DNA therapeutic agents encoding antibodies or antigen binding fragments

Abstract

The present invention relates to systems for expressing antibodies or antigen-binding fragments in a subject, and methods of treating diseases therewith.

Description

Background Art

[0001] Infusion of antibody therapeutics is an established method for treating acute life-threatening infectious diseases and for protecting immunocompromised individuals or as a therapy for other ailments. Many therapeutic antibodies require careful storage to maintain their therapeutic activity. In addition, the concentration of infused antibodies may decrease over time due to serum protein turnover or due to anti-drug antibody responses that neutralize the infused antibodies. Improved methods are needed to provide clinically relevant and long-lasting antibody levels to subjects.

[0002] Brief description of the invention

[0003] Described herein is a platform and related methods for producing the antibody or Fab required in a subject. In some cases, after transfecting a subject's tissue (e.g., muscle tissue) with a stable recombinant DNA (e.g., a plasmid or multiple plasmids) encoding the antibody or Fab required or its Fab, the subject expresses the antibody or Fab. In some cases, the recombinant DNA is transfected using a preparation that allows efficient transfection of the test tissue. In some cases, the preparation comprises a lipid vesicle that encapsulates DNA and contains a small fusogenic protein, which causes the target cells in the tissue of the efficient transfection subject. Then, the encoded antibody is expressed and secreted by the subject's own cells at a level sufficient to be clinically relevant (e.g., having therapeutic or preventive activity). Surprisingly, the platform has sufficient adaptability so that a variety of antibodies and different antibody forms (e.g., V H H form, etc.) is encoded into a DNA vector (e.g., plasmid) and introduced into a subject to produce clinically relevant antibody or antigen-binding fragment titers in the subject without the need for a large amount of vector optimization. The system and method provided herein are superior to administering exogenous antibodies to subjects because a large amount of protein expression, purification, and quality control schemes required for developing protein antibodies do not need to be developed. In addition, the cost of administering antibodies using this novel method is expected to be much lower than the conventional administration of infusion antibodies. Therefore, the flexibility of the system and method provided herein provides a promising platform that can be used to quickly and easily develop antibody therapies for various indications.

[0004] In one aspect, provided herein is a system for expressing an antibody or an antigen-binding fragment thereof in a subject, the system comprising: a plasmid comprising a polynucleotide sequence encoding a heavy chain variable domain of the antibody or an antigen-binding fragment thereof; and wherein the plasmid is encapsulated in a lipid vesicle.

[0005] In one aspect, provided herein is a system for expressing an antibody or antigen-binding fragment thereof in a subject, the system comprising: a plasmid comprising a polynucleotide sequence encoding a heavy chain variable domain of the antibody or antigen-binding fragment thereof; wherein the plasmid is encapsulated in a lipid vesicle; and wherein when the plasmid encapsulated in the lipid vesicle is administered, the subject produces a peak plasma level of the antibody or antigen-binding fragment thereof of at least 50 ng / mL.

[0006] In some embodiments, the antibody or antigen-binding fragment thereof is a single domain antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a V H H antibody. In some embodiments, the heavy chain variable domain is fused to the Fc domain, optionally via a peptide linker.

[0007] In some embodiments, the plasmid encodes the full-length heavy chain of an antibody. In some embodiments, the plasmid also comprises a polynucleotide sequence encoding a light chain or an antigen-binding fragment of an antibody. In some embodiments, the plasmid encodes the full-length light chain of an antibody. In some embodiments, the polynucleotide sequence encoding the heavy chain variable domain and the polynucleotide sequence encoding the light chain are operatively coupled so that the sequence is transcribed into a single transcript. In some embodiments, the polynucleotide sequence encoding the heavy chain and the polynucleotide sequence encoding the light chain are separated by a self-cleaving peptide coding sequence.

[0008] In some embodiments, the system comprises a second plasmid comprising a second polynucleotide sequence encoding a light chain of an antibody. In some embodiments, the light chain of the antibody is a kappa chain or a lambda chain. In some embodiments, the second plasmid is also encapsulated in a lipid vesicle.

[0009] In some embodiments, the lipid vesicle comprises a fusion-associated small transmembrane (FAST) protein. In some embodiments, the FAST protein comprises a domain from one or more FAST proteins, the FAST protein being selected from p10, p14, p15, and p22. In some embodiments, the FAST protein comprises an amino acid sequence having at least 80% sequence identity with the following sequence:

[0010]

[0011] In some embodiments, the vector comprises a promoter operably linked to the polynucleotide sequence, the promoter selected from CAG, CMV, EF1A, CBh, CBA and SFFV. In some embodiments, the plasmid comprises a CAG promoter. In some embodiments, the plasmid is a DNA plasmid.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgG1, IgG2a, IgG2b, IgG3, IgG4, IgD, IgM, IgA1, IgA2, or IgE heavy chain. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgG1, IgG2a, IgG2b, IgG3, or IgG4 heavy chain. In some embodiments, the antibody comprises an IgG1 heavy chain. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 80% sequence identity with the following sequence:

[0013]

[0014] wherein each X is independently absent or is any amino acid.

[0015] In some embodiments, the antibody or antigen-binding fragment comprises an Fc domain having one or more mutations or a combination of mutations selected from the following: Arg435His (His435), Asn434Ala (A), Met428Leu / Asn434Ser (LS), Thr252Leu / Thr253Ser / Thr254Phe (LSF), Glu294Δ / Thr307Pro / Asn434Tyr (C6A-66), Thr256Asn / Ala378Val / Ser383Asn / Asn434Tyr (C6A-78), and Glu294Δ (Del), wherein the residue position numbers are based on the EU numbering convention. In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc domain having one or more mutations selected from M252Y, S254T, T256E, and any combination thereof, wherein the residue position numbers are based on the EU numbering convention.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a viral protein. In some embodiments, the viral protein is from a virus selected from the group consisting of a parvovirus, a picornavirus, a rhabdovirus, a paramyxovirus, an orthomyxovirus, a bunyavirus, a calicivirus, an arenavirus, a polyomavirus, a reovirus, a togavirus, a bunyavirus, a herpes simplex virus, a poxvirus, an adenovirus, a coxsackievirus, a flavivirus, a coronavirus, an astrovirus, an enterovirus, a rotavirus, a norovirus, a retrovirus, a papillomavirus, a parvovirus, an influenza virus, a hemorrhagic fever virus, and a rhinovirus. In some embodiments, the viral protein is from a virus selected from the group consisting of: Hantavirus, rabies virus, Nipah virus, Hendra virus, Rift Valley fever virus, Lassa virus, Marburg virus, Crimean-Congo hemorrhagic fever virus, hMPV, RSV, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, norovirus, monkeypox virus, vaccinia virus, Japanese encephalitis virus, yellow fever virus, HSV-1, HSV-2, ME RS, varicella virus, hand, foot and mouth virus, CMV (HHV-5), equine encephalitis virus, EBV (HHV-4), human metapneumovirus, norovirus, enterovirus, smallpox virus, West Nile virus, paramyxoviridae, rhinovirus, mononucleosis virus, coxsackie virus B, influenza virus, poliovirus, measles virus, rubella virus, HPV, Zika virus, mumps virus, herpesviridae, chikungunya virus, Haemophilus influenzae and SARS-CoV-2 virus. In some embodiments, the viral protein is from SARS-CoV-2. In some embodiments, the viral protein is a SARS-CoV-2 spike protein. In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the antibody shown in Table 3.

[0017] In some embodiments, the antibody or antigen binding fragment specifically binds to a cancer antigen. In some embodiments, the antibody or antigen binding fragment specifically binds to a protein or component of a bacterium. In some embodiments, the antibody or antigen binding fragment specifically binds to a protein or component of a parasite. In some embodiments, the antibody or antigen binding fragment specifically binds to an allergen. In some embodiments, the antibody or antigen binding fragment specifically binds to an immune checkpoint molecule. In some embodiments, the antibody or antigen binding fragment specifically binds to an antigen involved in an inflammatory disease.

[0018] In some embodiments, the peak plasma level of the antibody or antigen-binding fragment thereof produced by administration is at least 75 ng / mL, at least 100 ng / mL, at least 150 ng / mL, at least 200 ng / mL, at least 250 ng / mL, at least 300 ng / mL, at least 400 ng / mL, at least 500 ng / mL, at least 600 ng / mL, at least 700 ng / mL, at least 800 ng / mL, at least 900 ng / mL, or at least 1000 ng / mL. In some embodiments, administration is performed without electroporation or hydroporation.

[0019] In some embodiments, the plasmid is a DNA plasmid.

[0020] Another aspect provided herein is a method of inducing antibody production in a subject, the method comprising administering to the subject a system provided herein. In some embodiments, administering to the subject a plasmid encapsulated in a lipid vesicle produces an antibody or antigen-binding fragment thereof at a plasma level of at least 50 ng / mL.

[0021] In some embodiments, administration is intramuscular, subcutaneous, intradermal, intranasal, oral, intrathecal or intravenous. In some embodiments, administration is intramuscular. In some embodiments, administration is intravenous. In some embodiments, administration is performed without electroporation or water perforation. In some embodiments, the peak plasma level of the antibody or its antigen-binding fragment produced by administration is at least 75ng / mL, at least 100ng / mL, at least 150ng / mL, at least 200ng / mL, at least 250ng / mL, at least 300ng / mL, at least 400ng / mL, at least 500ng / mL, at least 600ng / mL, at least 700ng / mL, at least 800ng / mL, at least 900ng / mL or at least 1000ng / mL.

[0022] In some embodiments, administration is performed once or twice. In some embodiments, the method comprises administering 2 doses of plasmid to a subject. In some embodiments, 2 doses are administered intravenously. In some embodiments, 2 doses are administered at intervals of about 2 weeks to about 12 weeks. In some embodiments, administration of the second dose results in a peak plasma level of the antibody or antigen-binding fragment that is more than 2 times higher than the peak plasma level reached after the first dose. In some embodiments, administration of the second dose results in a peak plasma level of the antibody or antigen-binding fragment that is at least 3 times, at least 4 times, or at least 5 times higher than the peak plasma level reached after the first dose.

[0023] In some embodiments, administration includes delivering a plasmid of about 0.1 mg / kg to about 20 mg / kg to a subject. In some embodiments, administration includes delivering a plasmid of about 0.1 mg / kg to about 20 mg / kg per dose to a subject. In some embodiments, the plasma level of the antibody or antigen-binding fragment maintains at least 50 ng / mL, at least 100 ng / mL, at least 200 ng / mL, at least 300 ng / mL, at least 400 ng / mL, at least 500 ng / mL, at least 500 ng / mL, at least 600 ng / mL, at least 700 ng / mL, at least 800 ng / mL, at least 900 ng / mL or at least 1000 ng / mL for a period of at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks or at least 20 weeks after administration. In some embodiments, the plasma level of the antibody or antigen binding fragment is maintained at a concentration of at least 50% of the peak plasma concentration achieved for a period of at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks after administration. In some embodiments, the plasma level of the antibody or antigen binding fragment is maintained at a concentration of at least 25% of the peak plasma concentration achieved for a period of at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks after administration. In some embodiments, the plasma level of the antibody or antigen binding fragment is maintained at a concentration of at least 10% of the peak plasma concentration achieved for a period of at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks after administration. In some embodiments, the concentration maintained by the antibody is reached after a single administration. In some embodiments, the concentration maintained by the antibody is reached after two administrations.

[0024] Other aspects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, wherein only exemplary embodiments of the present invention are shown and described. As will be appreciated, the present invention is capable of other and different embodiments, and some details can be modified in various obvious aspects, all without departing from the present invention. Therefore, the drawings and description are illustrative in nature, rather than restrictive.

[0025] Content Incorporated by Reference

[0026] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting content. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The novel features of the present invention are particularly described in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and the accompanying drawings, which show exemplary embodiments in which the principles of the present invention are used, and in which:

[0028] Figure 1A Shown are the plasma concentrations of human antibodies in Rag2 knockout mice 9 days after administration of the DNA-encoded antibody system provided herein. A total of 10 mice were transfected using the same protocol (antibody expression construct, transfection route, and DNA amount).

[0029] Figure 1B Shown are plasma concentrations of human antibodies in Rag2 knockout mice 16 days after administration of the antibody system encoded by the DNA provided herein.

[0030] Figure 1C Shown are plasma concentrations of human antibodies in Rag2 knockout mice 23 days after administration of the antibody system encoded by the DNA provided herein.

[0031] Figure 1D Shown are plasma concentrations of human antibodies in Rag2 knockout mice 30 days after administration of the antibody system encoded by the DNA provided herein.

[0032] Figure 1E Shown are plasma concentrations of human antibodies in Rag2 knockout mice 37 days after administration of the antibody system encoded by the DNA provided herein.

[0033] Figure 1F Shown are plasma concentrations of human antibodies in Rag2 knockout mice 44 days after administration of the antibody system encoded by the DNA provided herein.

[0034] Figure 1G Plasma antibody concentrations are shown for single and double doses of the indicated antibody formats. For both IV formats, antibody levels increased after the second administration on day 60.

[0035] Figure 1H Plasma antibody concentrations over time are shown for the indicated dosing regimens.

[0036] Fig. 1I Shown are plasma antibody concentrations over time for the indicated dosing regimens measured using a commercially available IgG1 standard.

[0037] Figure 1J Plasma antibody concentrations over time for the indicated dosing regimens, measured using an in-house produced IgG1 standard, are shown, which included re-measurement of the samples shown in Figure 1 P. Use of the in-house standard showed antibody levels to be approximately 25-fold lower than the commercial standard.

[0038] Figure 2 The time course of antibody expression for the indicated routes of administration is shown in Rag2 knockout mice.

[0039] Figure 3A The domain structure of the SARS-CoV-2 spike protein is shown.

[0040] Figure 3B Schematic showing mAb1 and mAb2 binding to the SARS-CoV-2 spike protein receptor binding domain (RBD) at non-overlapping sites.

[0041] Figure 4A Displays monoclonal antibodies, heavy chain only antibodies and V H Domain arrangement of the H antibody.

[0042] Figure 4B Display V H Chain arrangement of the H variable region.

[0043] Figure 5A A vector map showing the expression plasmid for the construct of the single transcript T2A form of mAbl.

[0044] Figure 5B Vector map of the expression plasmid encoding the heavy chain of mAbl is shown for the construct in a two-plasmid format (HC+LC).

[0045] Figure 5C Vector map of the expression plasmid encoding the light chain of mAbl is shown for the construct in a two-plasmid format (HC+LC).

[0046] Figure 6 Binding of antibodies to the receptor binding domain of the SARS-CoV-2 strain in the plasma of Rag2 knockout mice 44 days after administration of the DNA-encoded antibody system provided herein is shown.

[0047] Fig. 7A Shown are plasma antibody concentrations following a single dose of the indicated antibody forms at the indicated doses calculated using an in-house generated human IgG1 standard.

[0048] Figure 7B Showing that the more sensitive assay measured Fig. 7A Plasma antibody concentrations of some of the same samples.

[0049] Figure 8 Plasma antibody concentrations of samples with and without the SV40e element are shown.

[0050] Fig. 9 Display V H Plasma antibody concentrations of H-form antibodies in ng / mL (left) and nM (right). DETAILED DESCRIPTION OF THE INVENTION

[0052] The following description and examples describe embodiments of the present invention in detail. It should be understood that the present invention is not limited to the specific embodiments described herein, and therefore may be different. It will be appreciated by those skilled in the art that the present invention has many variations and modifications, which are all encompassed within its scope.

[0053] Although various features of the invention may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the invention may be described in the context of separate multiple embodiments for clarity, the invention may also be implemented in a single embodiment. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0054] I. Antibody or antigen-binding fragment expression system

[0055] Provided herein are systems for expressing antibodies or antigen-binding fragments. In some embodiments, the system is configured to express therapeutically relevant amounts of the antibody or antigen-binding fragment when administered to a subject.

[0056] In one aspect, provided herein is a system for expressing an antibody or its antigen binding fragment. In some embodiments, the system is configured to express an antibody or its antigen binding fragment when administered to a subject. In some embodiments, the system comprises a plasmid. In some embodiments, the plasmid comprises a polynucleotide sequence encoding a heavy chain variable domain of an antibody or its antigen binding fragment. In some embodiments, the plasmid is encapsulated in a lipid vesicle. In some embodiments, a lipid vesicle is administered to a subject. In some embodiments, the peak plasma level of the antibody or antigen binding fragment produced by the administered lipid vesicle is at least 50 ng / ml.

[0057] On the other hand, provided herein is a system for expressing an antibody or its antigen-binding fragment, wherein the system comprises a vector. In some embodiments, the vector comprises a polynucleotide sequence encoding a heavy chain variable domain of an antibody or its antigen-binding fragment. In some embodiments, the vector is encapsulated in a lipid vesicle. In some embodiments, the lipid vesicle is administered to a subject. In some embodiments, the peak plasma level of the antibody or antigen-binding fragment produced by the administered lipid vesicle is at least 50 ng / ml.

[0058] On the other hand, provided herein is a system for expressing an antibody or its antigen-binding fragment in a subject's tissue. In some embodiments, the system comprises a DNA molecule. In some embodiments, the DNA molecule comprises a polynucleotide sequence encoding a heavy chain variable domain of an antibody or its antigen-binding fragment. In some embodiments, the DNA molecule is encapsulated in a lipid vesicle. In some embodiments, the lipid vesicle is administered to the subject. In some embodiments, the peak plasma level of the antibody or antigen-binding fragment produced by the administered lipid vesicle is at least 50 ng / ml.

[0059] Carrier

[0060] In one aspect, the vector provided herein comprises a polynucleotide sequence encoding an antibody or antigen binding fragment with affinity for an antigen associated with a disease. Some embodiments herein are DNA vectors comprising a polynucleotide sequence encoding an antibody or antigen binding fragment with affinity for an antigen associated with a disease. The disease-associated antigen can be an antigen associated with a disease, wherein an example of such an antigen is a protein or other component of a virus, bacterium, parasite, or cancer, or an antigen to which another disease such as an autoimmune disease or an inflammatory disease is related. In some embodiments, the DNA vector is a plasmid, a viral vector, a cosmid, or an artificial chromosome. In some embodiments, the DNA vector is a plasmid.

[0061] In the embodiment where carrier is plasmid, plasmid may be advantageous at or below a certain size. In some embodiments, smaller plasmid provides the advantage that carrier is loaded into the preparation (for example, proteolipid vehicle provided herein) needed better, and due to the cross activity of less potential attributed to larger size and enhanced expression. In some embodiments, plasmid comprises at most about 50,000 base pairs (bp), at most about 45,000bp, at most about 40,000bp, at most about 35,000bp, at most about 30,000bp, at most about 25,000bp, at most about 20,000bp, at most about 15,000bp, at most about 10,000bp, at most about 9,000bp, at most about 8,000bp, at most about 7,000bp, at most about 6,000bp, at most about 5,000bp or at most about 4,000bp (for double-stranded DNA plasmid). In some embodiments, plasmid comprises at most about 5,000bp. In some embodiments, the plasmid comprises up to about 4,000 bp. In some embodiments, the plasmid is 4,000 bp to 5,000 bp. In some embodiments, the plasmid is 3,000 bp to 5,000 bp. In some embodiments, the plasmid is 3,000 bp to 4,000 bp. In some embodiments, the plasmid is 2,500 bp to 5,000 bp.

[0062] In some embodiments, the plasmid is or is derived from a bacterial or fungal plasmid. In some embodiments, the plasmid is or is derived from a yeast plasmid. In some embodiments, the plasmid is or is derived from a bacterium. In some embodiments, the plasmid backbone is derived from a bacterium or a fungus. In some embodiments, the plasmid backbone is derived from a bacterium. In some embodiments, the plasmid backbone is derived from a yeast.

[0063] In some embodiments, the plasmid comprises an R6K origin of replication. In some embodiments, the plasmid comprises a 140 bp RNA-based sucrose selective antibiotic-free marker (RNA-OUT). In some embodiments, the plasmid backbone (e.g., the portion of the plasmid that is not directly involved in the expression of the encoded gene (e.g., coding sequence, polyadenylation sequence, signal peptide coding sequence, and promoter)) is less than 1000 bp, less than 900 bp, less than 800 bp, less than 700 bp, less than 600 bp, or less than 500 bp. In some embodiments, the plasmid consists essentially of an origin of replication, a selective marker, and the portion of the plasmid that is directly involved in the expression of the encoded gene.

[0064] In some embodiments, the plasmid backbone is the NTC9385R plasmid. The NTC9835R plasmid is an expression vector comprising a bacterial backbone comprising a 140 bp RNA-based sucrose selective antibiotic-free marker (RNA-OUT). The NTC9385R plasmid is described in U.S. Patent No. 9,550,998, which is incorporated herein by reference in its entirety. NTC9835R is commercially available from Nature Technology Corporation under the trade name Nanoplasmid TM For commercial sale.

[0065] In some embodiments, the vector contains a polynucleotide sequence encoding a secretory signal peptide. In some embodiments, the polynucleotide encoding the secretory signal peptide is fused in frame to the 5' end of the polynucleotide encoding the antibody heavy chain, the antibody heavy chain antigen binding fragment, the antibody light chain and / or the antibody light chain antigen binding fragment. In some embodiments, the polynucleotide encoding the secretory signal peptide is fused to the polynucleotide sequence encoding the heavy chain. In some embodiments, the polynucleotide encoding the secretory signal peptide is fused in frame to the 5' end of the polynucleotide sequence encoding the therapeutic antibody light chain or light chain antigen binding fragment. In some embodiments, the polynucleotide encoding the secretory signal peptide is fused to the polynucleotide sequence encoding the light chain. In some embodiments, the secretory signal peptide is fused to the V H H antibody fusion.

[0066] In some embodiments, the vectors encoding antibodies or antigen binding fragments provided herein include one or more promoters, which contribute to the transcription of sequences encoding antibodies or antigen binding fragments. In some embodiments, the vector includes a promoter operably connected to a polynucleotide sequence encoding an antibody or antigen binding fragment. In some embodiments, the promoter allows the enhanced expression of the mRNA transcripts of the antibody or antigen binding fragment. In some embodiments, the vector includes a eukaryotic promoter. In some embodiments, the promoter is selected from CAG promoter, cytomegalovirus (CMV) promoter, human elongation factor-1α (EF1A) promoter, CBh promoter (see, e.g., Hum Gene Ther. 2011 Sep; 22 (9): 1143-53. doi: 10.1089 / hum.2010.245), chicken β-actin (CBA) promoter or spleen focus forming virus (SFFV) promoter.

[0067] In some embodiments, the vector comprises a CAG promoter. In some embodiments, the CAG promoter comprises a cytomegalovirus (CMV) early enhancer element, a promoter, the first exon and the first intron of a chicken β-actin gene, and a splice acceptor of a rabbit β-globin gene.

[0068] In some embodiments, the vector comprises one or more enhancers (e.g., an enhancer that replaces the CAG promoter or an enhancer in addition to the CAG promoter). In some embodiments, the vector comprises the SV40 enhancer (SV40e). In some embodiments, SV40e is incorporated upstream of the region encoding the antibody or its antigen-binding fragment. In some embodiments, SV40e is incorporated upstream of the promoter of the region encoding the antibody or its antigen-binding fragment. In some embodiments, SV40e is directly upstream of the promoter. In some embodiments, SV40e is directly upstream of the CAG promoter. In some embodiments, SV40e has a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% sequence identity with the following sequence:

[0069]

[0070] In some embodiments, the vector includes a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the WPRE is located downstream of the region encoding the antibody or antigen-binding fragment thereof. In some embodiments, the WPRE is located downstream of the region encoding the antibody or antigen-binding fragment thereof, but upstream of the polyadenylation signal. In some embodiments, the WPRE has a sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% sequence identity with the following sequence:

[0071]

[0072] Single transcript expression vector (e.g., plasmid) for light and heavy chain antibodies

[0073] In some embodiments, the system for expressing an antibody or antigen-binding fragment provided herein comprises a single vector (e.g., a DNA plasmid) encoding a heavy chain or fragment thereof of an antibody and a light chain or fragment thereof of an antibody. In some embodiments, the heavy chain or fragment thereof and the light chain or fragment thereof are encoded so that both chains or fragments thereof are transcribed in a single transcript, thereby simultaneously expressing both chains or fragments thereof in the same cell at the same concentration yield. Figure 5A An exemplary vector illustrating such a construct is shown in .

[0074] In some embodiments, the polynucleotide sequences encoding the heavy and light chains of the antibodies or their antigen-binding fragments are configured to be read as a single transcript. In some embodiments, the encoded fusion antibody heavy and light chains are separated by a self-cleaving peptide coding sequence. In some embodiments, the encoded cleavage peptide is a furin-T2A self-cleavage sequence. In some embodiments, the furin-T2A cleavage peptide sequence is RRKRGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 104). In some embodiments, the furin-T2A cleavage peptide sequence has at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity to the peptide sequence RRKRGSGEGRGSLLTCGDVEENPGP (SEQID NO: 104). In some embodiments, these polypeptide transport vesicle membranes are assembled and secreted in the lumen space of the extracellular vesicle system.

[0075] In some embodiments, the heavy chain of an antibody or a fragment thereof comprises a variable heavy chain domain and a CH1 domain. In some embodiments, the heavy chain or a fragment thereof further comprises a CH2 domain, a CH3 domain, or both. In some embodiments, the light chain of an antibody or a fragment thereof comprises a variable light chain domain and a constant light chain domain.

[0076] Split antibody expression system

[0077] In some embodiments, the system for antibodies or antigen-binding fragments expressed in a subject provided herein is configured to produce antibodies or antigen-binding fragments by translating two separate transcripts. In some embodiments, the heavy chain of an antibody or a fragment thereof and the light chain of an antibody or a fragment thereof are encoded on one or more vectors (e.g., plasmids) such that each vector is transcribed separately. In some embodiments, the heavy chain of an antibody or a fragment thereof and the light chain of an antibody or a fragment thereof are encoded on separate vectors. In some embodiments, separate plasmids are formulated together so that the vectors can be delivered to the same cell (e.g., both are encapsulated in the same lipid vesicle). In some embodiments, the heavy chain or a fragment thereof and the light chain or a fragment thereof are expressed in the same cell. In some embodiments, the heavy chain or a fragment thereof and the light chain or a fragment thereof are expressed in different cells. Exemplary DNA plasmid vectors encoding the heavy and light chains of antibodies, respectively, are as follows Figure 5B and Figure 5C As shown. Figure 5A Compared to the vectors shown, the vectors shown have essentially the same non-coding portions (ie, only the coding regions are altered).

[0078] In some embodiments, the vectors encoding the heavy chain or fragments thereof of the antibody and the vectors encoding the light chain or fragments thereof are used in equal mass ratios in the systems provided herein. In some embodiments, the vectors encoding the heavy chain or fragments thereof of the antibody and the vectors encoding the light chain or fragments thereof are used in equal molar ratios in the systems provided herein. In some embodiments, the molar ratio of the vectors encoding the heavy chain or fragments thereof and the vectors encoding the light chain or fragments thereof is about 2:1 to about 1:2. In some embodiments, the molar ratio of the vector encoding the heavy chain or fragment thereof to the vector encoding the light chain or fragment thereof is about 2: 1, about 1.9: 1, about 1.8: 1, about 1.7: 1, about 1.6: 1, about 1.5: 1, about 1.4: 1, about 1.3: 1, about 1.2: 1, about 1.1: 1, about 1: 1, about 1: 1.1, about 1: 1.2, about 1: 1.3, about 1: 1.4, about 1: 1.5, about 1: 1.6, about 1: 1.7, about 1: 1.8, about 1: 1.9, or about 1: 2. In some embodiments, the molar ratio of the vector encoding the heavy chain or fragment thereof to the vector encoding the light chain or fragment thereof is about 1.5: 1 to about 2: 1. In some embodiments, the molar ratio of the vector encoding the heavy chain or fragment thereof to the vector encoding the light chain or fragment thereof is about 1.5: 1 to about 2: 1. In some embodiments, the molar ratio of the vector encoding the heavy chain or a fragment thereof to the vector encoding the light chain or a fragment thereof is about 1.6: 1 to about 1.8: 1. In some embodiments, the molar ratio of the vector encoding the heavy chain or a fragment thereof to the vector encoding the light chain or a fragment thereof is about 1.7:1.

[0079] V H H expression

[0080] In some embodiments, the system provided herein for expressing an antibody or antigen-binding fragment comprises a vector encoding a heavy chain variable domain of an antibody or antigen-binding fragment thereof. In some embodiments, the vector does not encode the entire antibody heavy chain. In some embodiments, the vector encodes a V H H antibody. V H An exemplary cartoon diagram of the H antibody structure is shown in Figure 4B In some embodiments, the vector encodes a V fused to an Fc region. H H. V fused to the Fc region H An example diagram of H is Figure 4A (middle) Figure 4A Also shown are the intact antibody (left) and the V H H (right). In some embodiments, the vector encodes a V region fused to an Fc region via a linker peptide. H H. In some embodiments, the vector encodes a V region fused to an Fc region via a hinge region or a modified hinge region. H H. In some embodiments, the antibody is a camelized antibody containing only the heavy chain. In some embodiments, the vector encodes only the variable domain of the heavy chain to produce a single chain V H H antibody.

[0081] Antibodies and antigen-binding fragments

[0082] In some embodiments, the antibody or antigen binding fragment of the present invention specifically binds to the target antigen. If the antibody or antigen binding fragment binds with greater affinity, avidity, more easily and / or with a longer duration than it binds to other substances, the antibody or antigen binding fragment selectively binds or preferentially binds to the target. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, referring to specific binding means preferential binding, wherein the affinity of the antibody or its antigen binding fragment is at least 2 times higher than the affinity of the antibody to an unrelated substance, at least 3 times higher, at least 4 times higher, at least 5 times higher, at least 6 times higher, at least 7 times higher, at least 8 times higher, at least 9 times higher, at least 10 times higher, at least 20 times higher, at least 30 times higher, at least 40 times higher, at least 50 times higher, at least 60 times higher, at least 70 times higher, at least 80 times higher, at least 90 times higher, at least 100 times higher or at least 1000 times higher.

[0083] As used herein, the term "antibody" refers to an immunoglobulin (Ig), polypeptide or protein having a binding domain as an antigen binding domain or a binding domain homologous to an antigen binding domain. The term also includes other interchangeable terms for "antigen binding fragments" and similar binding fragments as described below. Natural antibodies and natural immunoglobulins (Ig) are generally heterotetrameric glycoproteins consisting of two identical light chains and two identical heavy chains of about 150,000 daltons. Each light chain is usually connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy chain and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable domain ("VH"), followed by multiple constant domains ("CH"). One end of each light chain has a variable domain ("VL") and the other end has a constant domain ("CL"); the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.

[0084] In some cases, the antibody or antigen-binding fragment comprises an isolated antibody or antigen-binding fragment, a purified antibody or antigen-binding fragment, a recombinant antibody or antigen-binding fragment, a modified antibody or antigen-binding fragment, or a synthetic antibody or antigen-binding fragment.

[0085] The antibodies and antigen binding fragments herein can be produced partially or entirely synthetically.Antibodies or antigen binding fragments can be polypeptides or proteins having a binding domain, which can be an antigen binding domain or can be homologous to an antigen binding domain.In one case, antibodies or antigen binding fragments can be produced in a suitable in vivo animal model and then separated and / or purified.

[0086] Depending on the amino acid sequence of the constant domain of the heavy chain of the antigen binding domain, immunoglobulin (Ig) can be divided into different categories. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. In some cases, Ig or a portion thereof can be human Ig. In some cases, the CH3 domain can be from an immunoglobulin. In some cases, the chain or portion of an antibody or antigen binding fragment, a modified antibody or antigen binding fragment or a binding agent can be from Ig. In this case, Ig can be IgG, IgA, IgD, IgE or IgM, or be derived from them. In the case where Ig is IgG, it can be a subtype of IgG, wherein the subtype of IgG can include IgG1, IgG2a, IgG2b, IgG3 or IgG4. In some cases, the CH3 domain may be from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE and IgM or derived from them. In some embodiments, the antibodies or antigen-binding fragments described herein comprise IgG or are derived from it. In some cases, the antibodies or antigen-binding fragments comprise IgG1 or are derived from it. In some cases, the antibodies or antigen-binding fragments comprise IgG4 or are derived from it. In some embodiments, the antibodies or antigen-binding fragments described herein comprise IgM or are derived from it, or are monomeric forms of IgM. In some embodiments, the antibodies or antigen-binding fragments described herein comprise IgE or are derived from it. In some embodiments, the antibodies or antigen-binding fragments described herein comprise IgD or are derived from it. In some embodiments, the antibodies or antigen-binding fragments described herein comprise IgA or are derived from it.

[0087] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ("κ" or "K") or lambda ("λ"), based on the amino acid sequence of the antibody constant domain. In some embodiments, the antibody or antigen-binding fragment comprises a κ light chain. In some embodiments, the antibody or antigen-binding fragment comprises a λ light chain.

[0088] The "variable region" of an antibody refers to the variable region of the light chain of an antibody or the variable region of the heavy chain of an antibody, alone or in combination. The variable regions of the heavy and light chains are each composed of four framework regions (FRs) connected by three complementary determining regions (CDRs), which are also called hypervariable regions. The CDRs in each chain are tightly held together by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md. (1991), pages 647-669; hereinafter referred to as "Kabat"); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-Iazikani et al., (1997) J. Molec. Biol. 273: 927-948)). As used herein, a CDR may refer to a CDR defined by either approach or by a combination of both approaches.

[0089] With respect to antibodies, the term "variable domain" refers to the variable domain of an antibody, which is used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed in the variable domains of an antibody. Instead, the variability is concentrated in three segments called hypervariable regions (also referred to as CDRs) in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are referred to as "framework regions" or "FRs". The variable domains of unmodified heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4), primarily in a β-folded configuration, with three CDRs interspersed during the period, the CDRs forming loops that connect portions of the β-folded structure in some cases. The CDRs in each chain are held together closely by the FRs, and together with the CDRs from other chains, contribute to the formation of the antigen binding site of the antibody (see Kabat).

[0090] The terms "hypervariable region" and "CDR" used herein refer to the amino acid residues of an antibody responsible for antigen binding. The CDR comprises amino acid residues from three sequence regions that bind to the antigen in a complementary manner, and the CDRs are referred to as CDR1, CDR2, and CDR3 for each VH and VL chain. According to Kabat, in the light chain variable domain, the CDRs generally correspond to about residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3), and in the heavy chain variable domain, the CDRs generally correspond to about residues 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3). It should be understood that the CDRs of different antibodies may contain insertions, so the amino acid numbering may be different. The Kabat numbering system uses a numbering scheme to explain this insertion, and the numbering scheme uses letters attached to specific residues (e.g., 27A, 27B, 27C, 27D, 27E, and 27F of CDRL1 in the light chain) to reflect any insertion in the numbering between different antibodies. Alternatively, in the light chain variable domain, according to Chothia and Lesk (J. Mol. Biol., 196:901-917 (1987)), the CDRs typically correspond to approximately residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3), and in the heavy chain variable domain, the CDRs typically correspond to approximately residues 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3).

[0091] As used herein, "framework region", "FW" or "FR" refers to the framework amino acid residues that form part of an antigen binding pocket or groove. In some embodiments, the framework residues form a loop that is part of an antigen binding pocket or groove, and the amino acid residues in the loop may or may not contact the antigen. The framework region generally includes the region between CDRs. According to Kabat, in the light chain variable domain, FR generally corresponds to about residues 0-23 (LFR1), 35-49 (LFR2), 57-88 (LFR3) and 98-109, and in the heavy chain variable domain, FR generally corresponds to about residues 0-30 (HFR1), 36-49 (HFR2), 66-94 (HFR3) and 103-133. As described above for the Kabat numbering of the light chain, the heavy chain also explains insertions in a similar manner (e.g., 35A, 35B of HCDR1 in the heavy chain). Alternatively, according to Chothia and Lesk, Id, in the light chain variable domain, FR generally corresponds to about residues 0-25 (LFR1), 33-49 (LFR2), 53-90 (LFR3) and 97-109 (LFR4), and in the heavy chain variable domain, FR generally corresponds to about residues 0-25 (HFR1), 33-52 (HFR2), 56-95 (HFR3) and 102-113 (HFR4). The cyclic amino acids of FR can be evaluated and determined by examining the three-dimensional structure of the antibody heavy chain and / or the antibody light chain. The solvent accessible amino acid positions of the three-dimensional structure can be analyzed because these positions may form loops and / or provide antigen contacts in the antibody variable domain. Some solvent accessible positions can tolerate amino acid sequence diversity, while other positions (e.g., structural positions) are generally less diverse. The three-dimensional structure of the antibody variable domain can be derived from crystal structure or protein modeling.

[0092] In the present invention, the following abbreviations (in brackets) are used according to custom and when necessary: ​​heavy chain variable region (HCVR), light chain variable region (LCVR), complementarity determining region (CDR), first complementarity determining region (CDR1), second complementarity determining region (CDR2), third complementarity determining region (CDR3), heavy chain first complementarity determining region (HCDR1), heavy chain second complementarity determining region (HCDR2), heavy chain third complementarity determining region (HCDR3), light chain first complementarity determining region (LCDR1), light chain second complementarity determining region (LCDR2) and light chain third complementarity determining region (LCDR3).

[0093] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The numbering of the residues in the Fc region is that of the EU index in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3.

[0094] The "antibodies" useful in the present invention encompass but are not limited to monoclonal antibodies, polyclonal antibodies, chimeric antibodies, bispecific antibodies, multispecific antibodies, heteroconjugate antibodies, humanized antibodies, human antibodies, transplanted antibodies, deimmunized antibodies, mutants thereof, fusions thereof, immunoconjugates thereof, antigen-binding fragments thereof and / or any other modified configurations of immunoglobulin molecules that contain the desired specific antigen recognition site, said other modified configurations including antibody glycosylation variants, antibody amino acid sequence variants and covalently modified antibodies.

[0095] In some cases, the antibody is a monoclonal antibody. As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for the naturally occurring mutations that may be present in small amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant (epitope) on the antigen. The modifier "monoclonal" indicates that the characteristic of the antibody is obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring the antibody to be produced by any particular method.

[0096] In some cases, antibody is a humanized antibody. As used herein, "humanized" antibody refers to an antibody in a non-human (e.g., mouse) form, and the antibody in the non-human form is a specific chimeric immunoglobulin, an immunoglobulin chain, or a fragment thereof, which contains a minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the complementary determining region (CDR) of the receptor are replaced by the residues of the CDR from non-human species (donor antibody) (such as from mice, rats, or rabbits, with the specificity, affinity, and biological activity required). In some cases, the Fv framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are neither found in the receptor antibody nor in the CDR or framework sequences imported, but are included to further improve and optimize antibody performance. Generally, humanized antibodies substantially comprise all of at least one (usually two) variable domains, wherein all or substantially all CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all FR regions are FR regions of human immunoglobulin consensus sequences. In the best case, humanized antibodies will also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically a portion of a human immunoglobulin. Antibodies can have the Fc region of the modification as described in, for example, WO 99 / 58572. Other forms of humanized antibodies have one or more (one, two, three, four, five or six) CDRs (also referred to as "deriving from" one or more CDRs from one or more CDRs of the original antibody) that change relative to the original antibody.

[0097] If desired, the immunogenicity of the antibodies or antigen-binding fragments described herein can be assessed and deimmunization (i.e., reducing the immune response of the antibody by altering one or more T cell epitopes) can be performed as desired. As used herein, "deimmunized antibody" means that one or more T cell epitopes in the antibody sequence have been modified so that the T cell response after administration of the antibody to a subject is reduced compared to the non-deimmunized antibody. Analysis of immunogenicity and T cell epitopes present in the antibodies and antigen-binding fragments described herein can be performed using software and specific databases. Exemplary software and databases include iTope TM , the iTope TM Developed by Antitope in Cambridge, UK. iTope TM iTope is a computer technology used to analyze peptide binding to human MHC class II alleles. TM The software predicts peptide binding to human MHC class II alleles and thereby provides an initial screen for the location of such "potential T cell epitopes". iTope TMThe software predicts favorable interactions between the amino acid side chains of the peptide and specific binding pockets within the binding groove of 34 human MHC class II alleles. The locations of key binding residues are achieved by computer-generated 9-mer peptides that overlap one amino acid spanning the variable region sequence of the test antibody. Each 9-mer peptide can be tested against each of the 34 MHC class II allotypes and scored based on its potential "fitness" and interaction with the MHC class II binding groove. High average binding scores are generated for >50% of the MHC class II alleles (in iTope TM The peptides with scores in the scoring function of >0.55) are considered as potential T cell epitopes. In this region, the core 9 amino acid sequence of peptide binding in the MHC II class groove is analyzed to determine the MHC II class pocket residues (P1, P4, P6, P7 and P9) and possible T cell receptor (TCR) contact residues (P-1, P2, P3, P5, P8). After identifying any T cell epitope, amino acid residue changes, substitutions, additions and / or deletions can be introduced to remove the identified T cell epitopes. This change can be made to retain antibody structure and function, while still removing the identified epitope. Exemplary changes can include but are not limited to conservative amino acid changes.

[0098] The antibody can be a human antibody. As used herein, "human antibody" means an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or prepared using any suitable technology for preparing human antibodies. This definition of human antibodies includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising a mouse light chain and a human heavy chain polypeptide. In one embodiment, the human antibody is selected from a phage library, wherein the phage library expresses human antibodies. Human antibodies can also be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated). Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes (such B lymphocytes can be recovered from individuals or can be immunized in vitro) that produce antibodies against target antigens.

[0099] Any antibody herein can be bispecific. Bispecific antibodies are antibodies with binding specificity to at least two different antigens, and can be prepared using the antibodies of the present invention. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chains-light chains, wherein the two heavy chains have different specificities. Bispecific antibodies can be composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain in another arm (providing a second binding specificity). This asymmetric structure with immunoglobulin light chain only in one half of the bispecific molecule promotes the separation of the required bispecific compounds from the unwanted immunoglobulin chain combination.

[0100] According to a method for preparing bispecific antibodies, an antibody variable domain (antibody-antigen binding site) with required binding specificity is fused to an immunoglobulin constant domain sequence. The fusion can have an immunoglobulin heavy chain constant domain, and the heavy chain constant domain comprises at least a portion of hinge, CH2 and CH3 regions. The first heavy chain constant region (CH1) containing the necessary site for light chain binding can be present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion and the immunoglobulin light chain (if necessary) is inserted into a separate expression vector and co-transfected into a suitable host organism. When the unequal ratios of the three polypeptide chains used in the construction provide optimal yields, this provides great flexibility for adjusting the mutual ratios of the three polypeptide chains in the embodiment. However, when expressing at least two polypeptide chains at equal ratios leads to high yields or when the ratio has no specific meaning, the coding sequences of two or all three polypeptide chains can be inserted into an expression vector.

[0101] In some cases, the antibodies herein are chimeric antibodies. "Chimeric" forms of non-human (e.g., mouse) antibodies include chimeric antibodies that contain minimal sequences derived from non-human Ig. In most cases, chimeric antibodies are mouse antibodies in which at least a portion of an immunoglobulin constant region (Fc) (usually a portion of a human immunoglobulin) is inserted to replace the mouse Fc.

[0102] Provided herein are antibodies and antigen-binding fragments thereof, modified antibodies and antigen-binding fragments thereof, and binding agents that specifically bind to one or more epitopes on one or more target antigens. In one case, the binding agent selectively binds to an epitope on a single antigen. In another case, the binding agent is bivalent and selectively binds to two different epitopes on a single antigen or binds to two different epitopes on two different antigens. In another case, the binding agent is multivalent (i.e., trivalent, tetravalent, etc.), and the binding agent binds to three or more different epitopes on a single antigen, or binds to three or more different epitopes on two or more (multiple) antigens.

[0103] Antigen-binding fragments of any antibody herein are also contemplated. The terms "antigen-binding portion of an antibody," "antigen-binding fragment," "antigen-binding domain," "antibody fragment," or "functional fragment of an antibody" are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Representative antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, bispecific F(ab')2, trispecific F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), dsFv, bispecific scFv, variable heavy chain domain, variable light chain domain, variable NAR domain, bispecific scFv, minibody, diabody, bispecific diabody, triabody, tetrabody, minibody, maxibody, camelid antibody, VHH, minibody, endosome, fusion protein comprising an antibody portion (e.g., domain antibody), single-chain binding polypeptide, scFv-Fc, Fab-Fc, bispecific T cell engager (BiTE; two scFvs produced as a single polypeptide chain, wherein each scFv comprises an amino acid sequence of a combination of CDRs or a combination of VL / VL as described herein), tetravalent tandem diabody (TandAb; non-covalent homodimer arranged in a head-to-tail manner) Antibody fragments produced by polymorphic folding, such as TandAb comprising scFv, wherein the scFv comprises an amino acid sequence of a combination of CDRs or a combination of VL / VL as described herein), dual affinity retargeting antibodies (DART; different scFvs linked by stable interchain disulfide bonds), bispecific antibodies (bscAb; two single-chain Fv fragments linked by a glycine-serine linker), single domain antibodies (sdAb), fusion proteins, bispecific disulfide-stabilized Fv antibody fragments (dsFv-dsFv': two different disulfide-stabilized Fv antibody fragments linked by a flexible linker peptide).

[0104] As used herein, the term "affinity" refers to the resistance of a complex of two or more agents to dissociation upon dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA) or any other suitable technique. Affinity can be determined by methods such as Scatchard analysis or any other suitable technique.

[0105] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents, which affinity is expressed as KD. The binding affinity (KD) of the antibodies or antigen binding fragments herein may be less than 500nM, 475nM, 450nM, 425nM, 400nM, 375nM, 350nM, 325nM, 300nM, 275nM, 250nM, 225nM, 200nM, 175nM, 150nM, 125nM, 100nM, 90nM, 80nM, 70nM, 50nM, 50nM, 49nM, 48nM, 47nM, 46nM, 45nM, 44nM, 43nM, 42nM, 41nM, 40nM, 39nM, 38nM, 37nM, 36nM, 375nM, 385nM, 395nM, 396nM, 397nM, 398nM, 399nM, 301nM, 302nM, 303nM, 304nM, 305nM, 306nM, 307nM, 308nM, 309nM, 310nM, 311nM, 312nM, 313nM, 314nM 5nM, 34nM, 33nM, 32nM, 31nM, 30nM, 29nM, 28nM, 27nM, 26nM, 25nM, 24nM, 23nM, 22nM, 21nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13n M, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 990pM, 980pM, 970pM, 960pM, 950pM, 940pM, 930pM, 920pM, 910pM, 900pM , 890pM, 880pM, 870pM, 860pM, 850pM, 840pM, 830pM, 820pM, 810pM, 800pM, 790pM, 780pM, 770pM, 760pM, 750pM, 740pM, 730pM, 720pM, 710 pM, 700pM, 690pM, 680pM, 670pM, 660pM, 650pM, 640pM, 630pM, 620pM, 610pM, 600pM, 590pM, 580pM, 570pM, 560pM, 550pM, 540pM, 530pM, 5 pM, 290pM, 280pM, 270pM, 260pM, 250pM, 240pM, 230pM, 220pM, 210pM, 200pM, 190pM, 180pM, 170pM, or any integer in between. Binding affinity can be measured using surface plasmon resonance (SPR), Biosensors, scintillation proximity assays, isothermal titration calorimetry (ITC) assays, enzyme-linked immunosorbent assays (ELISA), ORIGEN immunoassays (IGEN), fluorescence quenching, fluorescence transfer, yeast display, or any combination thereof. Binding affinity can also be screened using a suitable bioassay.

[0106] In some embodiments, the antibody or antigen-binding fragment comprises an IgG1, IgG2a, IgG2b, IgG3, IgG4, IgD, IgM, IgA1, IgA2, or IgE heavy chain or a portion thereof. In some embodiments, the antibody or antigen-binding fragment comprises an IgG1, IgG2a, IgG2b, IgG3, or IgG4 heavy chain or a portion thereof. In some embodiments, the antibody comprises an IgG1 heavy chain or a portion thereof.

[0107] In some embodiments, the antibody or its antigen-binding fragment comprises a modified Fc domain. In some embodiments, the modified Fc domain comprises one or more amino acid replacements relative to the wild-type Fc domain of an antibody of the relevant subtype or isotype. In some embodiments, the modified Fc domain has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the wild-type Fc domain of the corresponding antibody or antigen-binding fragment. In some embodiments, the modified Fc domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more replacements relative to the corresponding wild-type Fc domain of an antibody or antigen-binding fragment. In some embodiments, substitutions in the Fc domain allow the antibody or antigen-binding fragment to have desired physicochemical properties, such as enhanced half-life or in vivo stability or other pharmacokinetic parameters, altered binding to Fc receptors, altered glycosylation pattern, to introduce additional disulfide bonds or to disrupt one or more disulfide bonds, or to alter intra- or intermolecular interactions of the antibody or antigen-binding fragment. In some embodiments, the Fc domain of the antibodies or antigen-binding fragments provided herein (e.g., IgG1 heavy chain) comprises one or more substitutions or combinations of substitutions selected from the following: T250Q / M428L; M252Y / S254T / T256E+H433K / N434F; E233P / L234V / L235A / G236A+A327G / A330S / P331S; E333A; S239D / A330L / I332E; P257I / Q31I; K326W / E333S; S239D / I332E / G236A; N297A; L234A / L235A; N297A+M252Y / S254T / T256E; K322A and K444A (EU numbering). In some embodiments, the antibody or antigen-binding fragment comprises one or more mutations or combinations of mutations selected from the group consisting of Arg435His (His435), Asn434Ala (A), Met428Leu / Asn434Ser (LS), Thr252Leu / Thr253Ser / Thr254Phe (LSF), Glu294Δ / Thr307Pro / Asn434Tyr (C6A-66), Thr256Asn / Ala378Val / Ser383Asn / Asn434Tyr (C6A-78) and Glu294Δ (Del), wherein the residue position numbers are based on the EU numbering convention. In some embodiments, the Fc domain comprises one or more substitutions selected from the group consisting of M252Y, S254T and T256E (EU numbering).

[0108] In some embodiments, the antibody or antigen-binding fragment is V H H antibody. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable domain of a camelid antibody. In some embodiments, the heavy chain variable domain comprises a sequence having at least 80% sequence identity with the following sequence:

[0109] wherein each X is independently absent or is any amino acid. In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% sequence identity to the following sequence:

[0110]

[0111] wherein each X is independently absent or is any amino acid.

[0112] In some embodiments, V H The heavy chain variable domain of the H antibody comprises a V with Tyl, N3113V or N3130V H The H variable domain sequences (as shown in Table 17) have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical.

[0113] In some embodiments, the antibody or antigen-binding fragment is V H In some embodiments, the antibody or antigen-binding fragment is a V fused to an Fc domain. H In some embodiments, V H The H domain is fused to the IgG1 Fc domain. H The H domain is fused to the Fc domain via a linker peptide. In some embodiments, the linker peptide is an antibody hinge region peptide or a variant thereof. In some embodiments, the linker peptide comprises an amino acid sequence having at least 80% or at least 90% sequence identity with the sequence SDKTHTCP (SEQ ID NO: 105). In some embodiments, the Fc domain comprises a CH2 domain, a CH3 domain, or both. In some embodiments, the Fc domain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity with the following sequence:

[0114]

[0115] In some embodiments, V HThe H domain is fused to a modified hinge region and an Fc domain, and the Fc domain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to:

[0116]

[0117] Antigen bound by an antibody or antigen-binding fragment

[0118] In some cases, the antibodies or antigen-binding fragments encoded by the systems provided herein can be used to treat, prevent or alleviate one or more diseases associated with the antigen to which the antibody or antigen-binding fragment binds. In some embodiments, the antibody or antigen-binding fragment binds to an antigen associated with a disease.

[0119] Infectious Diseases

[0120] In some embodiments, the system provided herein for producing antibodies or antigen binding fragments in a subject can be used for treating, managing or preventing infectious diseases. Infectious diseases include but are not limited to viral infections, microbial infections, bacterial infections, parasitic infections, fungal infections, etc. In some embodiments, the system provided herein is effective for preventing acute infections (for example, reducing the risk of being infected by pathogens (such as viruses or bacteria) by a certain amount, such as at least 50%, at least 60%, at least 70%, at least 80% or at least 90%, or eliminating the risk of being infected by pathogens). In some embodiments, the system provided herein can be used for treating or managing chronic infections (for example, the system eliminates or reduces one or more symptoms associated with existing infections, or the system reduces the prevalence or frequency of such symptoms that symptoms recur from time to time).

[0121] In some embodiments, the systems provided herein comprise vectors encoding antibodies or antigen-binding fragments that bind to antigens associated with infectious diseases, and the systems are administered as prophylactic agents (e.g., before a subject is infected with a pathogenic pathogen). In some embodiments, the systems provided herein comprise vectors encoding antibodies or antigen-binding fragments that bind to antigens associated with infectious diseases, and the systems are administered for treating infections (e.g., treating acute infections shortly after infection or onset of symptoms, or treating chronic infections later after infection or onset of symptoms).

[0122] Viral infection

[0123] In some embodiments, the vector encoding antibody or antigen binding fragment of the system provided herein binds to an antigen associated with a virus. In some embodiments, the system effectively induces protection against viral infection. In some embodiments, the system effectively alleviates, reduces or eliminates viral infection. In some embodiments, the antigen associated with a virus is a component of the virus. In some embodiments, the antigen associated with a virus is a viral protein, a viral glycan, a viral lipid membrane or other component. In some embodiments, the antigen associated with a virus is a viral protein.

[0124] In some embodiments, the virus targeted by the antibody or antigen-binding fragment is selected from the group consisting of: parvovirus, picornavirus, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus, calicivirus, arenavirus, polyomavirus, reovirus, togavirus, bunyavirus, herpes simplex virus, poxvirus, adenovirus, coxsackievirus, flavivirus, coronavirus, astrovirus, enterovirus, rotavirus, norovirus, retrovirus, papillomavirus, parvovirus, influenza virus, hemorrhagic fever virus, and rhinovirus. In some embodiments, the virus targeted by the antibody or antigen-binding fragment is selected from the group consisting of: Hantavirus, rabies virus, Nipah virus, Hendra virus, Rift Valley fever virus, Lassa virus, Marburg virus, Crimean-Congo hemorrhagic fever virus, hMPV, RSV, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, norovirus, monkeypox virus, vaccinia virus, Japanese encephalitis virus, yellow fever virus, HSV-1, HSV-2, MERS, varicella virus, hand, foot and mouth virus, CMV (HHV-5), equine encephalitis virus, EBV (HHV-4), human metapneumovirus, norovirus, enterovirus, smallpox virus, West Nile virus, paramyxoviridae, rhinovirus, mononucleosis virus, coxsackie virus B, influenza virus, poliovirus, measles virus, rubella virus, HPV, Zika virus, mumps virus, herpesviridae, chikungunya virus, Haemophilus influenzae and SARS-CoV-2 virus. In some embodiments, the virus is SARS-CoV-2.

[0125] Systems for treating or preventing SARS-CoV-2

[0126] In some embodiments, the systems provided herein encode antibodies or antigen binding fragments for treating and / or preventing SARS-CoV-2 infection and related diseases. In some embodiments, the antibodies or antigen binding fragments bind to components of the SARS-CoV-2 virus (e.g., viral proteins of the SARS-CoV-2 virus).

[0127] In some embodiments, the antibodies or antigen binding fragments provided herein bind to one or more proteins expressed by the SARS-CoV-2 virus. The antibody or antigen binding fragment can bind to any SARS-CoV-2 protein. In a preferred embodiment, the antibody or antigen binding fragment binds to the SARS-CoV-2 protein that participates in the infection of the SARS-CoV-2 virus of the cell, thereby preventing the infection of the cell, or binds to the SARS-CoV-2 protein expressed on the surface of the infected cell, thereby targeting the infected cell to be killed by an immune cell (e.g., NK cell).

[0128] In some embodiments, the SARS-CoV-2 protein to which the antibody or antigen-binding fragment binds is the SARS-CoV-2 spike protein or the SARS-CoV-2 nucleocapsid protein. In some embodiments, the SARS-CoV-2 protein is the SARS-CoV-2 spike protein.

[0129] In some embodiments, the antibody or antigen-binding fragment binds to the full-length SARS-CoV-2 spike protein. In some embodiments, the antibody or antigen-binding fragment specifically binds to a portion (e.g., a specific subunit) of the SARS-CoV-2 spike protein. A schematic diagram of the SARS-CoV-2 spike protein domain is shown in Figure 3A As shown. In some embodiments, the portion of the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises one or more subunits of the SARS-CoV-2 spike protein. In some embodiments, the subunit to which the antibody or antigen-binding fragment binds is selected from the group consisting of an N-terminal domain (NTD), a receptor binding domain (RBD), an S1 domain, an S2 domain, a fusion peptide domain, a heptad repeat domain 1 (HR1), a heptad repeat domain 2 (HR2), and a transmembrane domain (TM), or any combination thereof. In some embodiments, the portion of the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises an RBD.

[0130] In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises one or more modifications to the sequence of SEQ ID NO: 200, which is the sequence of the originally identified SARS-CoV-2 spike protein sequence.

[0131]

[0132]

[0133] In some embodiments, the modification of the SARS-CoV-2 spike protein to which the antibody or antigen binding fragment binds is a modification identified in a variant form of the SARS-CoV-2 virus (e.g., a β, γ, δ, or ο (Omicron) variant). In some embodiments, the modification of the SARS-CoV-2 spike protein to which the antibody or antigen binding fragment binds is in the RBD of a variant form of the virus. Exemplary modifications of the SARS-CoV-2 spike protein to which the antibody or antigen binding fragment binds in the RBD of a selected variant can be found in Table 1 below. In some embodiments, additional modifications to the spike protein on the outside of the RBD are bound by the antibody or antigen binding fragment. In some embodiments, the additional modifications to which the antibody or antigen binding fragment binds are inside the RBD and outside the RBD.

[0134] Table 1: Selected RBD mutations of selected variants

[0135]

[0136]

[0137] In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises one or more mutations found in the α, β, γ, δ, ε, ζ, η, ι, θ, κ, λ, or ο (Omicron) variants. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises one or more mutations found in the RBD of the α, β, γ, δ, ε, ζ, η, ι, θ, κ, λ, or ο (Omicron) variants. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises each mutation found in the RBD of the α, β, γ, δ, ε, ζ, η, ι, θ, κ, λ, or ο (Omicron) variants. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises each mutation found in the α, β, γ, δ, ε, ζ, η, ι, θ, κ, λ, or ο (Omicron) variants.

[0138] In some embodiments, the SARS-CoV-2 spike protein bound by the antibody or antigen-binding fragment contains one or more mutations found in the β, γ, δ, or ο (Omicron) variants. In some embodiments, the SARS-CoV-2 spike protein bound by the antibody or antigen-binding fragment contains one or more mutations found in the RBD of the β, γ, δ, or ο (Omicron) variant. In some embodiments, the SARS-CoV-2 spike protein bound by the antibody or antigen-binding fragment contains each mutation found in the RBD of the β, γ, δ, or ο (Omicron) variant. In some embodiments, the SARS-CoV-2 spike protein bound by the antibody or antigen-binding fragment contains each mutation found in the RBD of the β variant. In some embodiments, the SARS-CoV-2 spike protein bound by the antibody or antigen-binding fragment contains each mutation found in the RBD of the γ variant. In some embodiments, the SARS-CoV-2 spike protein bound by the antibody or antigen-binding fragment contains each mutation found in the RBD of the δ variant. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises each mutation found in the RBD of the o(Omicron) variant. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations found in the RBD of the o(Omicron) variant.

[0139] In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises A67V, 69-70Del, T95I, 137-145Del, G142D, 143-145Del, Y145H, 211Del, L212I, ins214EPE, ins214TDR, A222V, G339D, R346K, R346S, V367F, S373P, S375F, P384L, N394S, Q414K, K417N, K417T, N439K, N440K, G446S, Y449H, Y449N, N45 69K, N764K, D796Y, N856K, Q954H, N969K or L981F modifications, or any combination thereof. In some embodiments, the SARS-CoV-2 spike protein bound by the antibody or antigen-binding fragment further comprises 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more of the following modifications: A67V, 69-70Del, T95I, 137-145Del, G142D, 143-145Del, Y145H, 211Del, L212I, ins214EPE, ins214TDR, A222V, G339D, R346K, R346S, V367F, S373P, S375F, P384L, N394S, Q414K, K417N, K417T, N439K, N 446S, Y449H, Y449N, N450K, L452R, L452Q, S477N, T478K, V483A, E484A, E484K, E484Q, E484Del, F490R, F490S, Q493K, S494P, G496S, Q498R, N501T, N501Y, E516Q, T547K, Q613H, A653V, H655Y, G669S, Q677H, N679K, ins679GIAL, P681H, P681R, A701V, N764K, D796Y, N856K, Q954H, N969K and L981F modifications.

[0140] In some embodiments, the SARS-CoV-2 spike protein bound by the antibody or antigen-binding fragment further comprises A67V, 69-70Del, T95I, 137-145Del, G142D, 143-145Del, Y145H, 211Del, L212I, ins214EPE, A222V, G339D, R346K, R346S, S371L, S373P, S375F, N394S, K417N, K417T, N440K, G446S , Y449H, Y449N, L452R, L452Q, S477N, T478K, E484A, E484K, E484Del, F490R, F490S, Q493K, G496S, Q498R, N501Y, T547K, Q613H, H655Y, Q677H, N679K, P681H, P681R, A701V, N764K, D796Y, N856K, Q954H, N969K or L981F modifications, or any combination thereof. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more of the following modifications: A67V, 69-70Del, T95I, 137-145Del, G142D, 143-145Del, Y145H, 211Del, L212I, ins214EPE, A222V, G339D, R346K, R346S, S371L, S373P, S375F, N39 4S, K417N, K417T, N440K, G446S, Y449H, Y449N, L452R, L452Q, S477N, T478K, E484A, E484K, E484Del, F490R, F490S, Q493K, G496S, Q498R, N501Y, T547K, Q613H, H655Y, Q677H, N679K, P681H, P681R, A701V, N764K, D796Y, N856K, Q954H, N969K and L981F modifications.

[0141] In some embodiments, the SARS-CoV-2 spike protein bound by the antibody or antigen-binding fragment further comprises A67V, 69-70Del, T95I, G142D, 143-145Del, Y145H, 211Del, L212I, ins214EPE, A222V, G339D, R346K, S371L, S373P, S375F, K417N, K417T, N440K, G446S, L452R, L452Q, S477N, T478K, E484A, E484K, F490S, Q493K, G496S, Q498R, N501Y, Y505H, T547K, H655Y, N679K, P681H, P681R, A701, N764K, D796Y, N856K, Q954H, N969K or L981F modifications, or any combination thereof. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more of the following modifications: A67V, 69-70Del, T95I, G142D, 143-145Del, Y145H, 211Del, L212I, ins214EPE, A222V, G339D, R346K, S371L, S373 P, S375F, K417N, K417T, N440K, G446S, L452R, L452Q, S477N, T478K, E484A, E484K, F490S, Q493K, G496S, Q498R, N501Y, Y505H, T547K, H655Y, N679K, P681H, P681R, A701, N764K, D796Y, N856K, Q954H, N969K and L981F modifications.

[0142] In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds further comprises A67V, 69-70Del, T95I, G142D, 143-145Del, 211Del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, K417T, N440K, G44 6S, S477N, L452R, T478K, E484A, E484K, Q493K, G496S, Q498R, N501Y, Y505H, T547K, H655Y, N679K, P681H, P681R, A701V, N764K, D796Y, N856K, Q954H, N969K or L981F modifications, or any combination thereof. In some embodiments, the SARS-CoV-2 spike protein bound by the antibody or antigen-binding fragment further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more of the following modifications: A67V, 69-70Del, T95I, G142D, 143-145Del, 211Del, L212I, ins214EPE, G339D, S371L, S373P, S 375F, K417N, K417T, N440K, G446S, S477N, L452R, T478K, E484A, E484K, Q493K, G496S, Q498R, N501Y, Y505H, T547K, H655Y, N679K, P681H, P681R, A701V, N764K, D796Y, N856K, Q954H, N969K, and L981F modifications. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds is at least partially aligned with the sequence set forth in SEQ ID NO: 200. In some embodiments, the SARS-CoV-2 spike protein comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 200. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises an amino acid sequence having at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the sequence set forth in SEQ ID NO: 200. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises an amino acid sequence having at least 99.5% sequence identity to the sequence set forth in SEQ ID NO: 200.In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises an amino acid sequence having at least 99.6% sequence identity to the sequence set forth in SEQ ID NO: 200. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises an amino acid sequence having at least 99.7% sequence identity to the sequence set forth in SEQ ID NO: 200. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises an amino acid sequence having at least 99.8% sequence identity to the sequence set forth in SEQ ID NO: 200. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds comprises an amino acid sequence having at least 99.9% sequence identity to the sequence set forth in SEQ ID NO: 1.

[0143] In some embodiments, anti-SARS-CoV-2 antibodies bind to multiple variants of the SARS-CoV-2 virus. In some embodiments, anti-SARS-CoV-2 antibodies bind to 2, 3, 4, 5, 6 or more variants of the SARS-CoV-2 strain (SEQ ID NO: 200). In some embodiments, anti-SARS-CoV-2 antibodies bind to 1, 2, 3, 4, 5, 6 or more variants of the SARS-CoV-2 strain selected from α, β, γ, δ, ε, ζ, ζ, η, ι, θ, κ, λ and ο (Omicron). In some embodiments, anti-SARS-CoV-2 antibodies bind to each of the β, δ, γ and ο (Omicron) variants. In some embodiments, anti-SARS-CoV-2 antibodies bind to the RBD of each of the β, δ, γ and ο (Omicron) variants. In some embodiments, anti-SARS-CoV-2 antibodies bind to δ and ο (Omicron) variants. In some embodiments, anti-SARS-CoV-2 antibodies bind to RBDs of both the delta and o (omicron) variants.

[0144] In some embodiments, the anti-SARS-CoV-2 antibody is an antibody or antigen-binding fragment provided herein.

[0145] In some embodiments, the SARS-CoV-2 antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) having an amino acid sequence of any one of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, or 97. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment heavy chain variable region comprises a sequence having at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity to any one of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, or 97.

[0146] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises a heavy chain complementary determining region 1 (HCDR1) having an amino acid sequence of any one of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, or 98. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment HCDR1 comprises a sequence having at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, or 94%) amino acid sequence identity to any one of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 57, 65, 73, 82, 90, or 98.

[0147] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises a heavy chain complementary determining region 2 (HCDR2) having an amino acid sequence of any one of SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, or 99. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment HCDR2 comprises a sequence having at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, or 94%) amino acid sequence identity to any one of SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 58, 66, 74, 83, 91, or 99.

[0148] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises a heavy chain complementary determining region 3 (HCDR3) having an amino acid sequence of any one of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, or 100. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment HCDR3 comprises a sequence having at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, or 94%) amino acid sequence identity to any one of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, or 100.

[0149] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises a light chain variable region (LCVR) having an amino acid sequence of any one of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, or 93. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment light chain comprises a sequence having at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity to any one of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, or 93.

[0150] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises a light chain complementary determining region 1 (LCDR1) having an amino acid sequence of any one of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, or 94. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment LCDR1 comprises a sequence having at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, or 94%) amino acid sequence identity to any one of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, or 94.

[0151] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises a light chain complementary determining region 2 (LCDR2) having an amino acid sequence of any one of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, or 95. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment LCDR2 comprises a sequence having at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, or 94%) amino acid sequence identity to any one of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, or 95.

[0152] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises a light chain complementary determining region 3 (LCDR3) having an amino acid sequence of any one of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, or 96. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment LCDR2 comprises a sequence having at least 70% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, or 94%) amino acid sequence identity to any one of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, or 96.

[0153] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:2, HCDR2 as set forth in SEQ ID NO:3, and HCDR3 as set forth in SEQ ID NO:4. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:2, HCDR2 as set forth in SEQ ID NO:3, and HCDR3 as set forth in SEQ ID NO:4, and a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to the sequence set forth in SEQ ID NO:1. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 as set forth in SEQ ID NO:6, LCDR2 as set forth in SEQ ID NO:7, and LCDR3 as set forth in SEQ ID NO:8. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7, and LCDR3 shown in SEQ ID NO:8, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97% or 98% sequence identity to the sequence shown in SEQ ID NO:5.

[0154] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO: 10, HCDR2 as set forth in SEQ ID NO: 11, and HCDR3 as set forth in SEQ ID NO: 12. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO: 10, HCDR2 as set forth in SEQ ID NO: 11, and HCDR3 as set forth in SEQ ID NO: 12, and a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to the sequence set forth in SEQ ID NO: 9. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 as set forth in SEQ ID NO: 14, LCDR2 as set forth in SEQ ID NO: 15, and LCDR3 as set forth in SEQ ID NO: 16. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 shown in SEQ ID NO:14, LCDR2 shown in SEQ ID NO:15, and LCDR3 shown in SEQ ID NO:16, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97% or 98% sequence identity to the sequence shown in SEQ ID NO:13.

[0155] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO: 18, HCDR2 as set forth in SEQ ID NO: 19, and HCDR3 as set forth in SEQ ID NO: 20. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO: 18, HCDR2 as set forth in SEQ ID NO: 19, and HCDR3 as set forth in SEQ ID NO: 20, and a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to the sequence set forth in SEQ ID NO: 17. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 as set forth in SEQ ID NO: 22, LCDR2 as set forth in SEQ ID NO: 23, and LCDR3 as set forth in SEQ ID NO: 24. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:24, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97% or 98% sequence identity to the sequence shown in SEQ ID NO:21.

[0156] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:26, HCDR2 as set forth in SEQ ID NO:27, and HCDR3 as set forth in SEQ ID NO:28. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:26, HCDR2 as set forth in SEQ ID NO:27, and HCDR3 as set forth in SEQ ID NO:28, and a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to the sequence set forth in SEQ ID NO:25. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 as set forth in SEQ ID NO:30, LCDR2 as set forth in SEQ ID NO:31, and LCDR3 as set forth in SEQ ID NO:32. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 shown in SEQ ID NO:30, LCDR2 shown in SEQ ID NO:31, and LCDR3 shown in SEQ ID NO:32, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97% or 98% sequence identity to the sequence shown in SEQ ID NO:29.

[0157] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:34, HCDR2 as set forth in SEQ ID NO:35, and HCDR3 as set forth in SEQ ID NO:36. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:34, HCDR2 as set forth in SEQ ID NO:35, and HCDR3 as set forth in SEQ ID NO:36, and a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to the sequence set forth in SEQ ID NO:33. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 as set forth in SEQ ID NO:38, LCDR2 as set forth in SEQ ID NO:39, and LCDR3 as set forth in SEQ ID NO:40. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 shown in SEQ ID NO:38, LCDR2 shown in SEQ ID NO:39, and LCDR3 shown in SEQ ID NO:40, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97% or 98% sequence identity to the sequence shown in SEQ ID NO:37.

[0158] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:42, HCDR2 as set forth in SEQ ID NO:43, and HCDR3 as set forth in SEQ ID NO:44. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:42, HCDR2 as set forth in SEQ ID NO:43, and HCDR3 as set forth in SEQ ID NO:44, and a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to the sequence set forth in SEQ ID NO:41. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 as set forth in SEQ ID NO:46, LCDR2 as set forth in SEQ ID NO:47, and LCDR3 as set forth in SEQ ID NO:48. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 shown in SEQ ID NO:46, LCDR2 shown in SEQ ID NO:47, and LCDR3 shown in SEQ ID NO:48, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97% or 98% sequence identity to the sequence shown in SEQ ID NO:45.

[0159] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:50, HCDR2 as set forth in SEQ ID NO:51, and HCDR3 as set forth in SEQ ID NO:52. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:50, HCDR2 as set forth in SEQ ID NO:51, and HCDR3 as set forth in SEQ ID NO:52, and a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to the sequence set forth in SEQ ID NO:49. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 as set forth in SEQ ID NO:54, LCDR2 as set forth in SEQ ID NO:55, and LCDR3 as set forth in SEQ ID NO:56. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:55, and LCDR3 shown in SEQ ID NO:56, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97% or 98% sequence identity to the sequence shown in SEQ ID NO:53.

[0160] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:58, HCDR2 as set forth in SEQ ID NO:59, and HCDR3 as set forth in SEQ ID NO:60. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:58, HCDR2 as set forth in SEQ ID NO:59, and HCDR3 as set forth in SEQ ID NO:60, and a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to the sequence set forth in SEQ ID NO:57. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 as set forth in SEQ ID NO:62, LCDR2 as set forth in SEQ ID NO:63, and LCDR3 as set forth in SEQ ID NO:64. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 shown in SEQ ID NO:62, LCDR2 shown in SEQ ID NO:63, and LCDR3 shown in SEQ ID NO:64, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97% or 98% sequence identity to the sequence shown in SEQ ID NO:61.

[0161] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:66, HCDR2 as set forth in SEQ ID NO:67, and HCDR3 as set forth in SEQ ID NO:68. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:66, HCDR2 as set forth in SEQ ID NO:67, and HCDR3 as set forth in SEQ ID NO:68, and a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to the sequence set forth in SEQ ID NO:65. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 as set forth in SEQ ID NO:70, LCDR2 as set forth in SEQ ID NO:71, and LCDR3 as set forth in SEQ ID NO:72. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 shown in SEQ ID NO:70, LCDR2 shown in SEQ ID NO:71, and LCDR3 shown in SEQ ID NO:72, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97% or 98% sequence identity to the sequence shown in SEQ ID NO:69.

[0162] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:74, HCDR2 as set forth in SEQ ID NO:75, and HCDR3 as set forth in SEQ ID NO:76. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:74, HCDR2 as set forth in SEQ ID NO:75, and HCDR3 as set forth in SEQ ID NO:76, and a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to the sequence set forth in SEQ ID NO:73. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 as set forth in SEQ ID NO:78, LCDR2 as set forth in SEQ ID NO:79, and LCDR3 as set forth in SEQ ID NO:80. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 shown in SEQ ID NO:78, LCDR2 shown in SEQ ID NO:79, and LCDR3 shown in SEQ ID NO:80, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97% or 98% sequence identity to the sequence shown in SEQ ID NO:77.

[0163] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:82, HCDR2 as set forth in SEQ ID NO:83, and HCDR3 as set forth in SEQ ID NO:84. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:82, HCDR2 as set forth in SEQ ID NO:83, and HCDR3 as set forth in SEQ ID NO:84, and a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to the sequence set forth in SEQ ID NO:81. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 as set forth in SEQ ID NO:86, LCDR2 as set forth in SEQ ID NO:87, and LCDR3 as set forth in SEQ ID NO:88. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 shown in SEQ ID NO:86, LCDR2 shown in SEQ ID NO:87, and LCDR3 shown in SEQ ID NO:88, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97% or 98% sequence identity to the sequence shown in SEQ ID NO:85.

[0164] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:90, HCDR2 as set forth in SEQ ID NO:91, and HCDR3 as set forth in SEQ ID NO:92. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO:90, HCDR2 as set forth in SEQ ID NO:91, and HCDR3 as set forth in SEQ ID NO:92, and a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to the sequence set forth in SEQ ID NO:89. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 as set forth in SEQ ID NO:94, LCDR2 as set forth in SEQ ID NO:95, and LCDR3 as set forth in SEQ ID NO:96. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises LCDR1 shown in SEQ ID NO:94, LCDR2 shown in SEQ ID NO:95, and LCDR3 shown in SEQ ID NO:96, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97% or 98% sequence identity to the sequence shown in SEQ ID NO:93.

[0165] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO: 98, HCDR2 as set forth in SEQ ID NO: 99, and HCDR3 as set forth in SEQ ID NO: 100. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 as set forth in SEQ ID NO: 98, HCDR2 as set forth in SEQ ID NO: 99, and HCDR3 as set forth in SEQ ID NO: 100, and a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to the sequence set forth in SEQ ID NO: 97.

[0166] In some embodiments, the therapeutic compositions provided herein provide for expression of multiple antibodies or antigen binding fragments in a subject. In some embodiments, the therapeutic composition comprises a vector or multiple vectors encoding 2, 3, 4, 5 or more antibodies or antigen binding fragments that bind to a SARS-CoV-2 protein. In some embodiments, each antibody binds to a SARS-CoV-2 spike protein. In some embodiments, each antibody or antigen binding fragment thereof is an antibody or antigen binding fragment provided herein.

[0167] In some embodiments, the therapeutic compositions provided herein provide expression of multiple antibodies or antigen-binding fragments in a subject. In some embodiments, the therapeutic composition comprises one or more vectors encoding 2, 3, 4, 5 or more antibodies or antigen-binding fragments that bind to SARS-CoV-2 proteins. In some embodiments, each antibody binds to the SARS-CoV-2 spike protein. In some embodiments, each antibody or its antigen-binding fragment is an antibody or antigen-binding fragment provided herein. In some embodiments, the system provided herein includes two antibodies (e.g., Ab1 and Ab2) or their antigen-binding fragments. In some embodiments, each of the two antibodies binds to the RBD of the SARS-CoV-2 spike protein. In some embodiments, the two antibodies are capable of simultaneously binding to the RBD of the SARS-CoV-2 spike protein. In some embodiments, the two antibodies bind to two separate epitopes of the SARS-CoV-2 spike protein. Figure 3B An exemplary schematic diagram showing two antibodies combined in this manner. Figure 3B In the study, two complementary antibodies bind to the RBD at non-overlapping sites. One of the antibodies is a Class I anti-SARS-CoV-2 antibody that binds to the RBD in an "up" orientation, and the second antibody is a Class IV that binds to RBD core region 1. Specifically, Ab1 belongs to Class I and binds to the "receptor binding motif" (RBM) or ACE2 region of the spike RBD and is classified as an "ACE2 blocker." Ab2 belongs to Class IV, which does not overlap with the ACE2 binding site, but instead binds to a conserved region in the RBD (core 1 region).

[0168] In some embodiments, the expressed antibodies or antigen-binding fragments comprise two or more antibodies, each antibody comprising a VH and VL pair selected from SEQ ID NOs: 1 and 5, SEQ ID NOs: 9 and 13, SEQ ID NOs: 17 and 21, SEQ ID NOs: 25 and 29, SEQ ID NOs: 33 and 37, SEQ ID NOs: 41 and 45, SEQ ID NOs: 49 and 53, SEQ ID NOs: 57 and 61, SEQ ID NOs: 65 and 69, SEQ ID NOs: 73 and 77, SEQ ID NOs: 81 and 85, and SEQ ID NOs: 89 and 93.

[0169] Microbial infection

[0170] In some embodiments, the vector encoding of the system provided herein is an antibody or antigen binding fragment that binds to an antigen associated with an infectious microorganism. In some embodiments, the system effectively induces protection against microbial infection. In some embodiments, the system can effectively alleviate, reduce or eliminate the infection of a microorganism. In some embodiments, the antigen associated with a microorganism is a component of the microorganism. In some embodiments, the antigen associated with a microorganism is a protein, a polysaccharide, a lipid membrane, a cell wall or other component. In some embodiments, the antigen associated with a microorganism is a protein. In some embodiments, the microorganism is a bacterium. In some embodiments, the bacterium is a eukaryotic. In some embodiments, the bacterium is a prokaryotic. In some embodiments, the microorganism is a fungus.

[0171] In some embodiments, the microorganism targeted by the antibody or antigen-binding fragment is Bacillus anthracis, Corynebacterium diphtheria, Bordetella pertussis, Streptococcus pneumonia, Haemophilus influenza, Salmonella typhimurium, Shigella species, Streptococcus species, Chlamydia trachomatis, Yersinia pestis, Methicillin-resistant Staphylococcus aureus,MRSA), Staphylococcus aureus, Clostridium tetani, Vibrio cholera, Escherichia coli, Klebsiella pneumonia, Borrelia burgdorferi, Borreliamayonii, Clostridioides difficile, Pseudomonas aeruginosa, Helicobacter pylori, Streptococcus pyogenes, Francisella tularensis, Acinetobacter species, Neisseria gonorrhoeae, Leptospira species, Coxiella burnetii, Clostridium botulinum botulinum, Burkholderia pseudomallei, Gram-negative bacteria, Salmonella paratyphi, Mycobacterium leprae, Brucella species, Campylobacter species, Listeria monocytogenes, Mycobacterium avium, Mycoplasma pneumonia, Rickettsia species, Anaplasma species, Ehrlichia species, Neorickettsia species, Neoehrlichia species, Orientia species, Mycobacterium tuberculosis tuberculosis, Anaplasma phagocytophilum, Orientia tsutsugamushi, or Bartonella species.

[0172] parasitic infection

[0173] In some embodiments, the vector encoding antibody or antigen binding fragment of the system provided herein binds to an antigen associated with an infectious parasite. In some embodiments, the system effectively induces protection against parasitic infections. In some embodiments, the system can effectively alleviate, reduce or eliminate parasitic infections. In some embodiments, the antigen associated with the parasite is a component of the parasite. In some embodiments, the antigen associated with the parasite is a protein, polysaccharide, lipid membrane, cell wall or other component.

[0174] In some embodiments, the parasite is a Babesia species, Ancylostoma duodenale, Necator americanus, Sarcoptes scabiei, Ascaris lumbricoides, Schistosoma mansoni, Taeniasolium, Enterobius vermicularis, Wuchereria bancrofti, Toxoplasma gondii, Giardia lamblia, Entamoeba histolytica, Plasmodium species, Leishmania species, Trypanosoma cruzi cruzi, Schistosoma species, Cryptosporidium species, Trypanosoma brucei, Wuchereria bancrofti, Brugia malayi, Brugia timori, Entamoeba histolytica or Onchocerca volvulus.

[0175] Immune checkpoints for infectious diseases

[0176] In some embodiments, the vector encoding antibody or antigen binding fragment of the system provided herein is combined with an immune checkpoint molecule. In some embodiments, this immune checkpoint binding agent acts as an inhibitor of immune checkpoint. In some embodiments, immune checkpoint binding agents can be used to treat infectious diseases. In some embodiments, immune checkpoint molecules are PD-1, PD-L1, CTLA-4, TIM-3, TIGIT, 4-1BB (CD137), GITR (CD357) or killer IgG-like receptors (KIR). In some embodiments, immune checkpoint molecules are PD-1. In some embodiments, immune checkpoint molecules are PD-L1.

[0177] Other diseases and indications

[0178] Other indications or diseases that can be treated and / or prevented using the systems provided herein include any indication or disease that can be treated with antibodies. Non-limiting examples of such diseases or indications include cancer, autoimmune diseases, inflammatory diseases, autoinflammatory diseases, acute toxicity from environmental factors (e.g., toxins such as environmental toxins or other toxins, such as snake venom, etc.) or allergies.

[0179] In some embodiments, the antibodies or antigen-binding fragments of the systems provided herein are specific for a cancer antigen. In some embodiments, the cancer antigen is selected from the group consisting of programmed cell death 1 (PD1), programmed cell death ligand 1 (PDL1), CD5, CD20, CD19, CD22, CD30, CD33, CD40, CD44, CD52, CD74, CD103, CD137, CD123, CD152, carcinoembryonic antigen (CEA), integrin, epidermal growth factor (EGF) receptor family members, vascular epidermal growth factor (VEGF), proteoglycan, disialoganglioside, B7-H3, cancer antigen 125 (CA-125), epithelial cell adhesion molecule (EpCAM), vascular endothelial growth factor receptor 1, vascular endothelial growth factor receptor 2, tumor-associated glycoprotein, mucin 1 (MUC1), tumor necrosis factor receptor, insulin-like growth factor receptor, folate receptor alpha, transmembrane glycoprotein NMB, CC chemokine receptor, prostate specific membrane antigen (PSMA), recepteur d'origine nantais (RON) receptor, cytotoxic T lymphocyte antigen 4 (CTLA4), colon cancer antigen 19.9, gastric cancer mucin antigen 4.2, colorectal cancer antigen A33, ADAM-9, AFP carcinoembryonic antigen-alpha-fetoprotein, ALCAM, BAGE, β-catenin, carboxypeptidase M, B1, CD23, CD25, CD27, CD28, CD36, CD45, CD46, CD52, CD56, CD79a / CD79b, CD317, CDK4, CO-43 (Le b Blood type), CO-514 (Le ablood type), CTLA-1, cytokeratin 8, DR5, E1 series (B blood type), ephrin receptor A2 (EphA2), Erb (ErbB1, ErbB3, ErbB4), lung adenocarcinoma antigen F3, antigen FC10.2, GAGE-1, GAGE-2, GD2 / GD3 / GD49 / GM2 / GM3, GICA19-9, gp37, gp75, gp100, HER-2 / neu, human milk fat globule Antigen, human papillomavirus-E6 / human papillomavirus-E7, high molecular weight melanoma antigen (HMW-MAA), differentiation antigen (I antigen), I (Ma) found in gastric adenocarcinoma, integrin α-V-β-6, integrin β6 (ITGβ6), interleukin 13 receptor α2 (IL13Rα2), JAM-3, KID3, KID31, KS1 / 4 pan-cancer antigen, KSA (17-1A), human lung cancer antigen L6, human lung cancer antigen L2 0, LEA, LUCA-2, M1:22:25:8, M18, M39, MAGE-1, MAGE-3, MART, Myl, MUM-1, N-acetylglucosaminyltransferase, neoglycoprotein, NS-10, OFA-1 and OFA-2, oncostatin M (oncostatin receptor β), rhol5, prostate-specific antigen (PSA), PSMA, polymorphic epithelial mucin antigen (PEMA), PIPA, prostatic acid phosphate, R24, ROR1, SSEA-1, SSEA-3, SSEA-4, sTn, T cell receptor-derived peptide, T5A7, tissue antigen 37, TAG-72, TL5 (A blood type), TNF-α receptor (TNFαR), TNFβR, TNFγR, TRA-1-85 (H blood type), transferrin receptor, TSTA tumor-specific transplantation antigen, VEGF-R, Y hapten, Le y and 5T4.

[0180] In some embodiments, the antibodies or antigen-binding fragments of the systems provided herein are specific to allergens. In some embodiments, the allergens are derived from mites, insects, pollen, animal epithelium, mold, meat, fish, crustaceans, fruits, nuts, vegetables, flour or bran, milk, eggs, spices, hay, silk, cotton, latex, yeast, grass, tree, grain or animal hair. In some embodiments, the mite allergen is Der p 1, Der f 1 or tropical clawless mite (Blomia tropicalis). In some embodiments, the insect allergen is derived from cockroaches or locusts. In some embodiments, the pollen allergen is derived from mugwort, birch, nettle, chrysanthemum, alder, spruce, quinoa, goldenrod, Humulus japonicus, pine, orchard grass, dandelion, corn, poplar, sycamore, short ragweed, elm, English plantain, willow, wheat, timothy, queen palm, mulberry, rapeseed, or ryegrass. In some embodiments, the animal epithelial allergen is derived from dog epithelial, cat epithelial, goat epithelial, duck feathers or fur. In some embodiments, the mold allergen is derived from Alternaria tenuis, Botrytis c., Candida albicans, Cladosporium h., Curvularia l., Penicillium notatum, Pullalaria pullulans, Trichophyton mentagrophytes, Fusarium globosum, Helminthosporium halodes, Aspergillus f., Mucor mucedo, Rhizopus nigricans, or Serpulalacrymans. In some embodiments, the meat allergen is derived from lamb, chicken, beef, pork, duck, turkey, or goose. In some embodiments, the fish or crustacean allergen is derived from cod, carp, catfish, tuna, scallops, crab, shrimp, spiny lobster, or mussels. In some embodiments, the fruit or nut allergen is derived from pineapple, apple, citrus, banana, mango, strawberry, peanut, cashew, orange, paprika, peach, pear, tomato, walnut, grape, sunflower seed, almond, hazelnut, pistachio, pine nut, cocoa bean, chestnut, macadamia nut, Brazil nut, lupine, pecan, or pumpkin seed. In some embodiments, the vegetable allergen is derived from potato, parsley, spinach, soy, onion, leek, or cabbage. In some embodiments, the flour or bran allergen is derived from rice, corn flour, wheat flour, buckwheat, or mung bean.In some embodiments, the milk or egg allergen is derived from milk, whole eggs, egg whites, or egg yolks. In some embodiments, the spice allergen is derived from cocoa, cinnamon, paprika, black pepper, sesame, or garlic. In some embodiments, the allergen is derived from hay, silk fabric, cotton, latex, or yeast (e.g., baker's yeast). In some embodiments, the grass allergen is derived from velvet, orchard grass, ryegrass, timothy grass, bluegrass, or meadow fescue. In some embodiments, the tree allergen is derived from alder, hazel, poplar, elm, willow, birch, oak, or sycamore. In some embodiments, the grass allergen is derived from wormwood, nettle, dandelion, or English plantain. In some embodiments, the grain allergen is derived from grass, barley, oats, rye, or wheat. In some embodiments, the grass allergen is derived from Australian grass. In some embodiments, the grass allergen is derived from bahia grass, Johnson grass, Bermuda grass, velvet grass, or canary grass. In some embodiments, the animal hair allergen is derived from hamsters, dogs, rabbits, cats, or guinea pigs.

[0181] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an antigen involved in an inflammatory disease. In some embodiments, the inflammatory disease is allergy, asthma, celiac disease, glomerulonephritis, hepatitis, or inflammatory bowel disease. In some embodiments, the inflammatory disease is mast cell activation syndrome (MCAS).

[0182] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an antigen involved in an autoinflammatory disease. In some embodiments, the inflammatory disease is an autoinflammatory disease selected from familial Mediterranean fever (FMF), cryopyrin-associated periodic syndrome (CAPS), TNF receptor-associated periodic syndrome (TRAPS), IL-1 receptor antagonist deficiency (DIRA) or high IgD syndrome (HIDS).

[0183] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an antigen involved in an autoimmune disease. In some embodiments, the autoimmune disease is selected from rheumatoid arthritis, psoriasis, Guillain-Barré syndrome, Graves' disease, myasthenia gravis, vasculitis, lupus, type 1 diabetes, Hashimoto's disease, inflammatory bowel disease, celiac disease, or multiple sclerosis (MS).

[0184] Lipid vesicles

[0185] In some aspects, the antibody or Fab expression system provided herein comprises lipid vesicle.The lipid vesicle comprises one or more lipid components, and the lipid component can encapsulate the carrier (e.g., DNA plasmid) provided herein.In some embodiments, the lipid vesicle comprises one or more protein components, and the protein component is at least partially contacted or arranged in lipid.In some embodiments, the lipid vesicle includes lipid nanoparticle (LNP) compositions and wherein LNP encapsulates the composition of polynucleotide construct (e.g., carrier provided herein, such as plasmid DNA), and the polynucleotide construct comprises the coding region of the antibody or Fab provided herein.

[0186] In some embodiments, compositions comprising plasmid DNA encapsulated with LNP or other lipid vesicle formulations are non-toxic and non-immunogenic to animals at doses >15 mg / kg, and exhibit efficiencies greater than 80 times higher than those achievable with neutral lipid compositions, and 2-5 times higher than those achievable with cationic lipid compositions. In some embodiments, LNP or other lipid vesicle cargo is deposited directly into the cytoplasm, thereby bypassing the endocytic pathway.

[0187] In a further embodiment, the lipid vesicles of the present invention are used for targeted production of antibodies or antigen-binding fragments in target cells (which are then preferably secreted from the cells), the lipid vesicle composition comprising: (a) a lipid nanoparticle carrier for non-specific delivery of nucleic acids to mammalian cells, wherein the lipid nanoparticles comprise one or more lipids and one or more fusogenic membrane proteins, and (b) an expression construct for preferentially producing antibodies or antigen-binding fragments in target cells.

[0188] The lipid vesicle composition according to some aspects of these embodiments comprises one or more lipids at a concentration ranging from 1 mM to 100 mM, or 5 mM to 50 mM, or 10 mM to 30 mM, or 15 mM to 25 mM. The lipid vesicle formulations exemplified herein may comprise one or more lipids at a concentration of about 20 mM.

[0189] In some illustrative lipid vesicle compositions, one or more lipids are selected from 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG). The LNP composition may contain two or more lipids selected from the group consisting of DODAP, DOTAP, DOPE, cholesterol, and DMG-PEG.

[0190] Exemplified herein are lipid compositions comprising DODAP, DOTAP, DOPE, cholesterol and DMG-PEG in a molar ratio of 35-55 mol% DODAP: 10-20 mol% DOTAP: 22.5-37.5 mol% DOPE: 4-8 mol% cholesterol: 3-5 mol% DMG-PEG; or a molar ratio of about 45 mol% DODAP: about 15 mol% DOTAP: about 30 mol% DOPE: about 6 mol% cholesterol: about 4 mol% DMG-PEG. In some aspects, the lipid vesicle composition comprises DODAP, DOTAP, DOPE, cholesterol and DMG-PEG in a molar ratio of 45 mol% DODAP: 5 mol% DOTAP: 30 mol% DOPE: 6 mol% cholesterol: 4 mol% DMG-PEG.

[0191] According to other aspects of these embodiments, lipid vesicle preparations include one or more fusogenic membrane proteins, and the fusogenic membrane protein concentration range is 0.5 μM to 20 μM, or 1 μM to 10 μM or 3 μM to 4 μM. Illustrated herein are lipid vesicle preparations, wherein the concentration of fusogenic membrane proteins is about 3.5 μM, about 5 μM, about 7.5 μM, about 10 μM, about 12.5 μM, about 15 μM, about 20 μM. Exemplary suitable fusogenic membrane proteins include fusogenic membrane proteins provided herein, including p15x fusogenic membrane proteins (SEQ ID NO: 201), p14 fusogenic membrane proteins (SEQ ID NO: 202) and p14e15 fusogenic membrane proteins (SEQID NO: 203).

[0192] In additional aspects of these embodiments, the lipid vesicle formulation includes a vector comprising a polynucleotide sequence encoding one or more of the antibodies or antigen-binding fragments described above.

[0193] In some embodiments, the pharmaceutical compositions provided herein comprise a proteolipid vehicle (PLV). In some embodiments, the proteolipid vehicle encapsulates one or more other parts of the pharmaceutical composition (e.g., a DNA vector, such as any DNA vector provided herein).

[0194] Exemplified herein are lipid vesicle formulations that include a vector (eg, a DNA plasmid) at a concentration ranging from 20 μg / mL to 1.5 mg / mL, 100 μg / mL to 500 μg / mL, or at a concentration of about 250 μg / mL.

[0195] Suitable exemplary lipid vesicle formulations include the following: for each 1 mL of lipid vesicles, the lipid concentration is about 20 mM, the DNA content is about 250 μg, and the fusogenic protein (e.g., p14 or p14e15) is about 3.5 μM, wherein the lipid formulation comprises DODAP:DOTAP:DOPE:cholesterol:DMG-PEG in a molar % ratio of about 45:15:30:6:4, respectively.

[0196] The lipid vesicle comprises one or more lipid components. In some embodiments, the lipid of the lipid vesicle is a non-immunogenic lipid. In some embodiments, the lipid of the lipid vesicle comprises a naturally occurring lipid. In some embodiments, the lipid of the lipid vesicle comprises a naturally occurring mammalian lipid. In some embodiments, the lipid of the lipid vesicle comprises a naturally occurring human lipid.

[0197] In some embodiments, lipid vesicle comprises minimum cationic lipid.Cationic lipid is used in some lipid vesicle preparations, to promote the fusion of lipid vesicle and another film needed.However, in some embodiments, proteolipid vesicle provided herein uses alternative strategies to fuse lipid vesicle with required cell membrane (for example, fusogenic membrane protein).Therefore, in some cases, lipid vesicle provided herein uses less cationic lipid than other preparations, which makes lipid vesicle toxicity provided herein less.Due to their positive charge, cationic lipid has been used to concentrate negatively charged DNA molecules and promote DNA to be encapsulated in liposome.In some embodiments, lipid vesicle is included in proteolipid vehicle and is less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1% cationic lipid content (w / w of total lipid content). In some embodiments, the lipid vesicle comprises a molar ratio of ionizable lipid to carrier (e.g., plasmid) of less than 100: 1, less than 75: 1, less than 50: 1, less than 40: 1, less than 30: 1, less than 25: 1, or less than 20: 1. In some embodiments, the molar ratio of ionizable lipid to carrier (e.g., plasmid) is between 2.5: 1 and 20: 1.

[0198] Fusogenic membrane protein

[0199] In some embodiments, the proteolipid vehicle comprises a fusogenic membrane protein. A fusogenic membrane protein is a membrane-bound or associated protein that promotes lipid-lipid membrane fusion of two independent lipid membranes. Many such fusogenic membrane proteins are known in the art.

[0200] In some embodiments, the fusogenic membrane protein is derived from a virus. Examples of such viral-derived fusogenic membrane proteins include influenza virus hemagglutinin (HA) protein, Sendai virus F protein, Filoviridae Ebola virus glycoprotein, Retroviridae glycoprotein 41, Togaviridae alphavirus envelope protein E1, Flaviviridae flavivirus envelope protein, Herpesvirus glycoprotein B, Rhabdoviridae SVS G protein, Reoviridae fusion-associated small transmembrane protein (FAST) and derivatives thereof.

[0201] In other aspects of these embodiments, the lipid vesicle is a fusogenic lipid vesicle, such as a fusogenic lipid vesicle comprising a fusogenic membrane protein, such as the fusogenic p14 FAST membrane fusion protein from a reptilian reovirus, to catalyze lipid mixing between the lipid vesicle and the target cell plasma membrane. Suitable fusogenic membrane proteins are described in PCT Patent Publication Nos. WO2012 / 040825A1 and WO2002 / 044206A2, Lau, Biophys. Jg £: 272 (2004), Nesbitt, Master of Science Thesis (2012), Zijlstra, AACR (2017), Mrlouah, PAACRAM 77 / 13 Supnn: Abst 5143 (2017), Krabbe, Cancers 10: 216 (2018), Sanchez-Garria, Chem Comm 53: 4565 (2017), Clancy, / Virology 83 / 71: 2941 (2009), Sudo, J Control Release 255: 1 (2017), Wong, Cancer Gene Therapy 23:355 (2016), and Corcoran, JBC 281 / 421:31778 (2006), each of which is incorporated herein by reference in its entirety. Further example FAST membrane proteins, their fusion proteins and exemplary formulations can be found in PCT Publication No. WO2022 / 067446A1, which is incorporated herein by reference in its entirety.

[0202] Examples of fusion-promoting FAST proteins include p15 and p14e15 proteins with the amino acid sequences shown in Table 2. In some embodiments, the FAST proteins of lipid vesicles provided herein include sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% sequence identity with the sequences of the following p15x. In some embodiments, the FAST proteins of lipid vesicles provided herein include sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% sequence identity with the sequences of the following p14. In some embodiments, the FAST proteins of lipid vesicles provided herein include sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% sequence identity with the sequences of the following p14e15.

[0203] Table 2

[0204]

[0205] Preferred fusogenic membrane proteins are non-immunogenic (e.g., no specific immune response to the fusogenic membrane protein is produced after administration to a subject). In some cases, such fusogenic membrane proteins allow repeated administration of the pharmaceutical compositions provided herein and / or enhance the delivery of packaged materials (e.g., DNA vectors provided herein) to target cells.

[0206] In some embodiments, the fusogenic membrane protein is a FAST protein. Specific examples of FAST proteins are described in U.S. Patent No. 8,252,901 and U.S. Patent Application No. 2019 / 0367566, each of which is incorporated herein by reference in its entirety.

[0207] FAST proteins are a unique family of fusogenic membrane proteins encoded by fusogenic reoviruses. FAST proteins include: p10, p14, p15, and p22. FAST proteins range in size from 95 to 198 amino acids and are the smallest known viral membrane fusion proteins. FAST proteins do not mediate virus-cell fusion, but are non-structural viral proteins expressed on the surface of virus-infected or transfected cells, where they induce cell-cell fusion and the formation of multinuclear syncytia. When reconstituted into liposome membranes, purified FAST proteins induce liposome-cell and liposome-liposome fusion, indicating that FAST proteins are true membrane fusion proteins.

[0208] In contrast to most enveloped viral fusion proteins, in which the cytoplasmic tail is very short relative to the overall size of the protein, the FAST proteins all have an unusual topology that partitions the bulk of the protein into the membrane and cytoplasm, exposing only an ectodomain of 20 to 43 residues to the extracellular environment. Despite the small size of their ectodomains, both p14 and p10 encode a small hydrophobic segment (HP) that is hypothesized to induce lipid mixing similarly to fusion proteins encoded by enveloped viral fusion proteins. The p14 HP consists of the N-terminal 21 residues of the protein, but peptides corresponding to this sequence are required to include an N-terminal myristate moiety to mediate lipid mixing. Nuclear magnetic resonance (NMR) spectroscopy revealed that two proline residues within the p14 HP form a protruding ring structure that presents valine and phenylalanine residues at the apex and is connected to the rest of the protein by a flexible linker region. On the other hand, the p10 HP (flanked by two cysteine ​​residues forming intramolecular disulfide bonds) may be more similar to the internal fusion peptides of the Ebola virus and avian leukosis and sarcoma virus (ALSV) glycoproteins and may adopt a cystine lasso structure that forces the conserved valine and phenylalanine residues to be exposed to the solvent for membrane interactions. In contrast to p14 and p10, the 20-residue ectodomain of p15 completely lacks a hydrophobic sequence that could serve as a traditional fusion peptide. In the absence of such a motif, the p15 ectodomain instead encodes a polyproline helix that has been proposed to serve as a membrane destabilizing motif.

[0209] FAST proteins with improved properties to promote membrane fusion in the context of synthetic lipid vesicles, such as the proteolipid vehicles of the present invention, have been described previously (eg, US Pat. No. 10,227,386).

[0210] In some embodiments, the FAST protein comprises a domain from one or more FAST proteins selected from p10, p14, p15, and p22. In some embodiments, the FAST protein comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

[0211] In some embodiments, FAST protein comprises an intracellular domain having at least about 80%, at least about 85%, at least about 90% or at least about 95% sequence identity with an intracellular domain from p10, p14, p15 or p22. In some embodiments, FAST protein comprises an intracellular domain from p10, p14, p15 or p22. In some embodiments, FAST protein comprises an intracellular domain having at least about 80%, at least about 85%, at least about 90% or at least about 95% sequence identity with an intracellular domain from p15. In some embodiments, FAST protein comprises an intracellular domain from p15.

[0212] In some embodiments, FAST protein comprises a transmembrane domain from a wild-type FAST protein or a derivative thereof. In some embodiments, FAST protein comprises a transmembrane domain with at least about 80%, at least about 85%, at least about 90% or at least about 95% sequence identity with a transmembrane domain from p10, p14, p15 or p22. In some embodiments, the transmembrane domain comprises 23 amino acid residues (at least two hydrophobic β-branch residues adjacent to the extracellular domain, three consecutive serine residues adjacent to at least two hydrophobic β-branch residues, glycine residues at positions 7 and 13 of the connection between the extracellular domain and the first hydrophobic β-branch residue base). In some embodiments, the transmembrane domain comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90% or at least about 95% sequence identity with the sequence of IVSSSTGIIIAVGIFAFIFSFLY (SEQ ID NO: 204).

[0213] In some embodiments, FAST protein comprises an extracellular domain from a wild-type FAST protein or a derivative thereof. In some embodiments, FAST protein comprises an extracellular domain having at least about 80%, at least about 85%, at least about 90% or at least about 95% sequence identity with an extracellular domain from p10, p14, p15 or p22. In some embodiments, FAST protein comprises an extracellular domain from p10, p14, p15 or p22. In some embodiments, FAST protein comprises an intracellular domain having at least about 80%, at least about 85%, at least about 90% or at least about 95% sequence identity with an extracellular domain from p14. In some embodiments, FAST protein comprises an extracellular domain from p14.

[0214] In some embodiments, the FAST protein provided herein comprises an extracellular domain comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% sequence identity to the p14FAST protein (e.g., a sequence defined by the sequence MGSGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWE (SEQ ID NO: 205)), and comprising a functional myristoylation motif; a transmembrane domain comprising 23 amino acid residues (at least two hydrophobic β-branch residues adjacent to the extracellular domain, three consecutive serine residues adjacent to the at least two hydrophobic β-branch residues, glycine residues at positions 7 and 13 at the junction between the extracellular domain and the first hydrophobic β-branch residue base); and an intracellular domain comprising a sequence identical to the p15 intracellular domain (e.g., a sequence defined by the sequence KLLQWYNRKSKNKKRKEQIREQIELGLLSYGAGVASLPLLNVIAHNPGS (SEQ ID NO: NO:206) or VISATPIYKGPCTGVPNSRLLQITSGTAEENTRILNHDGRNPDGSINV (SEQ ID NO:207)) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% sequence identity.

[0215] In some embodiments, the FAST protein comprises amino acids having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to:

[0216]

[0217] In some embodiments, the FAST protein is provided by a commercial supplier. In some embodiments, the FAST protein is part of the Fusogenix platform produced by Entos Pharmaceuticals.

[0218] II. Application

[0219] The present invention relates to administering the system provided herein to a subject. In some embodiments, administration results in transfection of one or more cells of a subject. In some embodiments, the cells transfected by the system provided herein are long-acting cells (e.g., skeletal muscle cells), resulting in the presence of a stable level of antibody or AF or cells in the subject to produce antibody or AF over time. In some embodiments, this results in maintaining the level of treatment-related antibodies or AFs over time. This administration resulting in the need for or optimal pharmacokinetics of an antibody or AF can be effectively used for sustained treatment or prevention of related diseases. In some embodiments, administration is performed by injecting the lipid vesicle provided herein containing a carrier provided herein into a subject.

[0220] dose

[0221] In some embodiments, a prescribed dose of a vector (e.g., a DNA plasmid provided herein) is administered to a subject. In some embodiments, the prescribed dose is selected to elicit a desired level of antibody or antigen-binding fragment in a subject, which level will depend on the level of clinically or therapeutically relevant antibody or antigen-binding fragment.

[0222] In some embodiments, the dose of the vector administered to the subject is 0.1 mg / kg to 20 mg / kg. In some embodiments, the dose of the vector administered to the subject is 0.1 mg / kg to 0.5 mg / kg, 0.1 mg / kg to 1 mg / kg, 0.1 mg / kg to 2 mg / kg, 0.1 mg / kg to 3 mg / kg, 0.1 mg / kg to 4 mg / kg, 0.1 mg / kg to 5 mg / kg, 0.1 mg / kg to 7.5 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.1 mg / kg to 20 mg / kg, 0.5 mg / kg to 10 mg / kg, 0.1 mg / kg to 20 mg / kg, 0.5 mg / kg to 10 mg / kg, 0.1 mg / kg to 20 mg / kg, 0.5 mg / kg to 10 mg / kg, 0.1 mg / kg to 20 mg / kg, 0.1 mg / kg to 3 mg / kg, 0.1 mg / kg to 4 mg / kg, 0.1 mg / kg to 5 mg / kg, 0.1 mg / kg to 7.5 mg / kg, 0.1 ... g / kg to 1mg / kg, 0.5mg / kg to 2mg / kg, 0.5mg / kg to 3mg / kg, 0.5mg / kg to 4mg / kg, 0.5mg / kg to 5mg / kg, 0.5mg / kg to 7.5mg / kg, 0.5mg / kg to 10mg / kg, 0.5mg / kg to 20mg / kg, 1mg / kg to 2mg / kg, 1mg / kg to 3mg / kg, 1mg / kg to 4mg / kg, 1mg / kg to 5mg / kg, 1mg / kg to 7.5mg / kg, 1mg / kg to 10mg / kg, 1mg / kg to 20mg / kg, 2mg / kg to 3mg / kg, 2mg / kg to 4mg / kg, 2mg / kg to 5mg / kg, 2mg / kg to 7.5mg / kg, 2mg / kg to 10mg / kg, 2mg / kg to 20mg / kg, 3mg / kg to 4mg / kg, 3mg / kg to 5mg / kg, 3mg / kg to 7.5mg / kg In some embodiments, the dose of the vector administered to the subject is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, or about 20 mg / kg. In some embodiments, the dose of the vector administered to the subject is at least 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 7.5 mg / kg, or 10 mg / kg.In some embodiments, the dose of the vector administered to the subject is at most 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, or 20 mg / kg. In some embodiments, multiple doses of the same amount of vector are administered to the subject. In some embodiments, the subject receives a first dose and a second, lower dose (e.g., after an appropriate time period).

[0223] In some embodiments, the dose of the vector administered to the subject is about 10 micrograms to about 5,000 micrograms. In some embodiments, the dose of the vector administered to the subject is about 10 micrograms to about 50 micrograms, about 10 micrograms to about 100 micrograms, about 10 micrograms to about 250 micrograms, about 10 micrograms to about 500 micrograms, about 10 micrograms to about 1,000 micrograms, about 10 micrograms to about 5,000 micrograms, about 50 micrograms to about 100 micrograms, about 50 micrograms to about 250 micrograms, about 50 micrograms to about 500 micrograms, about 50 micrograms to about 1,000 micrograms, about 50 micrograms to about In some embodiments, the dose of the vector administered to a subject is about 10 micrograms, about 50 micrograms, about 100 micrograms, about 250 micrograms, about 500 micrograms, about 1,000 micrograms, about 100 micrograms to about 5,000 micrograms, about 250 micrograms to about 500 micrograms, about 250 micrograms to about 1,000 micrograms, about 250 micrograms to about 5,000 micrograms, about 500 micrograms to about 1,000 micrograms, about 500 micrograms to about 5,000 micrograms, or about 1,000 micrograms to about 5,000 micrograms. In some embodiments, the dose of the vector administered to a subject is about 10 micrograms, about 50 micrograms, about 100 micrograms, about 250 micrograms, about 500 micrograms, about 1,000 micrograms, or about 5,000 micrograms. In some embodiments, the dose of the vector administered to the subject is at least about 10 micrograms, about 50 micrograms, about 100 micrograms, about 250 micrograms, about 500 micrograms, or about 1,000 micrograms. In some embodiments, the dose of the vector administered to the subject is at most about 50 micrograms, about 100 micrograms, about 250 micrograms, about 500 micrograms, about 1,000 micrograms, or about 5,000 micrograms. In some embodiments, multiple doses of the same amount of vector are administered to the subject. In some embodiments, the subject receives a first dose and a second lower dose (e.g., after an appropriate period of time).

[0224] Dosage regimen

[0225] In some cases, a dosing regimen is used to reach and / or maintain the required antibody level in a subject. In some embodiments, the required antibody level and duration are reached after a single dose (e.g., for the treatment of acute infection). In some cases, repeated administration (e.g., 2, 3, 4 or more doses) (e.g., one or more higher sensitization doses followed by a lower maintenance dose) is required to reach the initial treatment of the antibody or Fab or a clinically relevant level.

[0226] In some embodiments, the subject is dosed once. In some embodiments, the subject is dosed twice, two weeks apart from the injections. In some embodiments, the subject is dosed twice, three weeks apart from the injections. In some embodiments, the subject is dosed twice, four weeks apart from the injections. In some embodiments, the subject is dosed twice, six weeks apart from the injections. In some embodiments, the subject is dosed twice, eight weeks apart from the injections. In some embodiments, the subject is dosed twice, 12 weeks apart from the injections.

[0227] In some cases, the subject is administered at regular intervals (for example, to continuously prevent infectious diseases, such as viruses). In some embodiments, the subject is administered approximately once a month, once every two months, once every three months, once every four months, once every six months, or once a year. In some embodiments, the dosing interval is selected so that the minimum level of the antibody or Fab is always reached (for example, plasma levels exceed 50ng / mL, 100ng / mL, 200ng / mL, 300ng / mL, 400ng / mL, 500ng / mL, 600ng / mL, 700ng / mL, 800ng / mL, 900ng / mL or 1000ng / mL). In some embodiments, after the initial sensitization stage (for example, two or more doses of relatively rapid succession, such as about 2-12 weeks apart), the subject is administered at regular intervals.

[0228] Where multiple doses are administered to a subject, the dose can optionally be varied in different doses (eg, an initial high dose followed by a lower maintenance dose).

[0229] In some embodiments, the subject receives multiple doses.

[0230] Route of administration

[0231] The antibody expression systems provided herein can be administered by a variety of administration routes. In some embodiments, the system is administered by intravenous injection. In some embodiments, the system is administered by subcutaneous injection. In some embodiments, the system is administered by intramuscular injection. In some embodiments, the system is administered by intradermal injection. In some embodiments, the system is administered intranasally. In some embodiments, the system is administered orally. In some embodiments, the system is administered by intrathecal injection. In preferred embodiments, the system is administered by intravenous or intramuscular administration.

[0232] In some embodiments, the systems provided herein can be administered without the need for any specialized equipment and achieve the desired therapeutic effect (e.g., the desired antibody or Fab level can be achieved). In some embodiments, the system is administered without electroporation or hydroporation. In some embodiments, the system is administered without electroporation. In some embodiments, the system is administered without hydroporation. In some embodiments, the system is administered using a standard needle and syringe setting (e.g., for intramuscular administration).

[0233] active

[0234] In some embodiments, the administered vector is capable of producing a plasma antibody or antigen-binding fragment concentration of 10 ng / ml to 20,000 ng / ml. In some embodiments, the administered vector is capable of producing a plasma antibody or antigen-binding fragment concentration of 10 ng / ml to 25 ng / ml, 10 ng / ml to 50 ng / ml, 10 ng / ml to 100 ng / ml, 10 ng / ml to 250 ng / ml, 10 ng / ml to 500 ng / ml, 10 ng / ml to 1,000 ng / ml, 10 ng / ml to 2,500 ng / ml, 10 ng / ml to 5,000 ng / ml, 10 ng / ml to 10,000 ng / ml, 10 ng / ml to 15,000 ng / ml, 10 ng / ml to 20,000 ng / ml, 2 5ng / ml to 50ng / ml, 25ng / ml to 100ng / ml, 25ng / ml to 250ng / ml, 25ng / ml to 500ng / ml, 25ng / ml to 1,000ng / ml, 25ng / ml to 2,500ng / ml, 25ng / ml to 5,000ng / ml, 25ng / ml to 10,000ng / ml, 25ng / ml to 15,000ng / ml, 25ng / ml to 20,000ng / ml, 50ng / ml to 100ng / ml, 50ng / ml to 250ng / ml, 50ng / ml to 500ng / ml, 50ng / ml to 1,000ng / ml, 50ng / ml to 2,500ng / ml, 50ng / ml to 5,000ng / ml, 50ng / ml to 10,000ng / ml, 50ng / ml to 15,000ng / ml, 50ng / ml to 20,000ng / ml, 100ng / ml to 250ng / ml, 100ng / ml to 500ng / ml, 100ng / ml to 1,000ng / ml, 100ng / ml to 2,500ng / ml, 100ng / ml to 5,000ng / ml, 100ng / ml to 10,000ng / ml, 100ng / ml to 15,000ng / ml, 100ng / ml to 20,000ng / ml, 250ng / ml to 500ng / ml, 250ng / ml to 1,000ng / ml, 250ng / ml to 2,500ng / ml, 250ng / ml to 5,000ng / ml, 250ng / ml to 10,000ng / ml, 250ng / ml to 15,000ng / ml, 250ng / ml to 20,000ng / ml, 500ng / ml to 1,000ng / ml, 500ng / ml to 2,500ng / ml, 500ng / ml to 5,1,000ng / ml, 500ng / ml to 10,000ng / ml, 500ng / ml to 15,000ng / ml, 500ng / ml to 20,000ng / ml, 1,000ng / ml to 2,500ng / ml, 1,000ng / ml to 5,000ng / ml, 1,000ng / ml to 10,000ng / ml, 1,000ng / ml to 15,000ng / ml, 1,000ng / ml to 20,000ng / ml, 2,500ng / ml to 5,000ng / ml, 2 , 500ng / ml to 10,000ng / ml, 2,500ng / ml to 15,000ng / ml, 2,500ng / ml to 20,000ng / ml, 5,000ng / ml to 10,000ng / ml, 5,000ng / ml to 15,000ng / ml, 5,000ng / ml to 20,000ng / ml, 10,000ng / ml to 15,000ng / ml, 10,000ng / ml to 20,000ng / ml or 15,000ng / ml to 20,000ng / ml. In some embodiments, the administered vector is capable of producing a plasma antibody or antigen-binding fragment concentration of 10 ng / ml, 25 ng / ml, 50 ng / ml, 100 ng / ml, 250 ng / ml, 500 ng / ml, 1,000 ng / ml, 2,500 ng / ml, 5,000 ng / ml, 10,000 ng / ml, 15,000 ng / ml, or 20,000 ng / ml. In some embodiments, the administered vector is capable of producing a plasma antibody or antigen-binding fragment concentration of at least 10 ng / ml, 25 ng / ml, 50 ng / ml, 100 ng / ml, 250 ng / ml, 500 ng / ml, 1,000 ng / ml, 2,500 ng / ml, 5,000 ng / ml, 10,000 ng / ml, or 15,000 ng / ml.

[0235] In some embodiments, the peak plasma level of the antibody or its antigen-binding fragment produced by administration is at least 75ng / mL, at least 100ng / mL, at least 150ng / mL, at least 200ng / mL, at least 250ng / mL, at least 300ng / mL, at least 400ng / mL, at least 500ng / mL, at least 600ng / mL, at least 700ng / mL, at least 800ng / mL, at least 900ng / mL or at least 1000ng / mL. In some embodiments, the peak plasma level of the antibody or its antigen-binding fragment produced by administration is at least 1000ng / mL. In some embodiments, the peak plasma level of the antibody or its antigen-binding fragment produced by administration is at least 1500ng / mL. In some embodiments, the peak plasma level of the antibody or its antigen-binding fragment produced by administration is at least 2000ng / mL. In some embodiments, the peak plasma level of the antibody or its antigen-binding fragment produced by administration is at least 2500ng / mL. In some embodiments, the peak plasma level of the antibody or its antigen-binding fragment produced by administration is at least 3000ng / mL. In some embodiments, the peak plasma level of the antibody or antigen binding fragment produced by administration is at least 4000ng / mL. In some embodiments, the peak plasma level of the antibody or antigen binding fragment produced by administration is at least 5000ng / mL. In some embodiments, the peak plasma level shown for the antibody or antigen binding fragment is reached after a single dose of the system provided herein. In some embodiments, the peak plasma level shown for the antibody or antigen binding fragment is reached after a single intramuscular dose of the system. In some embodiments, the peak plasma level shown for the antibody or antigen binding fragment is reached after two intramuscular doses of the system provided herein. In some embodiments, the peak plasma level shown for the antibody or antigen binding fragment is reached after two intramuscular doses of the system. In some embodiments, the peak plasma level shown for the antibody or antigen binding fragment is reached after two intravenous doses of the system.

[0236] In some embodiments, the plasma concentration of the antibody or antigen-binding fragment is maintained at a therapeutically or clinically relevant level (e.g., a level provided herein, such as at least about 50 ng / mL, 75 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1000 ng / mL, 2000 ng / mL, 3000 ng / mL, 4000 ng / mL, or 5000 ng / mL) for a long period of time. In some embodiments, the plasma level of the antibody or antigen-binding fragment is maintained for a period of 1 week to 206 weeks. In some embodiments, the plasma antibody or antigen-binding fragment concentration is maintained for at least 1 week to 2 weeks, 1 week to 4 weeks, 1 week to 8 weeks, 1 week to 13 weeks, 1 week to 26 weeks, 1 week to 52 weeks, 1 week to 104 weeks, 1 week to 206 weeks, 2 weeks to 4 weeks, 2 weeks to 8 weeks, 2 weeks to 13 weeks, 2 weeks to 26 weeks, 2 weeks to 52 weeks, 2 weeks to 104 weeks, 2 weeks to 206 weeks, 4 weeks to 8 weeks, 4 weeks to 13 weeks, 4 weeks to 26 weeks, 4 weeks to 26 weeks, 4 weeks to 52 weeks, 2 weeks to 104 weeks, 2 weeks to 206 weeks, 4 weeks to 8 weeks, 4 weeks to 13 ...2 In some embodiments, the plasma antibody or antigen-binding fragment concentration is maintained for a period of 1 week, 2 weeks, 4 weeks, 8 weeks, 13 weeks, 26 weeks, 52 weeks, 104 weeks, or 206 weeks. In some embodiments, the plasma antibody or antigen-binding fragment concentration is maintained for a period of at least 1 week, 2 weeks, 4 weeks, 8 weeks, 13 weeks, 26 weeks, 52 weeks, 104 weeks, or 206 weeks.

[0237] In some embodiments, the antibody or antigen-binding fragment plasma concentration is maintained above 50 ng / mL for at least 1 week, 2 weeks, 4 weeks, 8 weeks, 13 weeks, 26 weeks, 52 weeks, or 104 weeks. In some embodiments, the antibody or antigen-binding fragment plasma concentration is maintained above 75 ng / mL for at least 1 week, 2 weeks, 4 weeks, 8 weeks, 13 weeks, 26 weeks, 52 weeks, or 104 weeks. In some embodiments, the antibody or antigen-binding fragment plasma concentration is maintained above 100 ng / mL for at least 1 week, 2 weeks, 4 weeks, 8 weeks, 13 weeks, 26 weeks, 52 weeks, or 104 weeks. In some embodiments, the antibody or antigen-binding fragment plasma concentration is maintained above 250 ng / mL for at least 1 week, 2 weeks, 4 weeks, 8 weeks, 13 weeks, 26 weeks, 52 weeks, or 104 weeks. In some embodiments, the antibody or antigen-binding fragment plasma concentration is maintained above 500 ng / mL for at least 1 week, 2 weeks, 4 weeks, 8 weeks, 13 weeks, 26 weeks, 52 weeks, or 104 weeks. In some embodiments, the antibody or antigen-binding fragment plasma concentration is maintained above 750 ng / mL for at least 1 week, 2 weeks, 4 weeks, 8 weeks, 13 weeks, 26 weeks, 52 weeks, or 104 weeks. In some embodiments, the antibody or antigen-binding fragment plasma concentration is maintained above 1000 ng / mL for at least 1 week, 2 weeks, 4 weeks, 8 weeks, 13 weeks, 26 weeks, 52 weeks, or 104 weeks.

[0238] In some embodiments, the plasma level of the antibody or antigen binding fragment is maintained at a concentration of at least 50% of the peak plasma concentration achieved for a period of at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks after administration. In some embodiments, the plasma level of the antibody or antigen binding fragment is maintained at a concentration of at least 25% of the peak plasma concentration achieved for a period of at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks after administration. In some embodiments, the plasma level of the antibody or antigen binding fragment is maintained at a concentration of at least 10% of the peak plasma concentration achieved for a period of at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks after administration.

[0239] In some embodiments, the indicated concentrations of the antibody or antigen-binding fragment are achieved and maintained after a single dose of the vector. In some embodiments, the indicated concentrations of the antibody are achieved and maintained after multiple doses of the vector. In some embodiments, the indicated concentrations of the antibody or antigen-binding fragment are achieved and maintained after two doses of the vector. In some embodiments, the antibody or antigen-binding fragment concentrations are maintained without any additional administration of the vector (e.g., after one or two doses of the vector, depending on the regimen).

[0240] In some embodiments, the subject is administered 2 doses of the vector. In some embodiments, the second dose of the vector is administered about 2 weeks to about 26 weeks after the first dose. In some embodiments, 2 doses are administered about 2 weeks to about 12 weeks apart. In some embodiments, 2 doses are administered about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks apart. In some embodiments, 2 doses are administered about 4 weeks to about 12 weeks apart, about 6 weeks to about 12 weeks apart, about 8 weeks to about 12 weeks apart, about 4 weeks to about 10 weeks apart, about 6 weeks to about 10 weeks apart, or about 8 weeks to about 10 weeks apart. In some embodiments, 2 doses are administered at least 2 weeks apart, at least 4 weeks, or at least 6 weeks apart. In some embodiments, 2 doses are administered at most 26 weeks apart, at most 20 weeks apart, at most 16 weeks apart, at most 12 weeks apart, or at most 10 weeks apart. In some embodiments, the second dose is administered after a plateau in antibody or antigen-binding fragment concentration is reached.

[0241] In some embodiments, the two doses are the same. In some embodiments, the first dose is higher than the second dose.

[0242] In some embodiments, the second dose is administered to achieve a peak plasma level of the antibody or antigen-binding fragment that is higher than the predicted additive effect. In some embodiments, the peak plasma level of the antibody or antigen-binding fragment resulting from the administration of the second dose is more than 2 times higher than the peak plasma level reached after the first dose. In some embodiments, the peak plasma level of the antibody or antigen-binding fragment resulting from the administration of the second dose is at least 3 times, at least 4 times, or at least 5 times higher than the peak plasma level reached after the first dose. In some embodiments, the peak plasma level of the antibody or antigen-binding fragment resulting from the administration of the second dose is at least 3 times higher than the peak plasma level reached after the first dose. In some embodiments, the peak plasma level of the antibody or antigen-binding fragment resulting from the administration of the second dose is at least 4 times higher than the peak plasma level reached after the first dose. In some embodiments, the peak plasma level of the antibody or antigen-binding fragment resulting from the administration of the second dose is at least 5 times higher than the peak plasma level reached after the first dose. In some embodiments, each dose is administered by intravenous administration. In some embodiments, each dose is the same amount, or the second dose is a lower amount than the first dose.

[0243] Subjects

[0244] In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate, a feline, a canine, a bovine, a porcine, an ovine, a caprine, or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a child or an infant. In some embodiments, the subject is an adult.

[0245] III. Definitions

[0246] The understanding of all terms is intended to be the same as understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs.

[0247] The following definitions supplement the definitions in the art and relate to the present application, and are not attributed to any related or unrelated cases, such as any commonly owned patents or applications. Although any methods and materials similar or equivalent to those described herein can be used in the testing practice of the present invention, preferred materials and methods are described herein. Therefore, the terms used herein are only used for the purpose of describing specific embodiments and are not intended to be limiting.

[0248] The terms used herein are intended to describe specific situations only and are not intended to be limiting. In this application, unless otherwise specifically stated, the use of the singular includes the plural. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0249] In this application, unless otherwise stated, the use of "or" means "and / or". As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents may be used interchangeably. These terms may express that any combination is specifically contemplated. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C". The term "or" may be used in conjunction or in conjunction, unless the context specifically indicates otherwise.

[0250] The term "about" or "approximately" can mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5 times, or within 2 times of a value. If specific values ​​are described in the application and claims, it should be assumed that the term "about" means within an acceptable error range for the specific value unless otherwise stated.

[0251] As used in this specification and claims, the words "comprising" (and any form of "comprising," such as "including" and "containing"), "having" (and any form of "having," such as "having" and "having"), "including" (and any form of "including," such as "including" and "covering"), or "containing" (and any form of "containing," such as "containing" and "including") are inclusive or open-ended, and do not exclude additional, uncited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. In addition, the compositions of the invention can be used to implement the methods of the invention.

[0252] References in the specification to "some embodiments", "embodiments", "one embodiment" or "other embodiments" mean that a particular feature, structure or characteristic described in conjunction with the embodiments is included in at least some embodiments of the present invention, but not necessarily in all embodiments. To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0253] The scope provided herein is understood to be a shorthand for all values ​​in the range. For example, the scope of 1 to 50 is understood to include any number, combination of numbers or subranges consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, and all intermediate decimals between the above-mentioned integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9. About subranges, " nested subranges " extending from either end of the scope are particularly considered. For example, nested sub-ranges of the exemplary range 1 to 50 may include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0254] The term "subject" refers to an animal that is the object of treatment, observation or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, a cow, a horse, a dog, a sheep, or a cat.

[0255] The term "optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0256] Used herein is reference to the insertion and / or deletion of one or more nucleotides or amino acids from a sequence. As used herein with reference to a sequence, the term "ins" is located before a number, followed by a nucleotide or amino acid sequence, meaning that the listed nucleotide or amino acid sequence is inserted into the sequence after the residue shown. For example, "ins214TDR" means that the sequence "TDR" is inserted after residue 214 of the reference sequence. As used herein, the term "del" followed by a number or a numerical range means that the nucleotides or amino acids at the designated position number of the reference sequence are missing from the sequence. For example, 137-145del means that residues 137, 138, 139, 140, 141, 142, 143, 144 and 145 are missing from the reference sequence.

[0257] The term "V H "H" indicates that the heavy chain variable domain is obtained from, originates from or is derived from a heavy chain antibody. A heavy chain antibody is a functional antibody having two heavy chains and no light chain. Heavy chain antibodies are present in and can be obtained from camels (e.g., camels and alpacas), members of the biological taxonomy Camelidae. HH antibodies were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "antibodies without light chains"; Hamers-Casterman et al., Nature 363:446-448 (1993)). The term "V H The term “H domain” is intended to distinguish these variable domains from the heavy chain variable domains present in conventional four-chain antibodies (referred to herein as “VH domains” or “VH”) and the light chain variable domains present in conventional four-chain antibodies (referred to herein as “VL domains” or “VL”).

[0258] The term "camelization" V H refers to an immunoglobulin single-chain variable domain in which one or more amino acid residues from the amino acid sequence of a naturally occurring VH domain of a conventional four-chain antibody are replaced by those present in the V domain of a heavy chain antibody. H One or more amino acid residues at the corresponding positions of the H domain are substituted. Such "camelizing" substitutions may be inserted at amino acid positions that form and / or are present at the VH-VL interface, and / or at the so-called Camelidae Hallmark residues, as defined herein (see also, for example, WO9404678 and Davies and Riechmann (1994 and 1996)). Reference is made to Davies and Riechmann (FEBS 339: 285-290, 1994; Biotechnol. 13: 475-479, 1995; Prot. Eng. 9: 531-537, 1996) and Riechmann and Muyldermans (J. Immunol. Methods 231: 25-38, 1999).

[0259] IV. Sequence

[0260] In some embodiments, the antibodies or antigen-binding fragments in the systems provided herein comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the antibodies shown in the table below.

[0261] Table 3

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268] Although the present invention and its advantages have been described in detail, it should be understood that various modifications, substitutions and changes may be made herein without departing from the spirit and scope of the present invention as defined in the appended claims. The following examples further illustrate the present invention, which are only for illustrative purposes and are not intended to limit the present invention in any way. Example

[0269] Example 1 - Design of antibody or antigen-binding fragment expression system

[0270] Monoclonal antibody (mAb) sequences are constructed as single transcript (ST) or heavy chain / light chain (HC+LC) formats. Antibodies in ST format are one of two types: heavy chain (HC) and light chain (LC) linked by furin T2A (T2A) or V H H form. Codon optimization was performed using the Integrated DNA Technologies (IDT) codon optimization web tool (strategy 1) or the ThermoFisherGeneOptimizer TM Antibody encoding DNA sequences were codon optimized using web tools (Strategy 2).

[0271] The T2A format was designed by sequentially fusing nucleotide sequences encoding the following elements: Kozak sequence; HC signal peptide; immunoglobulin HC; furin cleavage site; T2A peptide derived from Thosea asigna virus; LC signal peptide; immunoglobulin LC; and stop codon. Antibody encoding DNA sequences were codon optimized using the Integrated DNA Technologies (IDT) codon optimization web tool (strategy 1) or the ThermoFisher GeneOptimizerTM web tool (strategy 2) to reduce the use of rare codons, balance GC content, and minimize RNA secondary structure. All immunoglobulin HCs used IGHG1 * 01 gene sequence, and LC uses IGKC * 01 (for mAb1) or IGLC2 * 01 (for mAb2) gene. The complete open reading frame is preceded by a CAG promoter. Figure 5A An exemplary vector map showing such a sequence is shown.

[0272] The HC+LC format was constructed using two plasmids (one encoding HC and one encoding LC) using the following elements in sequence: Kozak sequence; signal peptide (for HC or LC); immunoglobulin HC or LC; and stop codon. As with the ST format, the open reading frame was preceded by a CAG promoter and followed by a BGH polyadenylation signal. In some heavy chain sequences, YTE mutations [M252Y, S254T, T256E (EU numbering)] were introduced to increase serum / plasma half-life. Figure 5B and 5C Exemplary vector maps showing such sequences are shown.

[0273] Use the following sequence to design V H H antibody: Kozak sequence; HC signal peptide; V H H variable domain sequence; modified human hinge region; from IGHG1 * 01 human CH2 and CH3 domains; and a stop codon. The open reading frame is preceded by a CAG promoter and followed by a BGH polyadenylation signal. The V H The H variable domain sequence is Ty1, which is an anti-SARS-CoV-2 V isolated and disclosed in Hanke, et al., Nat. Comms. 2020 (doi: 10.1038 / s41467-020-18174-5). H H.

[0274] V H The H, T2A and HC+LC forms were constructed as circular nanoplasmids (Nature Technology Corporation). In addition to the above elements, the nanoplasmids include the RNA-OUT selectable marker plus the R6K origin to allow propagation in bacterial hosts. The nanoplasmids are marketed by Nature Technology Corporation under the trade name Nanoplasmid TM For commercial sale.

[0275] Table 4

[0276] Antibody ID mAb name Fc modification LC Type Ab1 Casirimab none K Ab2 Idezumab none λ Ab3 Tesagvir YTE K Ab4 Celgene YTE K Ab5 BMS-986414 YTE K Ab6 BMS-986413 YTE λ Ab7 ADG-20 YTE λ Ab8 MAD-0004J08 YTE K Ab9 Brii-198 YTE K Ab10 Brii-196 YTE λ Ab11 Enuzoyimab YTE K Ab12 DZIF-10c YTE K Ab15 Ty1 YTE N / A

[0277] Example 2 - In vitro testing of DNA-encoded antibodies

[0278] The antibody candidates encoded by the DNA described in Example 2 were tested for in vitro expression to verify protein production. HEK293T cells were plated at 2×10 5The density of cells was inoculated in Dulbecco's modified Eagle medium (supplemented with 10% fetal bovine serum and penicillin-streptomycin) in 12-well plates. One day after inoculation, 3.75 μl Lipofectamine3000, 2 μl P3000 and 1 μg DNA were used to transfect cells in each well. Plasmid DNA encoding GFP was transfected in parallel with each batch of mAb candidates, and GFP fluorescence was measured 24 hours after transfection as a control. Supernatants for mAb transfection were collected after 48 or 72 hours to measure IgG titers, which were quantified using sandwich ELISA. The plates were coated overnight with goat anti-human Fc polyclonal antibodies, and goat anti-human H+L polyclonal antibodies coupled to horseradish peroxidase were used to detect human IgG in the supernatant. Candidates were tested in the following configurations: 1) T2A nanoplasmid, 2) HC+LC nanoplasmid and 3) T2A plasmid. IgG expression values ​​are reported as the average of two repeated experiments. The sample was diluted at 1:10, 1:50, 1:250 and 1:1250 to accurately quantify titer compared with the standard curve of purified human IgG1 (concentration range of 3 μg / ml-1.3 ng / ml). The data provided below are produced using commercially available human IgG1 standards (IgG1 human ELISA standards (for uncoated ELISA kits), from ThermoFisherScientific, catalog number 39-50560-65). All samples described herein measured using commercially available standards refer to this standard. Nonlinear regression was used to fit the standard curve to a four-parameter sigmoidal dose-response curve, and the dilution of the cell supernatant in the linear dynamic range of ELISA was used to calculate the concentration. The IgG expression values ​​reported are the mean values ​​of two biological replicates.

[0279] Table 5: In vitro expression dose, codon optimization, antibody format and results

[0280]

[0281]

[0282] Example 3 - In vivo expression test study 1

[0283] Proteolipid vesicles (PLVs) containing T2A nanoplasmids were formulated at concentrations of 2.5, 2, 1, 0.6, and 0.33 mg / ml. PLVs containing co-formulated HC+LC nanoplasmids were generated with a total PLV concentration of 1 mg / ml (0.5 mg / ml HC nanoplasmid and 0.5 mg / ml LC nanoplasmid).

[0284] An exemplary process for preparing PLV is as follows: plasmid DNA material is encapsulated in fusion-associated small transmembrane protein (FAST)-PLV as a payload. Plasmid DNA is diluted in 10 mM sodium acetate buffer (pH 4.0) containing 5 nM FAST protein (Fusogenix from Entos Pharmaceuticals in San Diego, California). In addition, the PLV lipid component is dissolved in ethanol. The DNA-protein portion is mixed with the lipid portion at a 3:1 ratio and a flow rate of 12 mL / min in a NanoAssemblr desktop microfluidic instrument (Precision Nanosystems Inc, Vancouver, BC). The preparation was dialyzed against phosphate buffered saline (pH 7.4) in an 8000MWCO dialysis membrane (product code 12757486, BioDesign, Carmel, New York) for 3 hours, and the buffer was changed three times, then concentrated using an Amicon ultracentrifugal filter (EMDMillipore, Burlington, Massachusetts), and then passed through a 0.22 μm filter (GSWP04700, EMDMillipore). The resulting FAST-PLV DNA material was stored at 4°C until use.

[0285] Rag2 knockout mice were used to study antibody expression and titer because they are unable to mount an immune response against human antibodies. To optimize in vivo antibody expression, different vector strategies (T2A vs. HC+LC), doses, and routes of administration (intravenous (IV) vs. intramuscular (IM)) were compared, followed by a BGH polyadenylation signal.

[0286] Table 6: Injection of mice with PLV (including plasmid DNA dose, vector form, route of administration and injection volume).

[0287]

[0288]

[0289] Blood samples were collected at different time points as shown herein and processed into plasma. Human IgG titers were measured in mouse plasma using Meso Scale Discovery instrument by electrochemiluminescence assay (ECLIA). Human IgG titers in mice were quantitatively measured by measuring the ECLIA signal of plasma samples diluted 1:100, and calculated based on the standard curve of purified human IgG1 (concentration range of 3.2 μg / ml-0.78 ng / ml). The data provided in Table 7 were produced using commercially available human IgG1 standards. Standard curves were fitted to four-parameter S-shaped dose-response curves using nonlinear regression. The human IgG expression values ​​reported are the mean values ​​of each group on the 23rd day after injection.

[0290] Table 7

[0291]

[0292]

[0293] Figure 1A Shown are IgG plasma levels in mice at day 9 after administration of the indicated constructs. Figure 1B Shown are IgG plasma levels in mice at day 16 after administration of the indicated constructs. Figure 1C Shown are IgG plasma levels in mice at day 23 after administration of the indicated constructs. Figure 1D Shown are IgG plasma levels in mice at day 30 after administration of the indicated constructs. Figure 1E Shown are IgG plasma levels in mice at day 37 after administration of the indicated constructs. Figure 1F Shown are IgG plasma levels in mice at day 44 after administration of the indicated constructs. Figure 2 IgG concentrations in mouse plasma are shown for Ab1 HC+LC and Ab2 HC+LC administered by intravenous administration at different time points for individual animals (entries 4 and 7 of Table 6). Figures 1A-1F and Figure 2 The data presented in each were generated using a commercially available human IgG1 standard.

[0294] At the 44th day post-injection time point, mouse plasma was evaluated for binding to the SARS-CoV-2 RBD protein. Binding was measured by sandwich ELISA, in which ELISA plates were coated overnight with commercially available SARS-CoV-2 RBD protein (SinoBiological) at a concentration of 1 μg / ml. Plasma samples from five mice were evaluated, and 100 μg of Ab1 in the T2A form (number 11 from Tables 6 and 7) was administered to the mice by the intramuscular route. The plasma samples were incubated with RBD-coated plates at dilution factors of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640. Binding was detected using a goat anti-human Fc polyclonal antibody coupled to horseradish peroxidase. The results of this experiment are shown in Figure 6 As shown, the antibody concentration on the x-axis was determined by calculation based on the dilution ratio of the initial IgG concentration using a commercially available human IgG1 standard.

[0295] Table 8 below shows the human IgG concentration in ng / mL in mice administered at various time points using the system of antibodies or antigen-binding fragments described in Experiment No. 15 in Table 6 above. The data presented below were generated using a commercially available human IgG1 standard.

[0296] Table 8

[0297]

[0298] Table 9 below shows the human IgG concentration in ng / mL in mice administered at various time points with the system of antibodies or antigen-binding fragments described in Experiment No. 4 in Table 6 above. The data presented below were generated using a commercially available human IgG1 standard.

[0299] Table 9

[0300]

[0301] Table 10 below shows the human IgG concentration in ng / mL in mice administered at various time points with the system of antibodies or antigen-binding fragments described in Experiment No. 7 in Table 6 above. The data presented below were generated using a commercially available human IgG1 standard.

[0302] Table 10

[0303]

[0304] Table 11 below shows the human IgG concentration in ng / mL in mice administered at various time points using the system of antibodies or antigen-binding fragments described in Experiment No. 9 in Table 6 above. The data presented below were generated using a commercially available human IgG1 standard.

[0305] Table 11

[0306]

[0307] Table 12 below shows the human IgG concentration in ng / mL in mice administered at various time points using the system of antibodies or antigen-binding fragments described in Experiment No. 10 in Table 6 above. The data presented below were generated using a commercially available human IgG1 standard.

[0308] Table 12

[0309]

[0310]

[0311] Table 13 below shows the human IgG concentration in ng / mL in mice administered at various time points with the system of antibodies or antigen-binding fragments described in Experiment No. 17 in Table 6 above. The data presented below were generated using a commercially available human IgG1 standard.

[0312] Table 13

[0313]

[0314] Table 14 below shows the human IgG concentration in ng / mL in mice administered at various time points using the system of antibodies or antigen-binding fragments described in Experiment No. 18 in Table 6 above. The data presented below were generated using a commercially available human IgG1 standard.

[0315] Table 14

[0316]

[0317] Example 4 - In vitro expression - effect of multiple doses

[0318] On day 60 of the study described in Example 3 above, 5 of the 10 mice from study Experiment No. 4 (Ab1 HC+LC 100 μg IV), Experiment No. 7 (Ab2 HC+LC 100 μg IV), and Experiment No. 9 (Ab1 SC 100 μg IM) received a second booster dose of the same cargo previously delivered (i.e., same vehicle, same route of administration, same dose, etc.). The time course of antibody levels from this experiment in the form of Ab1 HC+LC 100 μg IV (Experiment No. 4), Ab2 HC+LC 100 μg IV (Experiment No. 7), and Ab1 SC 100 μg IM (Experiment No. 9) in single and multiple dose form (second dose received on day 60 of the study) is shown in Figure 2. Figure 1G middle. Figure 1GThe data presented in were generated using a commercially available human IgG1 standard. Although no substantial effect of boosting was observed in the IM format compared to the no-boost control, both IV formats showed greatly enhanced IgG levels after boosting. Surprisingly, for both IV multiple doses, the effect of the second dose produced antibody levels greater than the expected additive effect.

[0319] Example 5 - High Dose Ion Study

[0320] An additional group of mice was enrolled in the study described in Example 3 to determine the effects of higher doses administered IM (experiment numbers 15, 17, and 18 in Table 6 above). Figure 1H Shown are the time course antibody levels in mice for single dose forms of Ab1 HC+LC 100μg IV (Experiment No. 4), Ab1 HC+LC 250μg IV (Experiment No. 17), Ab1 HC+LC 500μg IV (Experiment No. 15), Ab1 T2A 30μg IM (Experiment No. 10), Ab1 T2A 100μg IM (Experiment No. 9), and Ab1 HC+LC 250μg IM (Experiment No. 18). Figure 1H The data provided in the were generated using commercially available human IgG1 standards. The results showed a clear dose response and improved kinetic response (i.e., faster antibody production rate) for higher doses. Antibody levels also remained relatively constant until the end of the study (approximately 300 days or longer).

[0321] Example 6 - Commercially available IgG1 standard versus in-house produced IgG1 standard

[0322] All results reporting the above IgG or antibody concentrations used the same commercially available human IgG1 standard (ThermoFisher IgG1 Human ELISA Standard (for uncoated ELISA kits), catalog number 39-50560-65). This commercial standard was then compared to an in-house produced IgG1 standard. An in-house IgG1 standard was prepared according to the following protocol: Purified Ab1 and Ab2 proteins were produced by transient transfection of Expi293 cells (ThermoFisher). Heavy and light chain nanoplasmids were co-transfected into 50 ml of suspended cell culture at a ratio of 25 μg per plasmid using the manufacturer's recommended protocol. Supernatants were collected on day 7 after transfection and sterilized by filtration with a 0.2 μm filter. IgG was purified from the supernatant using 2 ml of protein A resin (ThermoFisher). The supernatant was diluted with IgG binding buffer (ThermoFisher) and then applied to the resin. The resin was then washed with 5 column volumes (CV) of binding buffer, eluted with 2.5 CV of IgG elution buffer (ThermoFisher), and neutralized with 1 M Tris (pH 8.0). The sample buffer was exchanged into PBS. Sample concentration was measured using A280, purity was measured using SDS-PAGE, and functional activity was verified by antigen binding ELISA.

[0323] Figure 1J The results are shown for the internally generated IgG1 standard (including the difference between the measured values ​​and the commercially available IgG1 standard). Fig. 1I The data for Experiment Nos. 15, 17, and 18 analyzed with the internal standard (overlapping samples shown). The data show that the antibody concentration values ​​calculated with the internal standard are approximately 25 times lower than the commercial standard used in the above experiments. In the experiments provided below, the internal standard was used to calculate the antibody concentration, so this approximately 25-fold correlation should be considered when comparing the data generated by the two different standards (commercial standard and internal standard).

[0324] Example 7-In vivo expression test study 2

[0325] To further optimize in vivo antibody expression, additional in vivo mouse studies as described in Example 3 were performed using the following experimental groups shown in Table 15.

[0326] Table 15

[0327]

[0328] Blood samples were collected every 7 days after injection and processed into plasma. Human IgG titers in mouse plasma were measured by electrochemiluminescence assay (ECLIA) using a Meso Scale Discovery instrument. Human IgG titers in mice were quantified by measuring the ECLIA signal of plasma samples diluted 1:25-1:100 and calculated based on a standard curve of internally purified human IgG1 (concentration range of 200ng / ml-0.048ng / ml). The standard curve was fit to a four-parameter sigmoidal dose-response curve using nonlinear regression. Results from the initial time point of the experiment are shown in Table 1. Fig. 7A These results show that the 250-microgram and 500-microgram IM doses performed similarly, suggesting that increasing the dose above 250 micrograms has limited benefit. The liver preparation (high) did not provide a clear benefit compared with the standard preparation.

[0329] Although levels are expected to rise at later time points, the initial time points for Experiment Nos. 21, 22, and 26 were below the lower limit of quantification of the above assay. To better assess these earlier time points, a more sensitive assay was performed by coating the SARS-CoV-2 strain receptor binding domain (RBD) on the assay plate (Meso Scale Discovery) to achieve better quantification. The results of this experiment are shown in Figure 7B This experiment showed that the 1.7:1 HC:LC molar ratio group (experiment number 23) had a 60% increase in antibody levels at day 28 compared to the 1:1 HC:LC mas / mas ratio group (experiment number 21). It is expected that this trend will essentially continue at later time points as antibody levels continue to rise. This experiment also showed that at this time point, the liver preparation performed worse than the standard preparation.

[0330] Example 8 - Evaluation of SV40e Nuclear Localization Signal

[0331] Additional attempts to further increase antibody levels have been made by using the SV50 enhancer (SV40e) in nanoplasmid expression vectors (see, e.g., Hai-shan Li et al., “Enhancement of DNA Vaccine-Induced Immune Responses by a 72-Bp Element from SV40 Enhancer:,” Chinese Medical Journal 120, no. 6 (March 2007): 496-502, https: / / doi.org / 10.1097 / 00029330-200703020-00012; S Li et al., “Muscle-Specific Enhancement of Gene Expression by Incorporation of SV40 Enhancer in the Expression Plasmid,” Gene Therapy 8, no. 6 (March 1, 2001): 494-97, https: / / doi.org / 10.1038 / sj.gt.3301419; and Pontus Blomberg et al., “Electroporation of SV40 in vitro and in vivo” in vitro and in vivo (see, e.g., Hai-shan Li et al., “Enhancement of DNA Vaccine-Induced Immune Responses by a 72-Bp Element from SV40 Enhancer:,” Chinese Medical Journal 120, no. 6 (March 2007): 496-502, https: / / doi.org / 10.1097 / 00029330-200703020-00012; S Li et al., “Muscle-Specific Enhancement of Gene Expression by Incorporation of SV40 Enhancer in the Expression Plasmid,” Gene Therapy 8, no. 6 (March 1, 2001): 494-97, https: / / doi.org / 10.1038 / sj.gt.3301419; in Combination with a Plasmid Vector Containing SV40 Enhancer Elements Results in Increased and Persistent Gene Expression in Mouse Muscle,” Biochemical and Biophysical Research Communications 298, no. 4 (November 2002): 505-10, https: / / doi.org / 10.1016 / S0006-291X(02)02486-5). The sequence used in these experiments was TGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGAGCCTGGGGACTTTCCACACC (SEQ ID NO: 102). This element is proposed to increase transgene expression in DNA gene therapy by providing a nuclear localization signal to target plasmid DNA to the cell nucleus. The SV40e element was constructed by incorporating the SV40e cassette directly upstream of the CAG promoter. The cassette was incorporated into the heavy and light chain vectors in a split-vector configuration.

[0332] Initial attempts to use the SV40e element in in vitro experiments in HEK293 cells did not show significant differences in expression compared to cells without SV40e. Plasmids incorporating the SV40e element were then administered in vivo to Rag 2 mice (e.g., as described in the Examples above) in the groups shown in Table 16 below.

[0333] Table 16

[0334] Experiment number Payload dose way Mouse n 31 Ab1 WT HC+LC 250 μg IM Rag2 5 32 Ab1 SV40e HC+LC 250 μg IM Rag2 5

[0335] The results from this experiment are as follows Figure 8 Results were measured using an in-house generated IgG1 standard. Experiment No. 31 performed similarly to previous experiments testing the same payload at this dose. The SV40e vector showed an overall increase in expression of approximately 40%. This trend is expected to continue at later time points and with other dose forms.

[0336] Example 9-V H Evaluation of H-Fc fusion formats

[0337] Three V from camel species were also tested H H-Fc fusion antibody (see Table 17 below). H The H fragment was fused to the Fc domain from human IgG1 (referred to as V H H-Fc) to increase neutralization potency and in vivo half-life. These constructs are N3113V-Fc and N3130V-Fc (both described in Li, et al., 2022; doi 10.1016 / j.cell.2022.03.009) and Ty1-Fc (described in Hanke, et al., 2022; doi:10.1038 / s41467-020-18174-5). All three V H H-Fc antibody: Kozak sequence; HC signal peptide; V H H variable domain sequence; modified human hinge region; from IGHG1 * 01; and a stop codon. The open reading frame was preceded by a CAG promoter and followed by a BGH polyadenylation signal. All open reading frames were codon optimized using commercially available software from ThermoFisher to reduce the use of rare codons, balance GC content, and minimize RNA secondary structure.

[0338] Table 17

[0339]

[0340] All three VHH-Fc constructs were found to outperform the AB1 HC+LC format in in vitro expression in HEK293 cells. The purified N3113V-Fc and N3130V-Fc were found to bind to the RBD of both SARSCoV-2 strains with high affinity, while Ty-Fc had no substantial binding to the Omicron strain RBD.

[0341] Similar to the protocol in Example 3 above, 250 μg of payload of nanoplasmid vectors encoding VHH-Fc constructs (1 construct / vector) were administered to three independent groups of Rag2 mice (n=4 or 5) by IM injection. The results of this experiment are shown in Fig. 9 As shown. N3130V-Fc did not produce any detectable levels of antibody at any time point. Both N3113V-Fc and Ty1-Fc variants were better expressed than the Ab1 HC+LC format, with N3113V-Fc being about 3-fold better than Ab1 on a molar basis, and Ty1-Fc being about 10-15-fold better than Ab1 on a molar basis.

[0342] Example 10 - Evaluation of WPRE vectors in VHH Fc fusion formats

[0343] Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is constructed into nanoplasmid expression vector. It has been previously reported that this element increases transgenic expression in non-viral and viral vectors by improving transcription, stability, output and translation of mRNA transcripts (e.g., Reinhard Klein et al., "WPRE-Mediated Enhancement of Gene Expression Is Promoter and Cell Line Specific," Gene 372 (May 2006): 153-61, https: / / doi.org / 10.1016 / j.gene.2005.12.018; Lizheng Wang et al., "Enhancing Transgene Expression from Recombinant AAV8 Vectors in Different Tissues Using Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element," International Journal of Medical Sciences 13, no. 4 (2016): 286-91, https: / / doi.org / 10.7150 / ijms.14152). The WPRE vector was constructed by incorporating a WPRE cassette downstream of the antibody open reading frame and before the BGH polyadenylation signal. This cassette was incorporated into the heavy and light chain vectors in a split vector configuration, as well as the Ty1-Fc VHH construct. As described above, the WPRE vector showed approximately a 2-fold reduction in expression at day 7 when 100 μg of payload of the Ty1-Fc VHH construct was administered to Rag2 mice.

[0344] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided only as examples. Without departing from the present invention, those skilled in the art will now be aware of many variations, modifications and substitutions. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in the implementation of the present invention. The following claims are intended to define the scope of the present invention, and should therefore encompass methods and structures within the scope of these claims and their equivalents.

Claims

1. A system for expressing an antibody or an antigen-binding fragment thereof in a subject, the system comprising: a plasmid comprising a polynucleotide sequence encoding a heavy chain variable domain of an antibody or antigen-binding fragment thereof; wherein the plasmid is encapsulated in a lipid vesicle; and in, When the plasmid encapsulated in the lipid vesicles is administered, the subject produces a peak plasma level of the antibody or antigen-binding fragment thereof of at least 50 ng / mL.

2. A system for expressing an antibody or an antigen-binding fragment thereof in a subject, the system comprising: a plasmid comprising a polynucleotide sequence encoding a heavy chain variable domain of an antibody or antigen-binding fragment thereof; The plasmid is encapsulated in lipid vesicles.

3. The system of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a single domain antibody.

4. The system of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is V H H antibody.

5. The system of any one of claims 1-4, wherein the heavy chain variable domain is fused to an Fc domain, optionally via a peptide linker.

6. The system of any one of claims 1-3, wherein the plasmid encodes the full-length heavy chain of an antibody.

7. The system of any one of claims 1-6, wherein the plasmid further comprises a polynucleotide sequence encoding a light chain or an antigen-binding fragment of an antibody.

8. The system of claim 7, wherein the plasmid encodes the full-length light chain of an antibody.

9. The system of claim 7 or 8, wherein the polynucleotide sequence encoding the heavy chain variable domain and the polynucleotide sequence encoding the light chain are operably coupled such that the sequences are transcribed as a single transcript.

10. The system of claim 9, wherein the polynucleotide sequence encoding the heavy chain and the polynucleotide sequence encoding the light chain are separated by a self-cleaving peptide encoding sequence.

11. The system of any one of claims 1, 2 or 6, further comprising a second plasmid comprising a second polynucleotide sequence encoding a light chain of an antibody.

12. The system of claim 11, wherein the plasmid and the second plasmid are present in a ratio of about 1.7:

1.

13. The system of claim 11 or 12, wherein the light chain of the antibody is a kappa chain or a lambda chain.

14. The system of any one of claims 11-13, wherein the second plasmid is also encapsulated in a lipid vesicle.

15. The system of any one of claims 1-14, wherein the lipid vesicle comprises a fusion-associated small transmembrane (FAST) protein.

16. The system of claim 15, wherein the FAST protein comprises a domain from one or more FAST proteins selected from p10, p14, p15, and p22.

17. The system of claim 15 or 16, wherein the FAST protein comprises an amino acid sequence having at least 80% sequence identity to:

18. The system of any of the preceding claims, wherein the plasmid comprises a promoter selected from the group consisting of CAG, CMV, EF1A, CBh, CBA and SFFV operably linked to the polynucleotide sequence.

19. The system of claim 18, wherein the plasmid comprises a CAG promoter.

20. The system of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises an IgGl, IgG2a, IgG2b, IgG3, IgG4, IgD, IgM, IgAl, IgA2, or IgE heavy chain.

21. The system of claim 20, wherein the antibody or antigen-binding fragment thereof comprises an IgG1, IgG2a, IgG2b, IgG3, or IgG4 heavy chain.

22. The system of claim 20, wherein the antibody comprises an IgGl heavy chain.

23. The system of any of the preceding claims, wherein the heavy chain variable domain comprises a sequence having at least 80% sequence identity to: wherein each X is independently absent or is any amino acid.

24. The system of any of the preceding claims, wherein the antibody or antigen-binding fragment comprises an Fc domain having one or more mutations or a combination of mutations selected from the group consisting of Arg435His (His435), Asn434Ala (A), Met428Leu / Asn434Ser (LS), Thr252Leu / Thr253Ser / Thr254Phe (LSF), Glu294Δ / Thr307Pro / Asn434Tyr (C6A-66), Thr256Asn / Ala378Val / Ser383Asn / Asn434Tyr (C6A-78), and Glu294Δ (Del), wherein the residue position numbers are based on the EU numbering convention.

25. The system of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises an Fc domain having one or more mutations selected from the group consisting of M252Y, S254T, T256E, and any combination thereof, wherein residue position numbering is based on the EU numbering convention.

26. The system of any one of the preceding claims, wherein the plasmid comprises the SV40e element.

27. The system of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof specifically binds to a viral protein.

28. The system of claim 27, wherein the viral protein is from a virus selected from the group consisting of a parvovirus, a picornavirus, a rhabdovirus, a paramyxovirus, an orthomyxovirus, a bunyavirus, a calicivirus, an arenavirus, a polyomavirus, a reovirus, a togavirus, a bunyavirus, a herpes simplex virus, a poxvirus, an adenovirus, a coxsackievirus, a flavivirus, a coronavirus, an astrovirus, an enterovirus, a rotavirus, a norovirus, a retrovirus, a papillomavirus, a parvovirus, an influenza virus, a hemorrhagic fever virus, and a rhinovirus.

29. The system of claim 28, wherein the viral protein is from a virus selected from the group consisting of: Hantavirus, Rabies, Nipah, Hendra, Rift Valley Fever, Lassa, Marburg, Crimean-Congo, hMPV, RSV, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Norovirus, Monkeypox, Vaccinia, Japanese Encephalitis, Yellow Fever, HSV-1, HSV -2, MERS, varicella virus, hand, foot and mouth virus, CMV (HHV-5), equine encephalitis virus, EBV (HHV-4), human metapneumovirus, norovirus, enterovirus, smallpox virus, West Nile virus, Paramyxoviridae, rhinovirus, mononucleosis virus, coxsackievirus B, influenza virus, poliovirus, measles virus, rubella virus, HPV, Zika virus, mumps virus, herpesviridae, chikungunya virus, Haemophilus influenzae, and SARS-CoV-2 virus.

30. The system of claim 27, wherein the viral protein is from SARS-CoV-2.

31. The system of claim 30, wherein the viral protein is the SARS-CoV-2 spike protein.

32. The system of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with an antibody set forth in Table 3.

33. The system of any one of claims 1-26, wherein the antibody or antigen-binding fragment specifically binds a cancer antigen.

34. The system of claim 33, wherein the cancer antigen is selected from the group consisting of programmed cell death 1 (PD1), programmed cell death ligand 1 (PDL1), CD5, CD20, CD19, CD22, CD30, CD33, CD40, CD44, CD52, CD74, CD103, CD137, CD123, CD152, carcinoembryonic antigen (CEA), integrin, epidermal growth factor (EGF) receptor family members, vascular epidermal growth factor (VEGF), proteoglycan, disialylganglioside, B7-H3, cancer antigen 125 (CA-125), epithelial cell adhesion molecule (EpCAM), vascular endothelial growth factor receptor 1, vascular endothelial growth factor receptor 2, tumor associated glycoprotein, mucin 1 (MUC1), tumor necrosis factor receptor, insulin-like growth factor receptor, folate receptor alpha, transmembrane glycoprotein NMB, CC chemokine receptor, prostate specific membrane antigen (PSMA), recepteur d'originenantais (RON) receptor, cytotoxic T lymphocyte antigen 4 (CTLA4), colon cancer antigen 19.9, gastric cancer mucin antigen 4.2, colorectal cancer antigen A33, ADAM-9, AFP carcinoembryonic antigen-alpha-fetoprotein, ALCAM, BAGE, β-catenin, carboxypeptidase M, B1, CD23, CD25, CD27, CD28, CD36, CD45, CD46, CD52, CD56, CD79a / CD79b, CD317, CDK4, CO-43 (Le b Blood type), CO-514 (Le a blood type), CTLA-1, cytokeratin 8, DR5, E1 series (B blood type), ephrin receptor A2 (EphA2), Erb (ErbB1, ErbB3, ErbB4), lung adenocarcinoma antigen F3, antigen FC10.2, GAGE-1, GAGE-2, GD2 / GD3 / GD49 / GM2 / GM3, GICA 19-9, gp37, gp75, gp100, HER-2 / neu, human milk fat globule antigen, human papillomavirus-E6 / human papillomavirus-E7, high molecular weight melanoma antigen (HMW-MAA), differentiation antigen (I antigen), I (Ma) found in gastric adenocarcinoma, integrin α-V-β-6, integrin β6 (ITGβ6), interleukin 13 receptor α2 (IL13Rα2), JAM-3, KID3, KID31, KS1 / 4 pan-cancer antigen, KSA (17-1A), human lung cancer antigen L6, human lung cancer antigen L20, LEA, LUCA-2, M1:22:25:8, M18, M39, MAGE-1, MAGE-3, MART, Myl, MUM-1, N-acetylglucosaminyltransferase, pseudoglycoprotein, NS-10, OFA-1 and OFA-2, oncostatin M (oncostatin receptor β), rhol5, prostate-specific antigen (PSA), PSMA, polymorphic epithelial mucin antigen (PEMA), PIPA, prostatic acid phosphate, R24, ROR1, SSEA-1, SSEA-3, SSEA-4, sTn, T cell receptor-derived peptide, T5A7, tissue antigen 37, TAG-72, TL5 (A blood group), TNF-α receptor (TNFαR), TNFβR, TNFγR, TRA-1-85 (H blood group), transferrin receptor, TSTA tumor-specific transplantation antigen, VEGF-R, Y hapten, Le y and 5T4.

35. The system of any one of claims 1-26, wherein the antibody or antigen-binding fragment thereof specifically binds to a protein or component of a bacterium.

36. The system of claim 35, wherein the bacteria is Bacillus anthracis, Corynebacterium diphtheria, Bordetella pertussis, Streptococcus pneumonia, Haemophilus influenza, Salmonella typhimurium, Shigella species, Streptococcus species, Chlamydia trachomatis, Yersiniapestis, Methicillin-resistant Staphylococcus aureus,MRSA), Staphylococcus aureus, Clostridium tetani, Vibrio cholera, Escherichia coli, Klebsiella pneumonia, Borrelia burgdorferi, Borrelia mayonii, Clostridioides difficile, Pseudomonas aeruginosa, Helicobacter pylori, Streptococcus pyogenes, Francisella tularensis, Acinetobacter species, Neisseria gonorrhoeae, Leptospira species, Coxiella burnetii, Clostridium botulinum, Burkholderia pseudomallei, Gram-negative bacteria, Salmonella paratyphi, Mycobacterium leprae, Brucella species, Campylobacter species, Listeria monocytogenes, Mycobacterium avium, Mycoplasma pneumonia, Rickettsia species, Anaplasma species, Ehrlichia species, Neorickettsia species, Neoehrlichia species, Orientia species, Mycobacterium tuberculosis, Anaplasma phagocytophilum, phagocytophilum), Orientia tsutsugamushi, or Bartonella species.

37. The system of any one of claims 1-26, wherein the antibody or antigen-binding fragment thereof specifically binds to a protein or component of a parasite.

38. The system of claim 37, wherein the parasite is a Babesia species, Ancylostoma duodenale, Necator americanus, Sarcoptes scabiei, Ascaris lumbricoides, Schistosoma mansoni, Taenia solium, Enterobius vermicularis, Wuchereria bancrofti, Toxoplasma gondii, Giardia lamblia, Entamoeba histolytica, Plasmodium species, Leishmania species, Trypanosoma cruzi cruzi, Schistosoma species, Cryptosporidium species, Trypanosoma brucei, Wuchereria bancrofti, Brugia malayi, Brugia timori, Entamoeba histolytica or Onchocerca volvulus.

39. The system of any one of claims 1-26, wherein the antibody or antigen-binding fragment thereof specifically binds to an allergen.

40. The system of claim 39, wherein the allergen is derived from mites, insects, pollen, animal epidermis, mold, meat, fish, crustaceans, fruits, nuts, vegetables, flour or bran, milk, eggs, spices, hay, silk, cotton, latex, yeast, grass, trees, grains, or animal hair.

41. The system of any one of claims 1-26, wherein the antibody or antigen-binding fragment thereof specifically binds to an immune checkpoint molecule.

42. The system of claim 41, wherein the immune checkpoint molecule is PD-1, PD-L1, CTLA-4, TIM-3, TIGIT, 4-1BB (CD137), GITR (CD357) or killer IgG-like receptor (KIR).

43. The system of any one of claims 1-26, wherein the antibody or antigen-binding fragment thereof specifically binds an antigen involved in an inflammatory disease.

44. The system of claim 43, wherein the inflammatory disease is selected from allergy, asthma, celiac disease, glomerulonephritis, hepatitis, or inflammatory bowel disease.

45. The system of claim 43, wherein the inflammatory disease is mast cell activation syndrome (MCAS).

46. ​​The system of claim 43, wherein the inflammatory disease is an autoimmune disease selected from the group consisting of rheumatoid arthritis, psoriasis, Guillain-Barré syndrome, Graves' disease, myasthenia gravis, vasculitis, lupus, type 1 diabetes, Hashimoto's disease, inflammatory bowel disease, celiac disease, or multiple sclerosis (MS).

47. The system of claim 43, wherein the inflammatory disease is an autoinflammatory disease selected from the group consisting of familial Mediterranean fever (FMF), cryopyrin-associated periodic syndrome (CAPS), TNF receptor-associated periodic syndrome (TRAPS), IL-1 receptor antagonist deficiency (DIRA), or hyper IgD syndrome (HIDS).

48. The system of any of the preceding claims, wherein said administration results in a peak plasma level of the antibody or antigen-binding fragment thereof of at least 75 ng / mL, at least 100 ng / mL, at least 150 ng / mL, at least 200 ng / mL, at least 250 ng / mL, at least 300 ng / mL, at least 400 ng / mL, at least 500 ng / mL, at least 600 ng / mL, at least 700 ng / mL, at least 800 ng / mL, at least 900 ng / mL, or at least 1000 ng / mL.

49. The system of any of the preceding claims, wherein the administering is performed without electroporation or hydroporation.

50. The system of any one of the preceding claims, wherein the plasmid is a DNA plasmid.

51. A method of inducing antibody production in a subject, the method comprising administering to the subject the system of any one of the preceding claims.

52. A method of inducing antibody production in a subject, the method comprising administering to the subject: a plasmid comprising a polynucleotide sequence encoding a heavy chain variable domain of an antibody or antigen-binding fragment thereof; wherein the plasmid is encapsulated in a lipid vesicle; and Wherein administration of the plasmid encapsulated in the lipid vesicles to the subject produces a peak plasma level of the antibody or antigen-binding fragment thereof of at least 50 ng / mL.

53. The method of claim 51 or 52, wherein the administration is performed intramuscularly, subcutaneously, intradermally, intranasally, orally, intrathecally, or intravenously.

54. The method of any one of claims 51-53, wherein the administration is performed intramuscularly.

55. The method of any one of claims 51-53, wherein the administration is performed intravenously.

56. The method of any one of claims 51-55, wherein the administering is performed without electroporation or hydroporation.

57. The method of any one of claims 51-56, wherein the administration results in a peak plasma level of the antibody or antigen-binding fragment thereof of at least 75 ng / mL, at least 100 ng / mL, at least 150 ng / mL, at least 200 ng / mL, at least 250 ng / mL, at least 300 ng / mL, at least 400 ng / mL, at least 500 ng / mL, at least 600 ng / mL, at least 700 ng / mL, at least 800 ng / mL, at least 900 ng / mL, or at least 1000 ng / mL.

58. The method of any one of claims 51-57, wherein the administering is performed once or twice.

59. The method of any one of claims 51-57, wherein the method comprises administering 2 doses of a plasmid to the subject.

60. The method of claim 59, wherein the two doses are administered about 2 weeks to about 12 weeks apart.

61. The method of claim 59 or 60, wherein administration of the second dose results in a peak plasma level of the antibody or antigen-binding fragment that is more than 2-fold higher than the peak plasma level achieved after the first dose.

62. The method of claim 59 or 60, wherein administration of the second dose results in a peak plasma level of the antibody or antigen-binding fragment that is at least 3-fold, at least 4-fold, or at least 5-fold higher than the peak plasma level achieved after the first dose.

63. The method of any one of claims 51-62, wherein the administering comprises delivering about 0.1 mg / kg to about 10 mg / kg of the plasmid to the subject per dose.

64. The method of any one of claims 51-63, wherein the plasma level of the antibody or antigen-binding fragment is maintained at a concentration of at least 50 ng / mL, at least 100 ng / mL, at least 200 ng / mL, at least 300 ng / mL, at least 400 ng / mL, at least 500 ng / mL, at least 600 ng / mL, at least 700 ng / mL, at least 800 ng / mL, at least 900 ng / mL, or at least 1000 ng / mL for a period of at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 20 weeks after administration.

65. The method of any one of claims 51-64, wherein the plasma level of the antibody or antigen-binding fragment is maintained at a concentration of at least 50% of the peak plasma concentration achieved for a period of at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks after administration.

66. The method of any one of claims 51-64, wherein the plasma level of the antibody or antigen-binding fragment is maintained at a concentration of at least 25% of the peak plasma concentration achieved for a period of at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks after administration.

67. The method of any one of claims 51-64, wherein the plasma level of the antibody or antigen-binding fragment is maintained at a concentration of at least 10% of the peak plasma concentration achieved for a period of at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 20 weeks, at least 30 weeks, or at least 40 weeks after administration.

68. The method of any one of claims 65-67, wherein a maintained concentration of the antibody is achieved after a single administration.

69. The method of any one of claims 65-67, wherein the maintained concentration of the antibody or antigen-binding fragment is achieved after two administrations.

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