal allergic rhinitis or perennial allergic rhinitis or a chronic nasal or sinus condition.

History of asthma requiring treatment up to 1 year prior to IP administration.

Subjects with abnormal nasal structure including deviated septum and nasal polyps, chronic sinusitis, asthma, or COPD.

Current use of intranasal steroids.

The presence of significant uncontrolled medical or psychiatric illness (acute or chronic) as assessed by the investigator. This includes, but is not limited to, initiation of a new medical or surgical treatment, or a major dose change for uncontrolled symptoms, or drug toxicity within 3 months of screening and reconfirmation on day 0 before infection.

Positive serology for HIV-1 or HIV-2, or HBsAg or anti-HCV antibodies.

Malignancy or cancer treatment within 3 years, except for basal cell carcinoma or squamous cell carcinoma, which is acceptable.

The presence of immunosuppression or any medical condition that may be associated with impaired immunological reactivity, including but not limited to diabetes mellitus.

Current (or history of) receipt, during the previous 3 months, of any medication or other treatment that may undesirably affect the immune system, such as allergic injections, immunoglobulin, interferon, immunomodulators, cytotoxic drugs, or others medicinal products known to be frequently associated with significant major organ toxicity, or systemic corticosteroids (oral or injectable). Topical corticosteroids are acceptable.

History of drug or chemical dependence in the year before the study.

Intake of any investigational product or unregistered medicinal product within 30 days prior to IP administration, or current participation in, or intention to participate in, any investigational medicinal product study within a subsequent study period.

Receipt of blood or blood products within 6 months prior to IP administration or planned administration during the study period.

Acute illness within 72 hours prior to IP administration, defined as the presence of moderate or severe illness with or without fever (as determined by the investigator by history and physical examination) or the presence of fever >38°C orally.

Pregnant and/or lactating women.

Serum antibodies to AAV2/9 >1:80.

Any condition that, in the opinion of the investigator, may interfere with the main objectives of the study and assessments.

B. Determination of the dose level.

Starting dose levels were selected based on preclinical effect data and adjusted based on non-clinical safety assessments, with the highest dose limited by AAV aggregation properties. Doses range from 3.0 x 10 ¹² to 2.4 x 1013 kg/dose administered as 4 x 0.2 ml/nostril via IMAD. These doses are substantially lower than those given to monkeys, mice and ferrets when adjusted for total body weight. No significant toxicity was observed in any of the non-clinical studies that have been performed in these species at any dose using IP or IP structurally similar versions of AAV2/9 scheduled for clinical study.

C. Dose administration and duration.

Each dose group receives an IP of 0.4 ml per nostril via IMAD. Subjects are dosed one after the other. Treatment intervals in the same dose group are determined by non-clinical studies, but for reproduction purposes are 1 week. When all subjects in one cohort have completed day 14 of the study, consider preliminary safety data. If safety issues are not identified, dose escalation to the next dose is initiated. After completion of cohort 4 and preliminary demonstration of safety for the highest dose, the fifth and final cohort is included in the study to expand to 8 subjects in total (i.e. 4 additional subjects) at the optimal dose, which is the maximum tolerated dose or the highest dose that can be enter.

The product administered to patients is a mixture of two AAV2/9 vectors that were combined at the time of filling/completion. The administration of IP occurs in two consecutive doses of 0.2 ml in each nostril (i.e. a total of 0.8 ml of IP). IP is administered using a commercially approved device manufactured by Teleflex Medical (Teleflex.com). The device is called the LMA MAD NASAL™ (atomizing device for the lining inside the nose). Details on how to best use the device to deliver IP to the nasal mucosa are provided in the company's Procedure Guide. In general, the subject is placed in a supine position and the tip of the device is placed in the orifice of each nasal passage (one at a time), after which the IP is delivered by pushing the fluid through the atomizer via a syringe.

The maximum tolerated dose (MTD) is defined as the dose below the dose at which subjects exhibit dose-limiting toxicity (DLT), defined as either a Grade 3 treatment-related event in two subjects or one Grade 4 treatment-related event.

The application for this invention contains sequences and a list of sequences, which are thus incorporated by reference. Also incorporated herein by reference are U.S. Provisional Application No. 62/323,348, filed April 15, 2016, and U.S. Provisional Application No. 62/161,192, filed May 13, 2015. All publications, patents, and patent applications cited in this document , are hereby incorporated by reference in their entirety, as if each individual publication or patent application were specifically and individually incorporated by reference. Although the above invention has been described in some detail for purposes of illustration and example for the sake of clarity of understanding, those skilled in the art, in light of the teachings of this invention, will readily appreciate that certain changes and modifications can be made therein without departing from from the spirit or scope of the appended claims.

The following information is provided for sequences containing free text under the numeric identifier <223>.

Sequence list, free text

SEQID No. Free text in <223> 1 <220> <223> Plasmid carrying FI6cc germline light chain HC <220> < 221> repeat area < 222> (1)..(130) < 223> 5'ITR <220> < 221> repeat area <222> (198) . . (579) <223> IE CMV promoter <220> <221> promoter < 222> (582)..(862) <220> < 221> TATA signal < 222> (836)..(839) <220> <221> Intron <222> (956) . . (1928) <223> chicken beta-actin intron <220>

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<221> heterogeneous signs <222> (1940). . (1987) <223> c-myc 5'UTR <220> <221> heterogeneous signs <222> (1988). . (1992) <223> Kozak <220> <221> heterogeneous signs <222> (1993). . (2052) <223> leader <220> <221> CDS <222> (2053). . (2439) <223> variable severe FI6 <220> <221> CDS <222> (2440) . . (2760) <223> FI6 CHI <220> <221> CDS <222> (2761) . . (3426) <223> FI6 CH2-3 <220> <221> heterogeneous signs <222> (3427) . . (3438) <223> furin cleavage site <220> <221> heterogeneous signs <222> (3439)..(3510) <223> F2A <220> <221> heterogeneous signs <222> (3511)..(3570) <223> leader <220> <221> CDS <222> (3511)..(3570) <223> leader <220> <221> CDS <222> (3571)..(3909)

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< 223> kagga germline <220> <221> CDS < 222> (3910) .. (4230) <220> < 221> repeat area <222> (4308) . . (4432) <223> rabbit polyA globin <220> < 221> repeat area <222> (4521) . . (4650) < 223> 3'ITR <220> < 221> heterogeneous features <222> (4827) . . (5265) < 223> Fl ori on the complementary chain <220> < 221> heterogeneous features <222> (5294) . . (5936) <223> origin of replication <220> < 221> heterogeneous features <222> (6611) . . (7426) < 223> CDS for cap resistance located on complementary chain 8 <223> A cis plasmid containing the CR8033 heavy chain and germline kappa light chain <220> < 221> heterogeneous features < 222> (264)..(287) <223> CMV promoter end <220> <221> promoter < 222> (285)..(565) <223> CB promoter <220> < 221> heterogeneous features < 222> (288)..(318) <223> promoter start <220> < 221> TATA signal <222> (539) . . (542)

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<220> <221> Intron <222> (659) . . (1631) <223> chicken beta-actin intron <220> < 221> heterogeneous features <222> (1517) . . (1533) <223> end of intron <220> < 221> heterogeneous features < 222> (1643)..(1690) < 223> c-myc 5' UTR <220> < 221> heterogeneous features <222> (1691) . . (1698) < 223> Kozak <220> <221> CDS < 222> (1753)..(2133) < 223> variable heavy CR8033cc <220> <221> CDS <222> (2134) . . (2454) <223> CHI <220> <221> CDS < 222> (2455) .. (3120) <223>CH2-3 <220> < 221> heterogeneous features <222> (3121) . . (3132) <223> furin cleavage site <220> < 221> heterogeneous features <222> (3133) . . (3204) < 223> linker F2A <220> <221> CDS <222> (3265) . . (3603) < 223> mild germline kappa <220> <221> CDS

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Claims (19)

<222> (3604) . . (3924) < 223> constant light <220> < 221> polyA signal <222> (4000) . . (4126) <220> < 221> repeat area <222> (4215) . . (4344) < 223> 3' ITR (on complementary) <220> < 221> heterogeneous features < 222> (4521)..(4959) < 223> Fl ori on the complementary chain <220> < 221> heterogeneous features < 222> (4988)..(5630) <223> pUC origin region <220> < 221> heterogeneous features < 222> (7363) .. (7548) <223> Lac promoter <220> < 221> repeat area <222> (7603) . . (7732) < 223> 5'ITR <220> < 221> heterogeneous features <222> (7806) . . (7835) <223> promoter start 14 <223> contains leader-whole heavy chain-linker F2A furin-leader-whole light chain 15 <223> 1F16 1 leader-whole heavy chain-linker F2A furina-leader-whole light chain 16 <223> F16 26 17 <223> F16 28 18 <223> cDNA F16 30 19 <223> cDNA F16 35 21 <223> cDNA F16 22 <223> cDNA F16 42 23 <223> F16 B42 24 <223> mutated IL-2 25 <223> mutated IL-2 CLAIM 1. Composition for passive immunization against influenza infection, where the composition contains: (A) the first non-replicating recombinant adeno-associated virus with an AAV9 capsid (rAAV9) and a vector genome that contains: (a) AAV inverted terminal repeat (ITR), (b) enhancer, (c) chicken beta-actin promoter, (d) intron, (e) 5'-UTR, (f) nucleic acid sequence encoding the leader peptide, functionally associated with the FI6v3 heavy chain variable region (g), which together with (h) forms an immunoglobulin heavy chain, (g) a nucleic acid sequence encoding the FI6v3 heavy chain variable region, which contains nucleotides 2053-2439 of SEQ ID NO: 1, (h ) a nucleic acid sequence encoding a human Ch1 domain and a human IgG1 Fc chain - 36 041088 Lovec (CH2-3), (i) a nucleic acid sequence encoding a leader peptide operably linked to an immunoglobulin light chain (j), (j) a nucleic acid sequence encoding an immunoglobulin light chain, (k) a furin recognition site, and an F2A linker located between the heavy chain coding sequences and the light chain coding sequences, (l) a polyadenylation signal and (m) an AAV inverted terminal repeat; (B) a second non-replicating rAAV9, where the second rAAV9 has a vector genome that contains: (a) AAV ITR, (b) enhancer, (c) chicken beta-actin promoter, (d) intron, (e) 5'-UTR, (f) nucleic acid sequence encoding the leader peptide operably linked to the CR8033 heavy chain , (g) and (h), (g) a nucleic acid sequence encoding the heavy chain variable region of CR8033, which contains nucleotides 1753-2133 of SEQ ID NO: 8, (h) a nucleic acid sequence encoding a human CH1 domain and an Fc chain Human IgG1 (CH2-3), (i) nucleic acid sequence encoding a leader peptide operably linked to an immunoglobulin light chain (j), (j) nucleic acid sequence encoding an immunoglobulin light chain, (k) furin recognition site and F2A linker , located between the heavy chain coding sequences and the light chain coding sequences, (l) polyadenylation signal and (m) AAV ITR, and an aqueous liquid suspension base. 2. The composition of claim 1, wherein the vector genome in (A) is selected from SEQ ID NO: 1, 14, 15, or 16. 3. The composition of claim 1, wherein the vector genome in (B) contains SEQ ID NO: 8. 4. The composition according to claim 1, where the total concentration of rAAV9 in (A) and (B) is from about 1x1011 to about 6x10 ¹³ copies of the genome (kg)/ml, where k.g. determined by droplet digital PCR (ddPCR). 5. Composition according to claim 1, wherein the ratio of rAAV9 (A) and rAAV9 (B) is approximately 1:1. 6. The composition of claim 1 wherein the light chain (A) and/or (B) is a heterologous light chain. 7. The composition of claim 1, wherein the light chain (A) and the light chain (B) share a common source. 8. The composition of claim 1, wherein the light chain variable region is a germline antibody sequence with no defined specificity. 9. The composition of claim 8, wherein the light chain is a human germline kappa chain or kappa chain of IGKV4-1*101. 10. The composition of claim 1, wherein the 5'-UTR (A) or 5'-UTR (B) is independently selected from the human c-myc 5'-UTR. 11. The composition of claim 1, wherein the leader peptides (A) are heterologous with respect to immunoglobulin and wherein the leader peptides are different from each other. 12. The composition of claim 1, wherein the leader peptides (B) are heterologous with respect to immunoglobulin and wherein the leader peptides are different from each other. 13. A composition according to claim 11 or 12, wherein the leader peptide is the leader peptide of IL-2. 14. A method for inducing passive influenza immunization in a patient, comprising administering a composition according to any one of claims 1-13. 15. The method of claim 14 wherein the composition is formulated for intranasal, intramuscular, or intravenous administration. 16. The method of claim 14, wherein the patient is dosed in an amount of from about 1x10 ¹⁰ to about 3x1013 kg. 17. The method of claim 14 wherein the patient receives two consecutive injections of about 0.4 or about 0.2 ml/nostril. 18. A kit for passive immunization against influenza infection, containing a container with a composition according to any one of claims 1-13 and instructions for use. 19. Kit according to claim 18, also containing a diluent. -