Patent Application
20220332798
©2020 Oregon Health & Science University. A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. 37 CFR § 1.71(d).
Generally, the field relates to methods for producing antibodies against viral capsids. More specifically, the field relates to methods for producing monoclonal antibodies against adeno-associated virus (AAV), viruses in the family Parvoviridae, and other non-enveloped viruses by hydrodynamic-based transfection.
Monoclonal antibodies can serve as powerful scientific and therapeutic tools. However, effective animal immunization against an antigen, giving rise to the production of monoclonal antibodies, can be a very complicated, time-consuming, and expensive process. Generation of the antigen itself can also be similarly difficult process. Moreover, to be effective, traditional immunization methods often require that additional peptides, proteins or other adjuvants be added to the inoculum, risking side effects to the animal subject. And selecting an antibody with enough specificity to be deemed “monoclonal” requires the screening of large libraries of parent cells generated by the immunization. Thus, an immunization process that generates parent cells without the complexity, time, and expense of traditional methods facilitates the production of monoclonal antibodies, allowing for greater access to their scientific and therapeutic benefits.
The current disclosure provides a method to produce, in vivo, antibodies against viral capsids (VCs) that derive from a non-enveloped virus (NEV). Some of the embodiments are configured to antibodies against virus-like particles (VLPs). Step 1 of a preferred method administers, to a subject, by hydrodynamic-based transfection, a first set of genetic material encoding one or more NEV structural proteins that induces the subject's intracellular translation and assembly of the NEV proteins into one or more VCs. The subject, thereby, generates antibody-producing cells that secrete anti-VC antibodies. Step 2 of the preferred method includes optionally repeating step 1.
In addition, the disclosure provides a method containing the step of administering to the subject, by hydrodynamic-based transfection, a second set of genetic material encoding one or more NEV non-structural proteins for facilitating the subject's intracellular assembly of the NEV structural proteins into one or more VCs.
In addition, the disclosure provides a method containing the step of harvesting antibody-producing cells and/or anti-VC antibodies from the subject.
In addition, the disclosure provides a method containing the step of generating a plurality of hybridomas from the antibody-producing cells. The plurality of hybridomas is screened and one or more hybridomas from within the plurality is selected for culture and one or more of the selected hybridomas is cultured.
In some embodiments, step 1 of the method is repeated 1 to 10 times. In other embodiments, the time intervals between repeating step 1 are about 1 day to about 28 days.
In some embodiments, the first and second set of genetic material are at least one nucleic acid molecule chosen from a ssDNA, a dsDNA, a linear pDNA, a circular pDNA, a ssRNA, a dsRNA, a circRNA, a mRNA, an siRNA, a microRNA, and a sgRNA. In other embodiments, the first set of genetic material encodes at least one protein chosen from a NEV-based VP capsid protein, an AAV VP capsid protein, an AAV2 VP capsid protein, and an AAVAnc80 VP capsid protein. In further embodiments, the second set of genetic material encodes at least one protein chosen from NEV-based non-structural proteins that facilitate capsid assembly, an AAV-based AAP, an AAV2 AAP, and an AAPAnc80. In other embodiments, either the first or the second set of genetic material encode a gene expression cassette chosen from an enhancer, a promoter, or a polyadenylation signal.
In some embodiments, the non-enveloped virus is classified within the family Parvoviridae, or is an adeno-associated virus (AAV), a synthetic AAV, or an AAV vector. In other embodiments, the AAV vector is an AAVAnc80 vector, an AAV2 vector, or an AAV9 vector.
In some embodiments, the hydrodynamic-based transfection is performed substantially free of vaccine additive.
In some embodiments the subject is an animal. In other embodiments, the animal is a rodent. In further embodiments, the rodent is a mouse.
In some embodiments, anti-VC antibodies substantially neutralize a NEV's infectivity. In other embodiments, anti-VC antibodies do not substantially neutralize a NEV's infectivity. In further embodiments, the VCs constitute one or more substantially assembled, or partially assembled, NEV viral capsids. In still further embodiments, the anti-VC antibodies are mouse monoclonal antibodies.
Additional aspects and advantages will be apparent from the following detailed description of preferred embodiments, which proceeds with reference to the accompanying drawings.
Disclosed are novel DNA vaccine-based methods to immunize mice effectively without adjuvants. This method can be used for mouse monoclonal antibody production against assembled AAV viral capsids, and more broadly for any parvoviral or non-enveloped capsids as long as capsids can assemble in the mouse body by delivering genetic materials as nucleic acids coding viral components into the body. By delivering such genetic materials into hepatocytes hydrodynamically by tail vein injection in mice, a total of 19 hybridoma clones were identified that produced mouse monoclonal antibodies that recognized assembled capsids. Among the 19 monoclonal antibodies, 6 were found to possess the ability to neutralize AAV infection while the other 13 were found to bind assembled capsid but did not neutralize viral infectivity. All of the 6 neutralizing antibodies and 8 out of the 13 non-neutralizing monoclonal antibodies recognized only assembled capsids and did not bind capsid monomers while 5 of the 13 non-neutralizing monoclonal antibodies could recognize both assembled capsids and capsid monomers. Six monoclonal antibodies were affinity purified for downstream use for various purposes including ELISA, immunoblot, immunofluorescence microscopy, and so on. The sites of plasmid DNA delivery and subsequent viral capsid assembly could be organs and cell types other than the liver, such as skeletal muscles. The method reported here is extremely cost-effective and therefore could become a standard method for production of monoclonal antibodies against viruses in the family Parvoviridae including AAV and other non-enveloped viruses. In addition, the new reagents described herein could be used to create AAVAnc80-specific capsid titration ELISA kits, which do not currently exist.
The present disclosure provides novel methods to generate mouse monoclonal antibodies against assembled capsids of viruses in the family Parvoviridae that are neutralizing or non-neutralizing, by hydrodynamic-based transfection. “Hydrodynamic-based transfection,” as defined herein, is the process of introducing exogenous genetic materials into a subject by hydrodynamic delivery to enhance transfection of the genetic material and thereby facilitate intracellular transgene expression (Liu F. et al., 1999). In some embodiments, the hydrodynamic-based transfection results in the immunization of the subject as a consequence of the subject's intracellular transgene expression of the exogenous genetic material. “Hydrodynamic delivery”, as defined herein, is the application of controlled hydrodynamic pressure in capillaries to enhance endothelial and parenchymal cell permeability. (Suda T. et al., 2007). For example, hydrodynamic delivery includes the pressurized injection of a large volume of solution into a vasculature, such as by hydrodynamic tail vein injection in rodents. “Transfection,” as defined herein, is the process of introducing exogenous genetic material into eukaryotic cells. “Genetic material,” as defined herein, are nucleic acid molecules, examples of which include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), plasmid DNA (pDNA), linear pDNA, circular pDNA, single stranded RNA (ssRNA), double stranded RNA (dsRNA), circular RNA (circRNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), and single guide RNA (sgRNA).
In one embodiment, the hydrodynamic-based transfection relied on intravenous injection of plasmid DNAs expressing parvovirus structural proteins and non-structural viral proteins that are necessary and sufficient for viral capsid assembly in the body of a mouse, specifically in mouse liver and mouse hepatocytes in vivo. These methods do not require immunization of mice with viral particles or use of any adjuvant or proteins, which significantly simplifies the overall procedure of mouse monoclonal antibody production against assembled parvovirus capsids. Although AAV2 and AAVAnc80 were used for a proof-of-concept study, this disclosure provides methods that can be applied broadly to viruses in the family of Parvoviridae and other types of non-enveloped viruses that can be produced as virus-like particles (VLPs) in the body of a mouse, in mouse liver, and specifically in mouse hepatocytes, in vivo, by delivering genetic materials into the body of mice in vivo that code viral proteins. Although plasmid DNA was used for reduction purposes, genetic materials could be any forms of nucleic acids, including DNA and RNA. The method may also be used to produce mouse monoclonal antibodies against other non-enveloped viruses, and organs and cell types other than the liver and hepatocytes could be targeted for delivery of genetic materials to immunize mice and produce anti-viral capsid antibodies.
The present disclosure also provides antibodies that recognize assembled parvovirus capsids. For example, described herein are 19 novel mouse monoclonal antibodies, 1B11, 1C8, 2A7, 2F11, 3D6, 5B10, 1F10, 2B5, 2D9, 4A12, 4C2, 4D12, 4H10, 6E11, 1A11, 2E3, 4B7, 6F12, and 7F11, that were raised against assembled AAVAnc80 viral capsids and were produced by the method described above. The AAV capsids are composed of 60 subunits comprising VP1, VP2 and VP3 proteins at an approximately 1:1:10 stoichiometric ratio. The fully assembled capsids can be produced only with VP3 protein without packaging viral genomes, making VP3-only empty capsids, namely, virus-like particles (VLPs). VLPs have been widely used for immunizing animals and humans for the purpose of vaccination against infectious diseases and for the purpose of the production of monoclonal and polyclonal anti-viral neutralizing antibodies used as experimental reagents. It was discovered that AAV VP3-only particles (VLPs) can be produced in mouse hepatocytes and can be secreted in the blood circulation when hepatocytes are transfected with two plasmids, one expressing the VP3 protein and the other expressing the assembly-activating protein (AAP) under the control of the cytomegalovirus immediate early gene (CMV-IE) enhancer-promoter. The CMV-IE enhancer-promoter was selected because it is a widely-used ubiquitous and “strong” promoter; however, other enhancer-promoters can also be used for the purpose of immunization. Based on the discovery that AAV4, AAV5 and AAV11 capsid assembly does not require AAP proteins (Earley et al. 2017), it is presumed that VLPs of these serotypes can be produced by transfection of only one plasmid expressing the VP3 protein. It was also discovered that significant immune responses against assembled AAV capsids can be mounted by tail vein injection of the above-described plasmid DNAs without using any additional treatment such as administration of an adjuvant. Based on this discovery, a design was conceived of generating mouse monoclonal antibodies against assembled AAV viral capsids (and potentially any viruses in the family of Parvoviridae and more broadly any non-enveloped viral capsids) by hydrodynamic-based transfection of mice, which can simplify and streamline the procedure of producing mouse monoclonal anti-virus antibodies as well as reduce costs. Here, hydrodynamic-based transfection was employed to produce mouse monoclonal antibodies against AAVAnc80 capsids. As a result, the above-described 19 novel mouse monoclonal antibodies were identified to bind to assembled capsids. Among the 19 monoclonal antibodies, 6 were found to possess the ability to neutralize AAV infection. The other 13 were found to bind to assembled capsid but did not neutralize viral infectivity. All of the 6 neutralizing antibodies, and 8 out of the 13 non-neutralizing monoclonal antibodies, recognized assembled capsids only and did not bind to capsid monomers, while 5 of the 13 non-neutralizing monoclonal antibodies recognized both assembled capsids and capsid monomers. Isotypes have been determined for the following 9 monoclonal antibodies using Mouse Immunoglobulin Isotyping ELISA kit (BD Biosciences): 1B11 (IgG1-κ), 1C8 (IgG3-κ), 2A7 (IgG1-κ), 2F11 (IgG1-κ), 3D6 (IgG1-κ), 5B10 (IgA-κ), 1A11 (IgM-κ), 4B7 (IgG2a-κ), and 6F12 (IgM-κ). The following 6 monoclonal antibodies were purified by affinity chromatography using Thermo Scientific™ Pierce™ Protein G Agarose: 1B11, 2A7, 2F11, 3D6 and 5B10, which can react only with assembled AAVAnc80 capsids, do not recognize capsid monomers and have the ability to neutralize viral infectivity; and 4B7, which reacts with both assembled AAVAnc80 capsids and capsid monomers and is devoid of the ability to neutralize viral infectivity. Out of the 6 monoclonal antibodies, all but 2F11 did not cross-react with AAV2, AAV5 or AAV9 capsids. 2F11 showed cross-reactivity with AAV2 but did not bind to AAV5 or AAV9.
In certain embodiments, the present disclosure provides methods of producing antibodies against assembled viral capsid of a non-enveloped virus comprising the steps of (1) immunizing an animal with a first set of one or more genetic materials encoding one or more viral structural proteins for VLP formation and, when necessary or useful for VLP formation, a second set of genetic materials encoding one or more viral non-structural proteins that function in VLP formation and (2) harvesting antibodies and/or antibody-producing cells from the animal. A “set of genetic materials” may comprise one or more such sequences. In some embodiments, the animals are immunized by tail vein injection. In other embodiments, methods such as the use of lipid nanoparticles deliver the genetic materials to the animal's liver without using high pressure injection. In other embodiments, genetic materials are delivered to an animal's organ other than the liver such as skeletal muscle with high pressure injection or with methods such as the use of lipid nanoparticles. Methods of harvesting antibodies and antibody-producing cells from immunized animals are known in the art. The animal may be a rodent or other suitable animal. In certain embodiments, the animal is a mouse.
In certain embodiments, the antibodies produced by the methods described herein are monoclonal antibodies. Such methods may further comprise the steps of (3) generating a plurality of hybridomas from the antibody-producing cells harvested from the animal, (4) screening the plurality of hybridomas to determine which hybridomas produce antibodies that are able to bind to the fully-assembled viral capsid, and (5) culturing one or more hybridomas that are able to bind to the assembled viral capsid. Methods of producing and culturing hybridomas are known in the art.
In certain embodiments, the non-enveloped virus is a virus in the family Parvoviridae. In some embodiments, the non-enveloped virus is AAV. In particular embodiments, the AAV may be a primate serotype AAV vector. In some embodiments, a primate AAV may be derived from any known serotype, e.g., from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVhu37, AAVrh8, AAVrh10 and AAVrh74. In some embodiments, the non-enveloped virus is a synthetic AAV vector, for example, AAV-DJ, AAV-LK03, AAV-KP1 and AAVAnc80. In some embodiments, the non-enveloped virus is AAV and the second set of nucleic acid sequences encode one or more viral assembly activating proteins (AAPs).
In certain embodiments, at least one of the genetic materials used in the immunization are DNA molecules. In certain embodiments, at least one of the genetic materials are RNA molecules. In certain embodiments, the genetic materials encode a gene expression cassette such as an enhancer, a promoter, or a polyadenylation signal. “Gene expression cassette,” as defined herein, is a region of genetic material that has a sequence motif that regulates transcription, “Enhancer,” as defined herein, is a region of genetic material having a sequence motif that regulates transcription by increasing the likelihood that transcription will occur. “Promoter,” as defined herein, is a region of genetic material having a sequence motif that regulates transcription by binding to proteins involved in the initiation of transcription. “Polyadenylation signal,” as defined herein, regulates the addition of a poly(A) tail to mRNA.
In certain embodiments, the immunization is performed substantially free of vaccine additive. “Vaccine additive,” as defined herein, are ingredients included in an inoculum to enhance subject immunity, ensure vaccine safety and activity, and facilitate the vaccine's production. Vaccine additives may include preservatives such as Thimerosol, adjuvants such as aluminum salts, stabilizers such as sugars or gelatin, cell culture material such as egg protein, or inactivating ingredients such as formaldehyde.
In certain embodiments, the immunization is performed without using any adjuvant. In certain embodiments, material used for the immunization is substantially free from exogenous peptides and protein. “Substantially free from” means that any peptides or proteins are present only in inconsequential amounts, that is, in amounts that are not expected to have any effect on the generation of the animal's immune response. In certain embodiments, the animal is immunized using tail vein injection. In certain embodiments, the animal is immunized using intramuscular injection. In certain embodiments, the animal is immunized using intravascular injection via a vein other than the tail vein or via an artery. In certain embodiments, the animal is immunized using subcutaneous injection. In certain embodiments, the animal is immunized using intraperitoneal injection. In certain embodiments, the animal is immunized via injection into cerebrospinal fluid. In certain embodiments, the animal is immunized via retrograde intraductal injection. In certain embodiments, the animal is immunized by direct intraparenchymal injection.
The present disclosure also provides antibodies that recognize assembled viral capsids. Such antibodies may or may not have neutralizing ability. Neutralizing ability may be determined using methods known in the art. In certain embodiments, the antibodies are specific for a particular viral serotype, variant, or mutant, and do not substantially cross-react with other viral serotypes, variants, or mutants. The present disclosure also provides kits comprising such antibodies that may be used, for example, for ELISA, immunoblot, or immunofluorescence microscopy.
The following examples are for illustration only. In light of this disclosure, those of skill in the art will recognize that variations of these examples and other embodiments of the disclosed subject matter are enabled without undue experimentation.
AAV vector transduction mechanisms and the mechanisms of AAV capsid assembly were studied, including the roles and functions of AAP (assembly activating protein) in AAV virion formation. The present disclosure is based, in part, on the novel conception that AAV capsid assembly can take place in the mouse liver when AAV VP and AAP proteins are co-expressed in hepatocytes by DNA transfection and that assembled capsids can be secreted into the bloodstream and effectively immunize mice without infecting the mice with AAV viral particles. It was hypothesized that in vivo production of assembled capsids in the liver and their secretion into the bloodstream for AAP-independent serotypes (e.g., AAV4, AAV5 and AAV11) did not require co-expression of AAPs.
To investigate, 3 sixteen-week-old male C57BL/6J mice were injected via the tail vein using a hydrodynamic injection technique (e.g., HTV, which stands for Hydrodynamic Tail Vein injection) with a mixture of 50 μg of pCMV-AAV2VP3 and 50 μg of pCMV-AAP2 plasmids in a volume of 100 mL/kg saline. The injection was completed within less than 5 seconds (i.e., HTV). pCMV-AAV2VP3 and pCMV-AAP2 express AAV2 VP3 capsid protein and AAV2 AAP protein (i.e., AAP2) under the CMV-IE enhancer-promoter. There was one control animal that did not receive the treatment. Whole blood samples and the livers were collected three days post-injection. Using an AAV2 capsid-specific ELISA (AAV2 Titration ELISA, PROGEN), the concentrations of AAV2VP3 virus-like particles (VLPs) in the whole blood and the amount of VLPs in 100 mg liver tissue were determined. To prepare the liver tissue extract potentially containing VLPs, liver tissues were minced and incubated at 37° C. for 30 min in 140 mM NaCl, 5 mM KCl, 0.7 mM K2HPO4, 25 mM Tris-HCl, and 0.5% sodium deoxycholate according to the method reported by Walz et al, (Walz, Anisi et al. 1998). The liver extracts were adjusted to 200 mg liver tissue in 1 mL lysate. The data demonstrated that AAV2VP3 capsid assembly took place in the liver (as shown in
Next, it was investigated whether the above-described method could trigger a significant immune response and produce antibodies against AAV2VP3 VLPs, which may or may not neutralize AAV2 infectivity. To this end, 5 ten-week-old BALB/cJ female mice were injected by HTV with a mixture of pCMV-AAV2VP3 and pCMV-AAP2 plasmids in the same manner as described above. The same injection was repeated at day 14 and day 21. Following the third plasmid injection, serum samples were collected and anti-AAV2 neutralizing antibodies (NAbs) were analyzed by our standard NAb assay (Adachi et al. Sci Rep 2020). In brief, following heat-inactivation of the serum samples at 56° C. for 30 min, the resulting serum samples were diluted with Dulbecco's Modified Eagle Medium (DMEM) at 1:10, 1:100, 1:1,000 and 1:10,000 ratios. Ten μL of each diluted serum sample and 10 μL of AAV2-CMV-luc (i.e., an AAV2 vector expressing the firefly luciferase gene product under the control of the CMV-IE enhance-promoter) preparation containing 1×109 vg were incubated for 1 h at 37° C. After pretreatment with the wild-type adenovirus type 5, human embryonic kidney (HEK) 293 cells were treated with each test sample prepared as described above. Forty-eight hours later, the luciferase gene expression levels were quantified by the Bright-Glo™ Luciferase Assay kit (Promega). The neutralizing titer of the samples was determined as the highest dilution at which 50% or greater inhibition of the luciferase expression was observed.
As shown in Table 1, all five of the animals developed neutralizing antibodies against AAV2 following three plasmid injections. The titers ranged between dilutions of 1:1,000 and 1:10,000.
pCMV-AAVAnc80VP3 and pCMV-AAPAnc80 plasmids were constructed, and these plasmids were tested for AAVAnc80VP3 VLP production in HEK293 cells. It was found that AAVAnc80 VP3 only capsid can be assembled in HEK293 cells when AAV2 AAP (AAP2) or some of the other AAPs (AAP1, 3, 7, 8 and 9) are co-expressed, while the assembly did not take place in the absence of AAP (
Although AAPAnc80 and other AAPs can also support AAVAnc80 VP3 capsid assembly, it was found that AAP2 supplementation results in higher VLP production than AAPAnc80 supplementation (data not shown). Based on this, the combination of pCMV-AAVAnc80VP3 and pCMV-AAP2 were used for DNA vaccination to produce antibodies against AAVAnc80 capsids.
Example 3—DNA Vaccination to Immunize Mice With Assembled AAVAnc80 Viral Capsids
pCMV-AAVAnc80VP3 and pCMV-AAP2 plasmid DNAs were grown in E. coli using the standard method, extracted and purified by a commercially available plasmid extraction kit. The plasmids were then 0.45-μm filtered. To immunize mice with AAVAnc80 viral capsids, 10 nine-week-old BALB/cJ female mice were injected with pCMV-AAVAnc80VP3 and pCMV-AAP2 plasmid DNAs (50 μg each in 100 mL/kg of saline) via the tail vein using a hydrodynamic injection technique at days 0, 14, 21, 28 and 35, similar to the method reported by Bates et al. (Bates, Zhang et al. 2006). Three untreated animals were also included as controls. At the three-week time point after the third plasmid injection, serum samples were collected and anti-AAVAnc80 antibody titers were determined by an AAVAnc80 capsid antibody ELISA and a HEK293-based NAb assay using an AAVAnc80-CMV-luc vector. For the AAVAnc80 capsid antibody ELISA, Nunc MaxiSorp™ 96 well ELISA plates were coated with 1×109 vg of AAVAnc80 vector per well (either AAVAnc80-CMV-luc or AAVAnc80-CMV-GFP). Serum samples were diluted at 1:10 with an ELISA dilution buffer and used for ELISA in duplicate. The results demonstrated that all 10 animals developed ELISA-positive antibodies against AAVAnc80 capsids (
In the ELISA shown in
Table 2 represents the neutralizing antibody titer of the samples, which was determined as the highest dilution at which 50% or greater inhibition of the luciferase expression was observed.
At the 6-week time point, 2 mice showing high AAVAnc80 NAb titers were selected for generation of monoclonal antibody-producing hybridoma cell lines. The spleens harvested from these two mice were sent to the Monoclonal Antibody Core at Oregon Health & Science University (OHSU). Splenocytes were then isolated and fused with a mouse myeloma cell line using a standard method. Approximately 700 hybridoma clones were screened for antibody production by an AAVAnc80 capsid antibody ELISA and AAVAnc80 capsid monomer antibody ELISA, resulting in the identification of a total of 19 clones producing monoclonal antibodies (
Through further characterization of the 19 monoclonal antibodies, including a cell-based NAb assay, it was found that the monoclonal antibodies may be categorized into the following three distinct groups: (Category 1) Monoclonal antibodies that can recognize only assembled capsids and do not react with capsid monomers, and have the ability to neutralize viral infectivity (6 clones; 1B11, 1C8, 2A7, 2F11, 3D6, and 5B10); (Category 2) Monoclonal antibodies that recognize only assembled capsids and do not react with capsid monomers, and do not have the ability to neutralize viral infectivity (8 clones; 1F10, 2B5, 2D9, 4A12, 4C2, 4D12, 4H10, and 6E11); and (Category 3) Monoclonal antibodies that recognize both assembled capsids and capsid monomers, and do not have the ability to neutralize viral infectivity (5 clones; 1A11, 2E3, 4B7, 6F12, and 7F11) (
Immunoglobulin classes, isotypes, and light chains of nine monoclonal antibodies were determined (as shown in Table 3). Four of the monoclonal antibodies showed IgG1-κ, one showed IgG2a-κ, one showed IgG3-κ, one showed IgA-κ, and two showed IgM-κ. Anti-AAVAnc80 capsid monoclonal antibodies of IgA and IgM classes all showed weak binding to the antigen. The IgM class monoclonal antibodies showed cross reactivity to other serotypes. The IgG1 isotype was most commonly found among the identified monoclonal antibodies and all of them had the ability to neutralize infectivity. One monoclonal antibody that bound both assembled capsid and capsid monomer was an IgG2a antibody.
To purify 6 select monoclonal antibodies from hybridoma culture media, each corresponding hybridoma cell line was grown in 200 mL culture. Affinity chromatography purification of monoclonal antibodies was carried out at the OHSU Monoclonal Antibody Core using Thermo Scientific™ Pierce™ Protein G Agarose and according to the Core's standard purification protocol. Table 4 summarizes the concentration of immunoglobulins in each purified monoclonal antibody reagent.
Cross-reactivity of anti-AAVAnc80 capsid monoclonal antibodies was tested by anti-AAVx capsid antibody ELISAs (x=Anc80, 2, 5, and 9). Among 9 monoclonal antibodies tested, the two IgM monoclonal antibodies cross-reacted with AAV2, AAV5, and AAV9 capsids (Table 3). One IgG1 monoclonal antibody (2F11) cross-reacted with AAV2 capsid but did not bind to AAV5 or AAV9 capsids. In contrast, monoclonal antibodies 1B11, 2A7, 4B7, and 3D6 only reacted with AAVAnc80 capsids among the four AAV capsids tested (as shown in
Affinity-purified anti-AAVAnc80 capsid monoclonal antibodies 2A7 (2.6 mg/mL) and 1B11 (3.1 mg/ML) were diluted by 1,000, 10,000, 100,000, and 1,000,000-fold. Ten μL of the diluted moAb samples were reacted with 109 vg of AAVAnc80-CMV-luc in a 20 μL reaction volume and incubated at 37° C. for 1 h. Meanwhile, 2×104 CHO-K1 cells/well in a 96-well plate were infected with human adenovirus type 5 and incubated at 37oC for 1 h. The moAb/AAV mixtures were then added to the cells together with media to make a total of 200 μL/well. Cells were incubated two days to allow for AAVAnc80-CMV-luc infection and expression of luciferase. Luciferase expression levels were then determined by Bright-Glo Luciferase Assay detection kit (Promega) and expressed as relative light units (RLUs). RLU values were used to determine relative transduction efficiencies of tested conditions, which are values normalized with the RLU values obtained from the AAV only (no moAb) controls. NAb titer was defined as the highest dilution that suppressed the relative transduction efficiency by >50%. With this definition, the affinity-purified moAbs, 2A7 and 1 B11, had NAb titers of >1:10,000 (
With the purified and characterized anti-AAVAnc80 capsid monoclonal antibodies 2A7 and 1B11 in hand, it was sought to establish AAVAnc80 capsid titration ELISA kits. AAVAnc80 capsid can be recognized by anti-AAV2 capsid mouse monoclonal antibody A20 and anti-AAV8 capsid mouse monoclonal antibody ADK8 via these monoclonal antibodies' non-specific cross-reactivity across different serotypes. A20 and ADK8 are commercially available through several vendors including PROGEN. Although AAVAnc80 capsid titration by ELISA might be possible using these cross-reacting non-AAVAnc80 mouse monoclonal antibodies, no AAVAnc80 capsid titration ELISA kits existed that used anti-AAVAnc80 capsid-specific monoclonal antibodies. To establish anti-AAVAnc80 capsid moAb-based sandwich ELISAs for AAVAnc80 capsid titration, biotinylated 2A7 and 1B11 moAbs were first generated. To this end, purified 2A7 and 1B11 moAbs were dialyzed against phosphate-buffered saline (PBS) without calcium and magnesium (PBS(−)) to remove sodium azide (NaN3) contained in the moAb stocks. Each NaN3-free moAb was biotinylated by spontaneous reaction of its primary amines with Biotin-7-NHS, using a commercially available kit (Biotin Protein Labeling Kit, Millipore Sigma). The manufacturer's instructions for reaction incubation and column chromatography to elute biotinylated moAb were followed. Eluted protein concentration was measured by absorbance on a BioTek Epoch, using the “IgG” function. After pooling the top biotinylated protein-containing fractions, 1% bovine serum albumin (BSA) was added to increase the overall protein concentration and prevent loss of moAb due to nonspecific binding to tube walls, according to the manufacturer's recommendation. 0.05% NaN3 was then added to prevent growth of contaminants during storage at 4° C.
Sandwich ELISAs to quantify AAVAnc80 particles were then developed. In brief, an anti-AAVAnc80 moAb (either 2A7 or 1B11) was coated onto the bottom of MaxiSorp™ Immuno Clear Standard Modules in a 0.1M carbonate-bicarbonate buffer and incubated overnight at 4° C. (100 ng/well). After being washed, plates were blocked with the blocking/sample dilution buffer at 37° C. for 1 h. After the plates were washed, known amounts of AAVAnc80 particles in the blocking/sample dilution buffer were added to the wells and incubated at 37° C. for 1 h. After the plates were washed, biotinylated anti-AAVAnc80 moAb (either 2A7 or 1B11) diluted in the blocking/sample dilution buffer was added to the wells and incubated at 37° C. for 1 h. After the plates were washed, Pierce™ High Sensitivity Streptavidin-HRP was added and incubated at 37° C. for 1 h. Finally, a chromogenic substrate OPD was added and OD490 was determined for each well. Amounts and dilutions of primary moAb antibodies, biotinylated moAbs and Streptavidin-HRP, and type of the blocking/sample dilution buffer were experimentally optimized.
Using AAVAnc80 vector capsids purified by two cycles of CsCl gradient ultracentrifugation that could remove the majority of empty capsids, the sensitivity of the AAVAnc80 capsid titration of the developed ELISAs was determined. The ELISAs could detect at least 1×106 vg of AAVAnc80 vectors in 50 μL per well. OD490 values did not reach saturation even at 1×1010 vg of AAVAnc80 vectors in 50 μL per well, showing a 4-log dynamic range (
Using AAVAnc80 capsid standards prepared by a 2-fold serial dilution in a range between 7.8×105 and 1.0×108 vg per 50 μL per well, standard curves were drawn to determine the linear dynamic range of the ELISA to be used for capsid quantification. This assay revealed that an unambiguous linear range is observed between 3.2×106 and 1×108 vg in 50 μL per well with Pearson's correlation coefficient r=0.98234 for moAb 2A7 and r=0.98526 for moAb 1B11 (
All references cited in this disclosure are incorporated by reference in their entirety.
It will be obvious to those having skill in the art that many changes may be made to the details of the above-described embodiments without departing from the underlying principles of the invention. The scope of the present invention should, therefore, be determined only by the following claims.
This invention was made with government support under R01 NS088399 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/54151 | 10/2/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62909650 | Oct 2019 | US |
