DNA therapeutics encoding antibodies or antigen-binding fragments

Abstract

The present invention provides a system for the expression of antibodies or antibody-binding fragments in a target, and a method for treating a disease using the same. [Solution] A platform and related methods for producing a desired antibody or antigen-binding fragment in a subject are described herein, the antibody or antigen-binding fragment may be expressed by the subject after transfection of the subject tissue (e.g., muscle tissue) with stable recombinant DNA (e.g., one or more plasmids) encoding the desired antibody or antigen-binding fragment.

Description

[Background technology]

[0001] background Antibody therapy infusion is an established method for treating life-threatening acute infections, protecting immunocompromised individuals, or as a treatment for other diseases. Many therapeutic antibodies require careful storage to maintain their therapeutic activity. Furthermore, the concentration of infused antibodies may decrease over time due to serum protein turnover or anti-drug antibody responses that neutralize the infused antibodies. Improved modalities are needed to deliver clinically appropriate and sustained levels of antibodies to target patients. [Overview of the project] [Means for solving the problem]

[0002] Brief summary Platforms and related methods for producing desired antibodies or antigen-binding fragments in a subject are described herein. In some cases, the antibody or antigen-binding fragment is expressed by the subject after transfection of the subject tissue (e.g., muscle tissue) with stable recombinant DNA (e.g., one or more plasmids) encoding the desired antibody or antigen-binding fragment. In some cases, the recombinant DNA is transfected using a formulation that enables highly efficient transfection of the subject tissue. In some cases, the formulation contains lipid vesicles, which encapsulate the DNA and contain small fusion proteins that lead to highly efficient transfection of target cells in the subject tissue. Subsequently, the encoded antibody is expressed and secreted by the subject's own cells at levels sufficient to be clinically appropriate (e.g., having therapeutic or prophylactic activity). Surprisingly, this platform is compatible with a wide variety of antibodies and different antibody formats (e.g., V HThe H format (or similar) is encoded in a DNA vector (e.g., a plasmid), introduced into a target, and is highly adaptable to produce a clinically appropriate antibody or antigen-binding fragment titer in the target without requiring extensive vector optimization. The systems and methods provided herein are advantageous over administering exogenous antibodies to a target because they do not require the development of the large-scale protein expression, purification, and quality control protocols required for protein antibodies. Furthermore, the cost of administering antibodies using this novel approach is expected to be significantly lower than that of conventional administration of injected antibodies. Thus, the flexibility of the systems and methods provided herein presents a promising platform that can be used to rapidly and easily develop antibody therapies for a wide variety of indications.

[0003] In one embodiment, the Specified Information provides a system for expressing an antibody or an antigen-binding fragment thereof in a subject, comprising a plasmid containing a polynucleotide sequence encoding a heavy chain variable domain of the antibody or the antigen-binding fragment, wherein the plasmid is encapsulated in a lipid vesicle.

[0004] In one embodiment, the Specified Information Provides a system for expressing an antibody or an antigen-binding fragment in a subject, comprising a plasmid containing a polynucleotide sequence encoding a heavy chain variable domain of the antibody or the antigen-binding fragment, wherein the plasmid is encapsulated in a lipid vesicle, and when the plasmid encapsulated in the lipid vesicle is administered, the subject produces a peak plasma level of at least 50 ng / mL of the antibody or the antigen-binding fragment.

[0005] In some embodiments, the antibody or its antigen-binding fragment is a single-domain antibody. In some embodiments, the antibody or its antigen-binding fragment is V H This is an H antibody. In some embodiments, the heavy chain variable domain is fused to the Fc domain via a peptide linker as needed.

[0006] In some embodiments, the plasmid encodes the full-length heavy chain of the antibody. In some embodiments, the plasmid further comprises a polynucleotide sequence encoding the light chain or antigen-binding fragment of the antibody. In some embodiments, the plasmid encodes the full-length light chain of the antibody. In some embodiments, the polynucleotide sequence encoding the heavy chain variable domain and the polynucleotide sequence encoding the light chain are operably linked so that the sequences are transcribed as 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.

[0007] In some embodiments, the system includes a second plasmid containing a second polynucleotide sequence encoding the light chain of the 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.

[0008] In some embodiments, the lipid vesicle contains a fusion-associated small transmembrane (FAST) protein. In some embodiments, the FAST protein contains one or more domains derived from FAST proteins selected from p10, p14, p15, and p22. In some embodiments, the FAST protein has the following sequence: [ka] It contains an amino acid sequence that has at least 80% sequence identity with respect to [the given sequence].

[0009] In some embodiments, the vector includes a promoter operably ligated to a polynucleotide sequence selected from CAG, CMV, EF1A, CBh, CBA, and SFFV. In some embodiments, the plasmid includes a CAG promoter. In some embodiments, the plasmid is a DNA plasmid.

[0010] 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 is

化

[0011] 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 group consisting of Arg435His (His435), Asn434Ala (A), Met428Leu / Asn434Ser (LS), Thr252Leu / Thr253Ser / Thr254Phe (LSF), Glu294delta / Thr307Pro / Asn434Tyr (C6A-66), Thr256Asn / Ala378Val / Ser383Asn / Asn434Tyr (C6A-78), and Glu294delta (Del), wherein residue position numbering is based on the EU numbering scheme. In some embodiments, 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 scheme.

[0012] In some embodiments, the antibody or its antigen-binding fragment specifically binds to the viral protein. In some embodiments, the viral protein is derived from a virus 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 viral proteins include hantavirus, rabies virus, nipah virus, hendra virus, rift valley fever virus, lassa virus, Marburg virus, Crimean-Congo fever virus, hMPV, RSV, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, norovirus, monkeypox virus, cowpox virus (coxpox), Japanese encephalitis virus, yellow fever virus, HSV-1, HSV-2, MERS virus, The virus is derived from a virus selected from the group consisting of varicella virus, hand-foot-and-mouth disease virus, CMV (HHV-5), equine encephalitis virus, EBV (HHV-4), human metapneumovirus, norovirus, enterovirus, smallpox virus, West Nile virus, paramyxovirus, rhinovirus, mononucleosis virus, coxsackievirus B, influenza virus, poliovirus, measles virus, rubella virus, HPV, Zika virus, mumps virus, herpesvirus, chikungunya virus, Haemophilus influenzae (H. influenzae), and SARS-CoV-2 virus. In some embodiments, the viral protein is derived from SARS-CoV-2. In some embodiments, the viral protein is the SARS-CoV-2 spike protein.In some embodiments, 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 to the antibody set forth in Table 3.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a cancer antigen. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a bacterial protein or component. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a parasite protein or component. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an allergen. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an immune checkpoint molecule. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an antigen associated with an inflammatory disease.

[0014] In some embodiments, 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. In some embodiments, the administration is performed without electroporation or hydroporation.

[0015] In some embodiments, the plasmid is a DNA plasmid.

[0016] In another aspect, provided herein is a method of inducing antibody production in a subject, comprising the step of administering the system provided herein to the subject. In some embodiments, administration of the plasmid encapsulated in lipid vesicles to the subject results in a plasma level of the antibody or antigen-binding fragment thereof of at least 50 ng / mL.

[0017] In some embodiments, administration is performed intramuscularly, subcutaneously, intradermally, intranasally, or intrathecally. In some embodiments, administration is performed intramuscularly. In some embodiments, administration is performed intravenously. In some embodiments, administration is performed without electroporation or hydroporation. In some embodiments, administration results in peak plasma levels of the antibody or its antigen-binding fragment 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.

[0018] In some embodiments, the administration is performed once or twice. In some embodiments, the method includes a step of administering two doses of plasmid to the target. In some embodiments, the two doses are administered intravenously. In some embodiments, the two doses are administered approximately 2 weeks to 12 weeks apart. In some embodiments, the administration of the second dose results in a peak plasma level of antibody or antigen-binding fragment that is more than twice the peak plasma level achieved after the first dose. In some embodiments, the administration of the second dose results in a peak plasma level of antibody or antigen-binding fragment that is at least 3 times, at least 4 times, or at least 5 times the peak plasma level achieved after the first dose.

[0019] In some embodiments, the administration involves delivery of plasmid to a target at a dose of approximately 0.1 mg / kg to approximately 20 mg / kg. In some embodiments, the administration involves delivery of plasmid to a target at a dose of approximately 0.1 mg / kg to approximately 20 mg / kg. In some embodiments, plasma levels of the antibody or antigen-binding fragment persist at concentrations 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 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, plasma levels of the antibody or antigen-binding fragment persist 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, plasma levels of the antibody or antigen-binding fragment persist 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, plasma levels of the antibody or antigen-binding fragment persist 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 sustained concentration of the antibody is achieved after a single dose. In some embodiments, the sustained concentration of the antibody is achieved after two doses.

[0020] Further aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, which illustrates and describes only exemplary embodiments of the present disclosure. It will be understood that other different embodiments are possible, and some of their details can be modified in various obvious ways, all without departing from the present disclosure. Therefore, the drawings and description should be considered illustrative and not restrictive. Embedding by reference

[0021] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. To the extent that any publications and patents or patent applications incorporated by reference conflict with the disclosures contained herein, this specification is intended to supersede and / or take precedence over any such conflicting material.

[0022] Novel features of this disclosure are described in detail in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of this disclosure are utilized, and to the appended drawings. [Brief explanation of the drawing]

[0023] [Figure 1-1] Figure 1A shows the plasma concentrations of human antibodies in Rag2 knockout mice 9 days after administration of the DNA-coding antibody system provided herein. A total of 10 mice were transfected using the same protocol (antibody expression construct, transfection pathway, and amount of DNA).

[0024] Figure 1B shows the plasma concentrations of human antibodies in Rag2 knockout mice 16 days after administration of the DNA-coding antibody system provided herein.

[0025] [Figure 1-2] Figure 1C shows the plasma concentrations of human antibodies in Rag2 knockout mice 23 days after administration of the DNA-coding antibody system provided herein.

[0026] Figure 1D shows the plasma concentrations of human antibodies in Rag2 knockout mice 30 days after administration of the DNA-coding antibody system provided herein.

[0027] [Figure 1-3] Figure 1E shows the plasma concentrations of human antibodies in Rag2 knockout mice 37 days after administration of the DNA-coding antibody system provided herein.

[0028] Figure 1F shows the plasma concentrations of human antibodies in Rag2 knockout mice 44 days after administration of the DNA-coding antibody system provided herein.

[0029] [Figure 1-4] Figure 1G shows plasma antibody concentrations for single and double doses of the illustrated antibody formulations. For both IV formulations, antibody levels increased after the second dose on day 60.

[0030] Figure 1H shows the time course of plasma antibody concentrations for the illustrated drug regimen.

[0031] [Figure 1-5] Figure 1I shows the time course of plasma antibody concentrations for the illustrated drug regimens, measured using a commercially available IgG1 standard.

[0032] Figure 1J shows the time-course plasma antibody concentrations for the illustrated drug regimens, measured using an internally generated IgG1 standard, including remeasurements of the samples shown in Figure 1P. The use of the internal standard results in antibody levels approximately 1 / 25th lower than those of the commercially available standard.

[0033] [Figure 2]Figure 2 shows the time course of antibody expression for the illustrated administration routes in Rag2 knockout mice.

[0034] [Figure 3] Figure 3A shows the domain structure of the SARS-CoV-2 spike protein.

[0035] Figure 3B shows a schematic diagram of the binding of mAb1 and mAb2 to the SARS-CoV-2 spike protein receptor-binding domain (RBD) at non-overlapping sites.

[0036] [Figure 4] Figure 4A shows the domain configurations of a monoclonal antibody, a heavy-chain-only antibody, and a VHH antibody.

[0037] Figure 4B shows V H This shows the chain configuration of the H variable region.

[0038] [Figure 5-1] Figure 5A shows the vector map of the expression plasmid for the single transcript T2A construct of mAb1.

[0039] [Figure 5-2] Figure 5B shows the vector map of the expression plasmid encoding the heavy chain of mAb1 in the two-plasmid (HC+LC) construct.

[0040] Figure 5C shows the vector map of the expression plasmid encoding the light chain of mAb1 in the two plasmid (HC+LC) construct.

[0041] [Figure 6] Figure 6 shows the binding of antibodies in the plasma of Rag2 knockout mice to the SARS-CoV-2 Wuhan strain receptor-binding domain 44 days after administration of the DNA-coding antibody system provided herein.

[0042] [Figure 7]Figure 7A shows the plasma antibody concentrations for a single dose of the indicated antibody form at the indicated dose, calculated using a human IgG1 standard developed in-house.

[0043] Figure 7B shows the plasma antibody concentrations for some of the same samples from Figure 7A, measured using a more sensitive assay.

[0044] [Figure 8] Figure 8 shows the plasma antibody concentrations for samples containing and without the SV40e element.

[0045] [Figure 9] Figure 9 shows the plasma antibody concentrations of VHH-type antibodies in ng / mL units (left) and nM units (right). [Modes for carrying out the invention]

[0046] Detailed explanation The following description and examples illustrate embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and is therefore subject to change. Those skilled in the art will recognize that numerous variations and modifications exist within the scope of the present disclosure.

[0047] Various features of this disclosure may be described in relation to a single embodiment, but they may also be provided separately or in any preferred combination. Conversely, for clarity, this disclosure may be described herein in relation to separate embodiments, but it may also be implemented in a single embodiment. Section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described. I. Expression system of antibody or antigen-binding fragments

[0048] A system for the expression of an antibody or antigen-binding fragment is provided herein. In some embodiments, the system is configured to express a therapeutically appropriate amount of antibody or antigen-binding fragment when administered to a subject.

[0049] In one embodiment, a system for expressing an antibody or an antigen-binding fragment thereof is provided herein. In some embodiments, the system is configured to express an antibody or an antigen-binding fragment when administered to a subject. In some embodiments, the system includes a plasmid. In some embodiments, the plasmid includes a polynucleotide sequence encoding a heavy chain variable domain of the antibody or an antigen-binding fragment. In some embodiments, the plasmid is encapsulated in a lipid vesicle. In some embodiments, the lipid vesicle is administered to a subject. In some embodiments, the administered lipid vesicle produces a peak plasma level of at least 50 ng / ml of the antibody or antigen-binding fragment.

[0050] In another embodiment, a system for expressing an antibody or an antigen-binding fragment thereof, comprising a vector, is provided herein. In some embodiments, the vector comprises a polynucleotide sequence encoding a heavy chain variable domain of the antibody or the 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 administered lipid vesicle produces a peak plasma level of at least 50 ng / ml of the antibody or antigen-binding fragment.

[0051] In yet another embodiment, a system for expressing an antibody or an antigen-binding fragment in a target tissue is provided herein. In some embodiments, the system includes a DNA molecule. In some embodiments, the DNA molecule includes a polynucleotide sequence encoding the heavy chain variable domain of the 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 target. In some embodiments, the administered lipid vesicle produces a peak plasma level of at least 50 ng / ml of antibody or antigen-binding fragment. vector

[0052] In one embodiment, a vector comprising a polynucleotide sequence encoding an antibody or antigen-binding fragment having affinity for a disease-related antigen is provided herein. In some embodiments, a DNA vector comprising a polynucleotide sequence encoding an antibody or antigen-binding fragment having affinity for a disease-related antigen is provided herein. The disease-related antigen may be an antigen associated with a disease, and examples of such antigens are viruses, bacteria, parasites, or proteins or other components of cancer, or antigens associated with another disease such as an autoimmune disease or inflammatory disorder. 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.

[0053] In embodiments where the vector is a plasmid, it may be advantageous for the plasmid to be of a certain size or smaller. In some embodiments, smaller plasmids have the advantage of better loading of the vector into the desired formulation (e.g., the proteolipid vehicle provided herein), as well as enhanced expression due to a lower likelihood of cross-reactivity resulting from the larger size. In some embodiments, the plasmids include up to about 50,000 base pairs (bp), up to about 45,000 bp, up to about 40,000 bp, up to about 35,000 bp, up to about 30,000 bp, up to about 25,000 bp, up to about 20,000 bp, up to about 15,000, up to about 10,000, up to about 9,000, up to about 8,000, up to about 7,000, up to about 6,000, up to about 5,000 bp, or up to about 4,000 bp (for double-stranded DNA plasmids). In some embodiments, the plasmid contains up to approximately 5,000 bp. In some embodiments, the plasmid contains up to approximately 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.

[0054] In some embodiments, the plasmid is a bacterial or fungal plasmid or derived therefrom. In some embodiments, the plasmid is a yeast plasmid or derived therefrom. In some embodiments, the plasmid is a bacterium or derived from a bacterium. In some embodiments, the plasmid backbone is derived from a bacterium or fungus. In some embodiments, the plasmid backbone is derived from a bacterium. In some embodiments, the plasmid backbone is derived from a yeast.

[0055] In some embodiments, the plasmid includes an R6K origin of replication. In some embodiments, the plasmid includes a 140 bp RNA-based sucrose-selectable antibiotic-free marker (RNA-OUT). In some embodiments, the plasmid backbone (e.g., parts of the plasmid not directly related to the expression of the encoded gene, e.g., coding sequences, polyadenylated sequences, signal peptide coding sequences, and promoters) 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 essentially consists of an origin of replication, a selection marker, and a part of the plasmid directly related to the expression of the encoded gene.

[0056] In some embodiments, the plasmid backbone is the NTC9385R plasmid. The NTC9385R plasmid is an expression vector containing a bacterial backbone with a 140 bp RNA-based, sucrose-selectable, 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 as described herein. NTC9385R is commercially available from Nature Technology Corporation under the trade name Nanoplasmid®.

[0057] In some embodiments, the vector contains a polynucleotide sequence encoding a secretion signal peptide. In some embodiments, the polynucleotide encoding the secretion signal peptide is fused in-frame with the 5' end of a polynucleotide encoding an antibody heavy chain, an antibody heavy chain antigen-binding fragment, an antibody light chain, and / or an antibody light chain antigen-binding fragment. In some embodiments, the polynucleotide encoding the secretion signal peptide is fused with a polynucleotide sequence encoding the heavy chain. In some embodiments, the polynucleotide encoding the secretion signal peptide is fused in-frame with the 5' end of a polynucleotide sequence encoding a therapeutic antibody light chain or light chain antigen-binding fragment. In some embodiments, the polynucleotide encoding the secretion signal peptide is fused with a polynucleotide sequence encoding the light chain. In some embodiments, the secretion signal peptide is V H It is fused with the H antibody.

[0058] In some embodiments, the vectors encoding the antibody or antigen-binding fragment provided herein include one or more promoters that assist in the transcription of the sequence encoding the antibody or antigen-binding fragment. In some embodiments, the vector includes a promoter operably ligated to the polynucleotide sequence encoding the antibody or antigen-binding fragment. In some embodiments, the promoter enables enhanced expression of the mRNA transcript of the antibody or antigen-binding fragment. In some embodiments, the vector includes a eukaryotic promoter. In some embodiments, the promoter is selected from the CAG promoter, the cytomegalovirus (CMV) promoter, the human elongation factor-1 alpha (EF1A) promoter, the CBh promoter (see, e.g., Hum Gene Ther. 2011 Sep;22(9):1143-53. doi: 10.1089 / hum.2010.245), the chicken β-actin (CBA) promoter, or the spleen-focusing virus (SFFV) promoter.

[0059] In some embodiments, the vector includes a CAG promoter. In some embodiments, the CAG promoter includes a cytomegalovirus (CMV) early enhancer element, a promoter for the chicken β-actin gene, a first exon and a first intron, and a splice acceptor for the rabbit β-globin gene.

[0060] In some embodiments, the vector includes one or more enhancers (for example, in place of or in addition to the enhancer of the CAG promoter). In some embodiments, the vector includes an SV40 enhancer (SV40e). In some embodiments, the SV40e is incorporated upstream of the region encoding the antibody or its antigen-binding fragment. In some embodiments, the SV40e is incorporated upstream of the promoter of the region encoding the antibody or its antigen-binding fragment. In some embodiments, the SV40e is located immediately upstream of the promoter. In some embodiments, the SV40e is located immediately upstream of the CAG promoter. In some embodiments, the 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 sequence TGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACC (SEQ ID NO: 102).

[0061] 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 its antigen-binding fragment. In some embodiments, the WPRE is located downstream of the region encoding the antibody or its antigen-binding fragment but upstream of the polyadenylation signal. In some embodiments, the WPRE is sequence [ka] The sequence has at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, or 100% sequence identity. Single transcript expression vectors (e.g., plasmids) for light chain and heavy chain antibodies.

[0062] In some embodiments, the system for expressing an antibody or antigen-binding fragment provided herein includes a single vector (e.g., a DNA plasmid) encoding both the heavy chain or fragment of the antibody and the light chain or fragment of the antibody. In some embodiments, both the heavy chain or fragment and the light chain or fragment are transcribed into a single transcript, and therefore The expression of both chains or fragments occurs simultaneously and at the same concentration within the same cell. An exemplary vector demonstrating such a construct is shown in Figure 5A.

[0063] In some embodiments, the polynucleotide sequences encoding the heavy and light chains of an antibody or its antigen-binding fragment are configured to be read as a single transcript. In some embodiments, the encoded fused antibody heavy and light chains are separated by a self-cleaving peptide encoding sequence. In some embodiments, the encoded cleaving peptide is a furin-T2A self-cleaving sequence. In some embodiments, the furin-T2A cleaving peptide sequence is RRKRGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 104). In some embodiments, the furin-T2A cleaving peptide sequence has at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with the peptide sequence RRKRGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 104). In some embodiments, these polypeptides cross the vesicle membrane and assemble in the luminal space of the exocytosis vesicle system for secretion.

[0064] In some embodiments, the antibody heavy chain or fragment comprises a variable heavy chain domain and a CH1 domain. In some embodiments, the heavy chain or fragment further comprises a CH2 domain, a CH3 domain, or both. In some embodiments, the antibody light chain or fragment comprises a variable light chain domain and a constant light chain domain. Split antibody expression system

[0065] In some embodiments, the systems provided herein for expressing an antibody or antigen-binding fragment in a subject are configured to produce the antibody or antigen-binding fragment via translation of two separate transcripts. In some embodiments, the heavy chain or fragment of the antibody, and the light chain or fragment of the antibody, are encoded on one or more vectors (e.g., plasmids) so that each is transcribed separately. In some embodiments, the heavy chain or fragment of the antibody, and the light chain or fragment of the antibody, are encoded on separate vectors. In some embodiments, the separate plasmids are formulated together so that the vectors can be delivered to the same cells (e.g., both are encapsulated in the same lipid vesicle). In some embodiments, both the heavy chain or fragment and the light chain or fragment are expressed in the same cell. In some embodiments, the heavy chain or fragment and the light chain or fragment are expressed in different cells. Exemplary DNA plasmid vectors encoding the heavy and light chains of an antibody separately are shown in Figures 5B and 5C. The vectors depicted therein have substantially identical non-coding portions (i.e., only the coding region is changed) compared to the vector depicted in Figure 5A.

[0066] In some embodiments, an equimolar ratio of a vector encoding the heavy chain or a fragment of the antibody, and a vector encoding the light chain or a fragment of the antibody, are used in the system provided herein. In some embodiments, an equimolar ratio of a vector encoding the heavy chain or a fragment of the antibody, and a vector encoding the light chain or a fragment of the antibody, are used in the system provided herein. In some embodiments, the molar ratio of the vector encoding the heavy chain or a fragment to the vector encoding the light chain or a fragment is about 2:1 to about 1:2. In some embodiments, the molar ratio of the vector encoding the heavy chain or its fragments to the vector encoding the light chain or its fragments 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 its fragments to the vector encoding the light chain or its fragments is about 1.5:1 to about 2:1. In some embodiments, the molar ratio of the vector encoding the heavy chain or its fragments to the vector encoding the light chain or its fragments is about 1.5:1 to about 2:1. In some embodiments, the molar ratio of the vector encoding the heavy chain or its fragments to the vector encoding the light chain or its fragments is approximately 1.6:1 to approximately 1.8:1. In some embodiments, the molar ratio of the vector encoding the heavy chain or its fragments to the vector encoding the light chain or its fragments is approximately 1.7:1. V H H expression

[0067] In some embodiments, the system for expressing an antibody or antigen-binding fragment provided herein includes a vector encoding the heavy chain variable domain of the antibody or antigen-binding fragment. In some embodiments, the vector does not encode the entire antibody heavy chain. In some embodiments, the vector is V H Codes for H antibody. V HAn exemplary schematic diagram of the H antibody structure is shown in Figure 4B. In some embodiments, the vector is V fused to an Fc region H H encoding. V fused to an Fc region H An exemplary depiction of H is provided, along with an intact antibody (left) and V H H alone (right), in Figure 4A (center). In some embodiments, the vector is V fused to an Fc region via a linker peptide H H encoding. In some embodiments, the vector is V fused to an Fc region via a hinge region or a modified hinge region H H encoding. In some embodiments, the antibody is a camelized antibody containing only a heavy chain. In some embodiments, the vector encodes only the variable domain of a heavy chain to produce a single-chain V H H antibody. Antibodies and antigen-binding fragments

[0068] In some embodiments, the antibody or antigen-binding fragment of the present disclosure specifically binds to a target antigen. An antibody or antigen-binding fragment selectively or preferentially binds to a target if it binds to the target with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to specific binding means that the affinity of the antibody or antigen-binding fragment thereof is at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the affinity of the antibody for an unrelated substance, which refers to preferential binding.

[0069] As used herein, the term “antibody” refers to an immunoglobulin (Ig), polypeptide, or protein that has an antigen-binding domain or a binding domain homologous thereto. This term further includes “antigen-binding fragment” and other interchangeable terms for similar binding fragments, such as those described below. Natural antibodies and natural immunoglobulins (Ig) are generally heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains. Each light chain is typically linked to a heavy chain by one covalent disulfide bond, although the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has intrachain disulfide crosslinks at regular intervals. Each heavy chain has a variable domain ("VH") at one end, followed by several constant domains ("CH"). Each light chain has a variable domain ("VL") at one end and a constant domain ("CL") at the other end. The constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. Certain amino acid residues are thought to form an interface between the variable domain of the light chain and the variable domain of the heavy chain.

[0070] In some examples, the antibody or antigen-binding fragment includes isolated antibody or antigen-binding fragments, purified antibody or antigen-binding fragments, recombinant antibody or antigen-binding fragments, modified antibody or antigen-binding fragments, or synthetic antibody or antigen-binding fragments. The antibodies and antigen-binding fragments described herein can be produced partially or entirely synthetically. The antibody or antigen-binding fragment may be a polypeptide or protein having a binding domain that may be or may be homologous to an antigen-binding domain. In one example, the antibody or antigen-binding fragment may be produced in a suitable in vivo animal model and subsequently isolated and / or purified.

[0071] Immunoglobulins (Ig) can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Ig or a part of it may, in some cases, be human Ig. In some cases, the CH3 domain may be of immunoglobulin origin. In some cases, an antibody or antigen-binding fragment, a modified antibody or antigen-binding fragment, or a chain or part of a binder may be derived from Ig. In such cases, Ig may be or be derived from IgG, IgA, IgD, IgE, or IgM. If Ig is IgG, it may be a subtype of IgG, and the subtypes of IgG may include IgG1, IgG2a, IgG2b, IgG3, or IgG4. In some cases, the CH3 domain may be derived from, or be derived from, an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. In some embodiments, the antibody or antigen-binding fragment described herein contains or is derived from IgG. In some examples, the antibody or antigen-binding fragment contains or is derived from IgG1. In some examples, the antibody or antigen-binding fragment contains or is derived from IgG4. In some embodiments, the antibody or antigen-binding fragment described herein contains or is derived from IgM, or is in monomeric form of IgM. In some embodiments, the antibody or antigen-binding fragment described herein contains or is derived from IgE. In some embodiments, the antibody or antigen-binding fragment described herein contains or is derived from IgD. In some embodiments, the antibody or antigen-binding fragment described herein contains or is derived from IgA.

[0072] The “light chains” of antibodies (immunoglobulins) derived from any vertebrate species can be assigned to one of two distinct types, called kappa ("κ" or "K") or lambda ("λ"), based on the amino acid sequence of their constant domains. In some embodiments, the antibody or antigen-binding fragment contains a kappa light chain. In some embodiments, the antibody or antigen-binding fragment contains a lambda light chain.

[0073] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either individually or in combination. Each of the heavy and light chain variable regions consists of four framework regions (FRs) linked by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, together with CDRs from other chains, contribute to the formation of the antibody's antigen-binding site. There are at least two methods for determining CDRs: (1) an approach based on interspecific 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; hereafter referred to as "Kabat"); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948)). As used herein, CDR may refer to a CDR defined by either approach or a combination of both approaches.

[0074] In relation to antibodies, the term "variable domain" refers to the variable domain of an antibody used in the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed across the variable domain of an antibody. More precisely, it is concentrated in three segments called hypervariable regions (also known as CDRs) in both the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the "framework region" or "FR". The unmodified heavy and light chain variable domains each contain four FRs (FR1, FR2, FR3, and FR4), and primarily take the form of a β-sheet configuration with three scattered CDRs, which form connecting loops and, in some cases, form part of the β-sheet structure. The CDRs in each chain are held together in close proximity by the FRs, and together with the CDRs from other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat).

[0075] The terms “hypervariable region” and “CDR” as used herein refer to the amino acid residues of an antibody involved in antigen binding. A CDR contains amino acid residues from three sequence regions that bind complementary to the antigen, known as CDR1, CDR2, and CDR3 for the VH and VL chains, respectively. According to Kabat, in the light chain variable domain, the CDR typically corresponds to residues 24–34 (LCDR1), 50–56 (LCDR2), and 89–97 (LCDR3), while in the heavy chain variable domain, the CDR typically corresponds to residues 31–35 (HCDR1), 50–65 (HCDR2), and 95–102 (HCDR3). Since the CDRs of different antibodies may contain insertions, it will be understood that the amino acid numbering may therefore differ. The Kabat numbering system describes such insertions using a numbering scheme that reflects any insertions in numbering between different antibodies by utilizing letters assigned to specific residues (e.g., 27A, 27B, 27C, 27D, 27E, and 27F of CDRL1 in the light chain). Alternatively, according to Chothia and Lesk (J. Mol. Biol., 196: 901-917 (1987)), in the light chain variable domain, CDR typically corresponds to residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3), and in the heavy chain variable domain, CDR typically corresponds to residues 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3).

[0076] As used herein, “framework region,” “FW,” or “FR” refers to framework amino acid residues that form part of an antigen-binding pocket or antigen-binding groove. In some embodiments, framework residues form loops that are part of an antigen-binding pocket or antigen-binding groove, and the amino acid residues within the loop may or may not be in contact with the antigen. Framework regions generally include regions between CDRs. According to Kabat, in the light chain variable domain, FRs typically correspond to residues 0–23 (LFR1), 35–49 (LFR2), 57–88 (LFR3), and 98–109, and in the heavy chain variable domain, FRs typically correspond to residues 0–30 (HFR1), 36–49 (HFR2), 66–94 (HFR3), and 103–133. As discussed above for the light chain using Kabat numbering, insertions in the heavy chain are described in a similar manner (e.g., 35A and 35B of HCDR1 in the heavy chain). Alternatively, according to Chothia and Lesk, ibid., in the light chain variable domain, FR typically corresponds to residues 0-25 (LFR1), 33-49 (LFR2), 53-90 (LFR3), and 97-109 (LFR4), and in the heavy chain variable domain, FR typically corresponds to residues 0-25 (HFR1), 33-52 (HFR2), 56-95 (HFR3), and 102-113 (HFR4). The loop amino acids of FR can be evaluated and determined by examining the three-dimensional structure of the antibody heavy chain and / or antibody light chain. The three-dimensional structure can be analyzed for amino acid positions accessible by the solvent, as such positions are likely to form loops in the antibody variable domain and / or provide antigen contact. Some solvent-accessible positions can tolerate diversity in amino acid sequences, while other positions (e.g., structural positions) generally have less diversity. The three-dimensional structure of antibody variable domains can be derived from crystal structure or protein modeling.

[0077] In this disclosure, the following abbreviations (in parentheses), as used where necessary, are used in accordance with convention: 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), first complementarity-determining region of the heavy chain (HCDR1), second complementarity-determining region of the heavy chain (HCDR2), third complementarity-determining region of the heavy chain (HCDR3), first complementarity-determining region of the light chain (LCDR1), second complementarity-determining region of the light chain (LCDR2), and third complementarity-determining region of the light chain (LCDR3).

[0078] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be either a native sequence Fc region or a variant Fc region. While the boundaries of the Fc region of an immunoglobulin heavy chain can 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 residues in the Fc region is the EU index numbering, as in Kabat. The Fc region of an immunoglobulin generally contains two constant domains, CH2 and CH3.

[0079] In this disclosure, “antibodies” are useful and include, but are not limited to, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, bispecific antibodies, multispecific antibodies, heteroconjugate antibodies, humanized antibodies, human antibodies, grafted antibodies, deimmunized antibodies, their variants, their fusions, their immunoconjugates, their antigen-binding fragments, and / or glycosylated variants of antibodies, amino acid sequence variants of antibodies, and antibodies modified to be covalently bonded, any other modified configuration of immunoglobulin molecules containing an antigen-recognition site having the required specificity.

[0080] In some cases, the antibody is a monoclonal antibody. As used herein, “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies constituting this population are identical except for naturally occurring mutations that may be present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody is against a single determinant (epitope) on an antigen. The modifier “monoclonal” indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method.

[0081] In some cases, the antibody is a humanized antibody. As used herein, “humanized” antibody refers to a form of non-human (e.g., mouse) antibody that is a specific chimeric immunoglobulin, immunoglobulin chain, or fragment thereof containing a minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient’s complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, that have the desired specificity, affinity, and bioactivity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may include residues that are not found in either the recipient antibody or the transferred CDR or framework sequence, but are included to further improve and optimize the antibody’s performance. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, with all or substantially all of the CDR region corresponding to that of a non-human immunoglobulin, and all or substantially all of the FR region being from the human immunoglobulin consensus sequence. Humanized antibodies are also considered to optimally contain at least a portion of the immunoglobulin constant region or domain (Fc), typically that of human immunoglobulin. Antibodies may have modified Fc regions, for example, as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that have been modified relative to the original antibody, which are also called one or more CDRs "derived" from one or more CDRs from the original antibody.

[0082] If necessary, the antibodies or antigen-binding fragments described herein can be evaluated for immunogenicity and, if required, deimmunized (i.e., the antibody becomes less immunoreactive by modifying one or more T cell epitopes). As used herein, “deimmunized antibody” means that one or more T cell epitopes in the antibody sequence have been modified such that the T cell response after administration of the antibody to a subject is reduced compared to an unimmunized antibody. Analysis of immunogenicity and T cell epitopes present in the antibodies and antigen-binding fragments described herein can be carried out through the use of software and specific databases. An exemplary software and database is iTope®, developed by Antitope, Cambridge, UK. iTope® is an in silico technology for the analysis of peptide binding to human MHC class II alleles. The iTope® software predicts peptide binding to human MHC class II alleles, thereby providing an initial screening for the location of such “potential T cell epitopes.” iTope® software predicts favorable interactions between the amino acid side chains of peptides and specific binding pockets within the binding grooves of 34 human MHC class II alleles. The locations of key binding residues are obtained by in silico generation of 9-mer peptides with one amino acid overlap across 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 their potential "fit" and interaction with the MHC class II binding groove. Peptides that produce a high average binding score (above 0.55 on the iTope® scoring function) for more than 50% of the MHC class II alleles are considered potential T cell epitopes. In such regions, the core 9-amino acid sequence for peptide binding within the MHC class II groove is analyzed to determine MHC class II pocket residues (P1, P4, P6, P7, and P9) and potential T cell receptor (TCR) contact residues (P-1, P2, P3, P5, P8).After identifying any T cell epitope, the identified T cell epitope can be removed by introducing changes, substitutions, additions, and / or deletions of amino acid residues. Such changes can be made in a way that preserves the structure and function of the antibody while still removing the identified epitope. Exemplary changes may include, but are not limited to, conservative amino acid changes.

[0083] The antibody may 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 an antibody produced using any suitable method for producing a human antibody. This definition of a human antibody 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, which expresses the human antibody. Human antibodies can also be produced by introducing a human immunoglobulin locus into a transgenic animal, for example, a mouse in which the endogenous immunoglobulin gene is partially or completely inactivated. Alternatively, human antibodies can also be prepared by immortalizing human B lymphocytes that produce antibodies targeting a target antigen (such B lymphocytes may be recovered from an organism or immunized in vitro).

[0084] Any of the antibodies described herein may be bispecific. A bispecific antibody is an antibody that has binding specificity to at least two different antigens and can be prepared using the antibodies disclosed herein. Conventionally, recombinant production of bispecific antibodies has been based on the simultaneous expression of two immunoglobulin heavy-light chain pairs, in which the two heavy chains have different specificities. A bispecific antibody may consist of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy-light chain pair (providing a second binding specificity) in the other arm. This asymmetrical structure, in which only half of the bispecific molecule has an immunoglobulin light chain, facilitates the separation of the desired bispecific compound from undesirable immunoglobulin chain combinations.

[0085] One approach to producing bispecific antibodies involves fusing an antibody variable domain (antibody-antigen binding site) with the desired binding specificity to an immunoglobulin constant domain sequence. The fusion may be with an immunoglobulin heavy chain constant domain containing at least a portion of the hinge, CH2, and CH3 regions. A first heavy chain constant region (CH1) containing the site required for light chain binding may be present in at least one of the fusions. The immunoglobulin heavy chain fusions, and the DNA encoding the immunoglobulin light chain if desired, are inserted into separate expression vectors and simultaneously transfected into a suitable host organism. This offers great flexibility in adjusting the relative proportions of the three polypeptide fragments, in embodiments where the optimal yield is obtained when the ratios of the three polypeptide chains used in construction are not equal. However, if the expression of at least two polypeptide chains in equal ratios yields a high yield, or if the ratios are not particularly important, it is possible to insert two or all three polypeptide chain coding sequences into a single expression vector.

[0086] In some cases, the antibodies described herein are chimeric antibodies. “Chimera” forms of non-human (e.g., mouse) antibodies include chimeric antibodies containing a minimal sequence derived from non-human Ig. In most cases, chimeric antibodies are mouse antibodies in which at least a portion of the immunoglobulin constant region (Fc), typically that of human immunoglobulin, is inserted in place of the mouse Fc.

[0087] Antibodies and their antigen-binding fragments that specifically bind to one or more epitopes on one or more target antigens, modified antibodies and their antigen-binding fragments, and binders are provided herein. In one example, the binder selectively binds to an epitope on a single antigen. In another example, the binder is bivalent and either selectively binds to two different epitopes on a single antigen or to two different epitopes on two different antigens. In yet another example, the binder is polyvalent (i.e., trivalent, tetravalent, etc.) and binds to three or more different epitopes on a single antigen or to three or more different epitopes on two or more (multiple) antigens.

[0088] Any antigen-binding fragment of an antibody as used herein is also assumed. 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 Fab, Fab', F(ab')2, bispecific F(ab')2, triplicate 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, AVIMER®, minibody, diabody, bispecific diabody, triabody, tetrabody, minibody, maxibody, camelid, VHH, minibody, intrabody, fusion protein containing an antibody portion (e.g., domain antibody), single-chain binding polypeptide, scFv-Fc, Fab-Fc, bispecific T cell engager (BiTE; two scFv produced as a single polypeptide chain, where each scFv is a combination of CDRs or VL as described herein). Examples include, but are not limited to, those containing an amino acid sequence including a combination of / VL, tetravalent tandem diabolic antibodies (TandAb; antibody fragments produced as head-tail non-covalent homodimer folders, e.g., TandAb containing scFv, where scFv contains an amino acid sequence including a combination of CDR or VL / VL as described herein), biaffinity retargeting antibodies (DART; different scFv linked by a stabilizing interchain disulfide bond), bispecificity antibodies (bscAb; two single-chain Fv fragments linked via a glycine-serine linker), single-domain antibodies (sdAb), fusion proteins, and bispecificity disulfide-stabilized Fv antibody fragments (dsFv-dsFv': two different disulfide-stabilized Fv antibody fragments linked by a flexible linker peptide).

[0089] As used herein, the term "avidity" refers to the resistance of a complex of two or more drugs to dissociation after dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA) or any other suitable method. Avidity can be determined by methods such as scatchard analysis or any other suitable method.

[0090] As used herein, the term “affinity” refers to the equilibrium constant for the reversible binding of two drugs and is expressed as KD. The binding affinity (KD) of an antibody or antigen-binding fragment as used herein is given as follows: <500 nM, <475 nM, <450 nM, <425 nM, <400 nM, <375 nM, <350 nM, <325 nM, <300 nM, <275 nM, <250 nM, <225 nM, <200 nM, <175 nM, <150 nM, <125 nM, <100 nM, <90 nM, <80 nM, <70 nM, <50 nM, <50 nM, <49 nM, <48 nM, <47 nM, <46 nM, <45 nM) Less than M, less than 44nM, less than 43nM, less than 42nM, less than 41nM, less than 40nM, less than 39nM, less than 38nM, less than 37nM, less than 36nM, less than 35nM, less than 34nM, less than 33nM, less than 32nM, less than 31nM, less than 30nM, less than 29nM, less than 28nM, less than 27nM, less than 26nM, less than 25nM, less than 24nM, less than 23nM, less than 22nM, less than 21nM, less than 20nM, less than 19nM, less than 18nM, less than 17nM, less than 16nM, less than 15nM, less than 14nM, less than 13nM, less than 12nM , less than 11nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, less than 1nM, less than 990pM, less than 980pM, less than 970pM, less than 960pM, less than 950pM, less than 940pM, less than 930pM, less than 920pM, less than 910pM, less than 900pM, less than 890pM, less than 880pM, less than 870pM, less than 860pM, less than 850pM, less than 840pM, less than 830pM, less than 820pM, less than 810pM, less than 800pM, 790pM Less than 780 pM, less than 770 pM, less than 760 pM, less than 750 pM, less than 740 pM, less than 730 pM, less than 720 pM, less than 710 pM, less than 700 pM, less than 690 pM, less than 680 pM, less than 670 pM, less than 660 pM, less than 650 pM, less than 640 pM, less than 630 pM, less than 620 pM, less than 610 pM, less than 600 pM, less than 590 pM, less than 580 pM, less than 570 pM, less than 560 pM, less than 550 pM, less than 540 pM, less than 530 pM, less than 520 pM, less than 510 pM, less than 500 pM,It may be less than 490 pM, less than 480 pM, less than 470 pM, less than 460 pM, less than 450 pM, less than 440 pM, less than 430 pM, less than 420 pM, less than 410 pM, less than 400 pM, less than 390 pM, less than 380 pM, less than 370 pM, less than 360 pM, less than 350 pM, less than 340 pM, less than 330 pM, less than 320 pM, less than 310 pM, less than 300 pM, less than 290 pM, less than 280 pM, less than 270 pM, less than 260 pM, less than 250 pM, less than 240 pM, less than 230 pM, less than 220 pM, less than 210 pM, less than 200 pM, less than 190 pM, less than 180 pM, less than 170 pM, or any integer between those. Binding affinity can be determined using surface plasmon resonance (SPR), KINEXA® 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 suitable bioassays.

[0091] 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 its 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.

[0092] In some embodiments, the antibody or its antigen-binding fragment contains a modified Fc domain. In some embodiments, the modified Fc domain contains one or more amino acid substitutions compared to the wild-type Fc domain of the relevant subtype or isotype antibody. 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 amino acid sequence of the wild-type Fc domain of the corresponding antibody or antigen-binding fragment. In some embodiments, the modified Fc domain contains one, two, three, four, five, six, seven, eight, nine, ten, or more substitutions compared to the corresponding wild-type Fc domain of the 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 in vivo half-life or stability or other pharmacokinetic parameters, altered binding to the Fc receptor, altered glycosylation pattern, introduced additional disulfide bonds, or disruption of one or more disulfide bonds, or altered intramolecular or intermolecular interactions of the antibody or antigen-binding fragment. In some embodiments, the Fc domain of the antibody or antigen-binding fragment (e.g., IgG1 heavy chain) provided herein includes one or more substitutions or combinations of substitutions selected from 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 an Fc domain having one or more mutations or combinations of mutations selected from Arg435His (His435), Asn434Ala (A), Met428Leu / Asn434Ser (LS), Thr252Leu / Thr253Ser / Thr254Phe (LSF), Glu294delta / Thr307Pro / Asn434Tyr (C6A-66), Thr256Asn / Ala378Val / Ser383Asn / Asn434Tyr (C6A-78), and Glu294delta (Del), where the residue position numbers are based on EU numbering rules. In some embodiments, the Fc domain comprises one or more substitutions selected from M252Y, S254T, and T256E (EU numbering). In some embodiments, the Fc domain includes substitutions M252Y, S254T, and T256E (EU numbering).

[0093] In some embodiments, the antibody or antigen-binding fragment is V H It is an H antibody. In some embodiments, the antibody or antigen-binding fragment contains the heavy chain variable domain of a camelid antibody. In some embodiments, the heavy chain variable domain is sequence [ka] It comprises a sequence having at least 80% sequence identity with respect to, where each X is independently either absent or any amino acid. In some embodiments, the heavy chain variable domain is sequence [ka] The sequence 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 with respect to, where each X is independently either absent or any amino acid.

[0094] In some embodiments, V HThe heavy chain variable domain of the H antibody contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the VHH variable domain sequence of Ty1, N3113V, or N3130V (listed in Table 17).

[0095] In some embodiments, the antibody or antigen-binding fragment is V H It is an H-fusion protein. In some embodiments, an antibody or antigen-binding fragment is fused with a V-domain. H It is an H domain. H The H domain is fused with the IgG1 Fc domain. In some embodiments, V 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 contains an amino acid sequence having at least 80% or at least 90% sequence identity to the sequence SDKTHTCP (SEQ ID NO: 105). In some embodiments, the Fc domain contains a CH2 domain, a CH3 domain, or both. In some embodiments, the Fc domain is sequence [ka] It contains 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 respect to. In some embodiments, V H The H domain is a modified hinge region, and sequence [ka] It is fused with an Fc domain containing an amino acid sequence that is at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identical to it. Antigen to which the antibody or antigen-binding fragment binds.

[0096] In some examples, the antibody or antigen-binding fragment encoded by the system provided herein is useful for treating, preventing, or mitigating 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 a disease-related antigen. infectious disease

[0097] In some embodiments, the systems provided herein for producing antibodies or antigen-binding fragments in a subject are useful for the treatment, management, or prevention of infectious diseases. These infectious diseases include, but are not limited to, viral, microbial, bacterial, parasitic, and fungal infections. In some embodiments, the systems provided herein are effective for the prevention of acute infections (e.g., reducing the risk of infection by a pathogen such as a virus or bacteria by a specific amount, e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, or eliminating the risk of infection by the pathogen). In some embodiments, the systems provided herein are useful in the treatment or management of chronic infections (e.g., eliminating or mitigating one or more symptoms associated with an existing infection, or reducing the prevalence or frequency of symptoms when they recur occasionally).

[0098] In some embodiments, the systems provided herein, comprising a vector encoding an antibody or antigen-binding fragment that binds to an infection-related antigen, are administered as a preventive measure (for example, before the subject is infected with a pathogen). In some embodiments, the systems provided herein, comprising a vector encoding an antibody or antigen-binding fragment that binds to an infection-related antigen, are administered for the treatment of an infection (for example, treatment of an acute infection immediately after infection or the onset of symptoms, or treatment of a chronic infection some time after infection or the onset of symptoms). Viral infection

[0099] In some embodiments, the vector of the system provided herein encodes an antibody or antigen-binding fragment that binds to a virus-related antigen. In some embodiments, the system is effective in inducing protection against viral infection. In some embodiments, the system is effective in mitigating, reducing, or eliminating viral infection. In some embodiments, the virus-related antigen is a component of the virus. In some embodiments, the virus-related antigen is a viral protein, viral glycan, viral lipid membrane, or other component. In some embodiments, the virus-related antigen is a viral protein.

[0100] 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 viruses targeted by the antibody or antigen-binding fragment include hantavirus, rabies virus, nipah virus, hendra virus, rift valley fever virus, lassa virus, Marburg virus, Crimean-Congo fever virus, hMPV, RSV, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, norovirus, monkeypox virus, cowpox virus, Japanese encephalitis virus, yellow fever virus, HSV-1, HSV-2, MERS virus, varicella virus, and hand-foot-and-mouth disease. The virus is selected from the group consisting of viruses, CMV (HHV-5), equine encephalitis virus, EBV (HHV-4), human metapneumovirus, norovirus, enterovirus, smallpox virus, West Nile virus, paramyxovirus, rhinovirus, mononucleosis virus, coxsackievirus B, influenza virus, poliovirus, measles virus, rubella virus, HPV, Zika virus, mumps virus, herpesvirus, chikungunya virus, Haemophilus influenzae, and SARS-CoV-2 virus. In some embodiments, the virus is SARS-CoV-2. Systems for the treatment or prevention of SARS-CoV-2

[0101] In some embodiments, the systems provided herein encode antibody or antigen-binding fragments for the treatment and / or prevention of SARS-CoV-2 infection and related diseases. In some embodiments, the antibody or antigen-binding fragments bind to components of the SARS-CoV-2 virus, for example, viral proteins of the SARS-CoV-2 virus.

[0102] In some embodiments, the antibody or antigen-binding fragment provided herein binds to one or more proteins expressed by the SARS-CoV-2 virus. The antibody or antigen-binding fragment may bind to any SARS-CoV-2 protein. In preferred embodiments, the antibody or antigen-binding fragment binds to SARS-CoV-2 proteins involved in the infection of cells with the SARS-CoV-2 virus, thereby preventing cell infection, or binds to SARS-CoV-2 proteins expressed on the surface of infected cells, thereby targeting infected cells for death by immune cells (e.g., NK cells).

[0103] 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.

[0104] 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 of the SARS-CoV-2 spike protein (e.g., a specific subunit). A schematic diagram of the SARS-CoV-2 spike protein domain is shown in Figure 3A. 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 N-terminal domain (NTD), receptor-binding domain (RBD), S1 domain, S2 domain, fusion peptide domain, heptad repeat domain 1 (HR1), heptad repeat domain 2 (HR2), and 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 includes the RBD.

[0105] In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds is the initially identified Wuhan strain spike protein sequence. [ka] [ka] This includes one or more modifications to the sequence of Sequence ID No. 200, which is the sequence of the sequence. In some embodiments, the modification of the SARS-CoV-2 spike protein to which an antibody or antigen-binding fragment binds is a modification identified in the variant form of the SARS-CoV-2 virus (e.g., beta, gamma, delta, or omicron variants). In some embodiments, the modification of the SARS-CoV-2 spike protein to which an antibody or antigen-binding fragment binds is located in the RBD of the variant form of the virus. Exemplary modifications of the SARS-CoV-2 spike protein to which an antibody or antigen-binding fragment binds in the RBD of an excerpted variant can be found in Table 1 below. In some embodiments, further modifications to the spike protein outside the RBD to which an antibody or antigen-binding fragment binds are located. In some embodiments, further modifications to which an antibody or antigen-binding fragment binds are located inside and outside the RBD. Table 1: Selected RBD mutations from selected mutant strains [Table 1-1] [Table 1-2]

[0106] In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes one or more mutations found in the alpha, beta, gamma, delta, epsilon, zeta, eta, iota, theta, kappa, lambda, or omicron mutant strains. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes one or more mutations found in the RBD of the alpha, beta, gamma, delta, epsilon, zeta, eta, iota, theta, kappa, lambda, or omicron mutant strains. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes each of the mutations found in the RBD of the alpha, beta, gamma, delta, epsilon, zeta, eta, iota, theta, kappa, lambda, or omicron mutant strains. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes each of the mutations found in alpha, beta, gamma, delta, epsilon, zeta, eta, iota, theta, kappa, lambda, or omicron mutant strains.

[0107] In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes one or more mutations found in the beta, gamma, delta, or omicron mutant strains. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes one or more mutations found in the RBD of the beta, gamma, delta, or omicron mutant strains. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes each of the mutations found in the RBD of the beta mutant strain. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes each of the mutations found in the RBD of the gamma mutant strain. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes each of the mutations found in the RBD of the delta mutant. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes each of the mutations found in the RBD of the omicron mutant. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the mutations found in the RBD of the omicron mutant.

[0108] In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds is 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, N450K This includes 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, or modified L981F, or any combination thereof. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds is 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, N450K, L452R, L452Q, S477N This further includes one, two, three, four, six, seven, eight, nine, ten, eleven, twelfth, thirteen, fourteen, fifteen or more of the following: 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 modifications of L981F.

[0109] In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds is 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, Y44 This further includes 9H, 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 modified L981F, or any combination thereof. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds is 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, L45 Further including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more of the following: 2Q, 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.

[0110] In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds is A67V, 69-70Del, T95I, G142D, 143-145Del, Y145H, 211Del, L212I, ins214EPE, A222V, G339D, R346K, S371L, S373P, S375F, K417N, K417T, N440K, G446 This further includes S, L452R, L452Q, S477N, T478K, E484A, E484K, F490S, Q493K, G496S, Q498R, N501Y, Y505H, T547K, H655Y, N679K, P681H, P681R, A701, N764K, D796Y, N856K, Q954H, N969K, or modified L981F, or any combination thereof. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds is A67V, 69-70Del, T95I, G142D, 143-145Del, Y145H, 211Del, L212I, ins214EPE, A222V, G339D, R346K, S371L, S373P, S375F, K417N, K417T, N440K, G446S, L452R, L452Q, S477 Further including one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more of the following: N, T478K, E484A, E484K, F490S, Q493K, G496S, Q498R, N501Y, Y505H, T547K, H655Y, N679K, P681H, P681R, A701, N764K, D796Y, N856K, Q954H, N969K, and L981F modifications.

[0111] In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds is A67V, 69-70Del, T95I, G142D, 143-145Del, 211Del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, K417T, N440K, G446S, S This further includes 477N, L452R, T478K, E484A, E484K, Q493K, G496S, Q498R, N501Y, Y505H, T547K, H655Y, N679K, P681H, P681R, A701V, N764K, D796Y, N856K, Q954H, N969K, or modified L981F, or any combination thereof. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds is A67V, 69-70Del, T95I, G142D, 143-145Del, 211Del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, K417T, N440K, G446S, S477N, L452R, T478K, E The following further include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, four, fifteen, or more, of the following modifications: 484A, E484K, Q493K, G496S, Q498R, N501Y, Y505H, T547K, H655Y, N679K, P681H, P681R, A701V, N764K, D796Y, N856K, Q954H, N969K, and L981F. 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 described 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 with the sequence described in SEQ ID NO: 200.In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes 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 with the sequence described in SEQ ID NO: 200. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes an amino acid sequence having at least 99.5% sequence identity with the sequence described in SEQ ID NO: 200. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes an amino acid sequence having at least 99.6% sequence identity with the sequence described in SEQ ID NO: 200. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes an amino acid sequence having at least 99.7% sequence identity with the sequence described in SEQ ID NO: 200. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes an amino acid sequence having at least 99.8% sequence identity with the sequence described in SEQ ID NO: 200. In some embodiments, the SARS-CoV-2 spike protein to which the antibody or antigen-binding fragment binds includes an amino acid sequence having at least 99.9% sequence identity with the sequence described in SEQ ID NO: 1.

[0112] In some embodiments, the anti-SARS-CoV-2 antibody binds to multiple variants of the SARS-CoV-2 virus. In some embodiments, the anti-SARS-CoV-2 antibody binds to two, three, four, five, six, or more variants of the Wuhan strain of SARS-CoV-2 (SEQ ID NO: 200). In some embodiments, the anti-SARS-CoV-2 antibody binds to one, two, three, four, five, six, or more variants of the Wuhan strain of SARS-CoV-2, selected from alpha, beta, gamma, delta, epsilon, zeta, zeta, eta, iota, theta, kappa, lambda, and omicron. In some embodiments, the anti-SARS-CoV-2 antibody binds to the beta, delta, gamma, and omicron variants, respectively. In some embodiments, the anti-SARS-CoV-2 antibody binds to the RBDs of the beta, delta, gamma, and omicron variants, respectively. In some embodiments, the anti-SARS-CoV-2 antibody binds to the delta and omicron mutants. In some embodiments, the anti-SARS-CoV-2 antibody binds to the RBD of the delta and omicron mutants.

[0113] In some embodiments, the anti-SARS-CoV-2 antibody is an antibody or antigen-binding fragment provided herein.

[0114] In some embodiments, the SARS-CoV-2 antibody or antigen-binding fragment includes a heavy chain variable region (HCVR) having one of the amino acid sequences of SEQ ID NOs: 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 includes a sequence having at least 70 percent (e.g., at least 80 percent, 85 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or more) amino acid sequence identity with one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, or 97.

[0115] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a heavy chain complementarity-determining region 1 (HCDR1) having one of the amino acid sequences of SEQ ID NOs: 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 includes a sequence having at least 70% (e.g., at least 80 percent, 85 percent, 90 percent, 91 percent, 92 percent, 93 percent, or 94 percent) amino acid sequence identity with one of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 57, 65, 73, 82, 90, or 98.

[0116] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a heavy chain complementarity-determining region 2 (HCDR2) having one of the amino acid sequences of SEQ ID NOs: 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 includes a sequence having at least 70% (e.g., at least 80 percent, 85 percent, 90 percent, 91 percent, 92 percent, 93 percent, or 94 percent) amino acid sequence identity with one of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 58, 66, 74, 83, 91, or 99.

[0117] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a heavy chain complementarity-determining region 3 (HCDR3) having one of the amino acid sequences of SEQ ID NOs: 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 includes a sequence having at least 70% (e.g., at least 80 percent, 85 percent, 90 percent, 91 percent, 92 percent, 93 percent, or 94 percent) amino acid sequence identity with one of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, or 100.

[0118] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a light chain variable region (LCVR) having one of the amino acid sequences of SEQ ID NOs. 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 includes at least 70 percent (e.g., at least 80 percent, 85 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or more) of amino acid sequence identity with respect to SEQ ID NOs. 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, or 93.

[0119] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a light chain complementarity-determining region 1 (LCDR1) having one of the amino acid sequences 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 includes a sequence having at least 70% (e.g., at least 80 percent, 85 percent, 90 percent, 91 percent, 92 percent, 93 percent, or 94 percent) amino acid sequence identity with one of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, or 94.

[0120] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a light chain complementarity-determining region 2 (LCDR2) having one of the amino acid sequences of SEQ ID NOs: 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 includes a sequence having at least 70% (e.g., at least 80 percent, 85 percent, 90 percent, 91 percent, 92 percent, 93 percent, or 94 percent) amino acid sequence identity with one of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, or 95.

[0121] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a light chain complementarity-determining region 3 (LCDR3) having one of the amino acid sequences of SEQ ID NOs: 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 includes a sequence having at least 70% (e.g., at least 80 percent, 85 percent, 90 percent, 91 percent, 92 percent, 93 percent, or 94 percent) amino acid sequence identity with one of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, or 96.

[0122] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 2, HCDR2 according to SEQ ID NO: 3, and HCDR3 according to SEQ ID NO: 4. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 2, HCDR2 according to SEQ ID NO: 3, and HCDR3 according to SEQ ID NO: 4, as well as VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 1. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes LCDR1 according to SEQ ID NO: 6, LCDR2 according to SEQ ID NO: 7, and LCDR3 according to SEQ ID NO: 8. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 6 (LCDR1), SEQ ID NO: 7 (LCDR2), and SEQ ID NO: 8 (LCDR3), as well as the sequence described in SEQ ID NO: 5.

[0123] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 10, HCDR2 according to SEQ ID NO: 11, and HCDR3 according to SEQ ID NO: 12. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 10, HCDR2 according to SEQ ID NO: 11, and HCDR3 according to SEQ ID NO: 12, as well as VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 9. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes LCDR1 according to SEQ ID NO: 14, LCDR2 according to SEQ ID NO: 15, and LCDR3 according to SEQ ID NO: 16. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 14, LCDR2 according to SEQ ID NO: 15, and LCDR3 according to SEQ ID NO: 16, as well as the sequence described in SEQ ID NO: 13.

[0124] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 18, HCDR2 according to SEQ ID NO: 19, and HCDR3 according to SEQ ID NO: 20. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 18, HCDR2 according to SEQ ID NO: 19, and HCDR3 according to SEQ ID NO: 20, as well as VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 17. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes LCDR1 according to SEQ ID NO: 22, LCDR2 according to SEQ ID NO: 23, and LCDR3 according to SEQ ID NO: 24. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes LCDR1 according to SEQ ID NO: 22, LCDR2 according to SEQ ID NO: 23, and LCDR3 according to SEQ ID NO: 24, as well as VL having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 21.

[0125] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 26, HCDR2 according to SEQ ID NO: 27, and HCDR3 according to SEQ ID NO: 28. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 26, HCDR2 according to SEQ ID NO: 27, and HCDR3 according to SEQ ID NO: 28, as well as VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 25. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes LCDR1 according to SEQ ID NO: 30, LCDR2 according to SEQ ID NO: 31, and LCDR3 according to SEQ ID NO: 32. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 30, LCDR2, SEQ ID NO: 31, and LCDR3, SEQ ID NO: 32, as well as the sequence described in SEQ ID NO: 29.

[0126] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 34, HCDR2 according to SEQ ID NO: 35, and HCDR3 according to SEQ ID NO: 36. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 34, HCDR2 according to SEQ ID NO: 35, and HCDR3 according to SEQ ID NO: 36, as well as VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 33. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes LCDR1 according to SEQ ID NO: 38, LCDR2 according to SEQ ID NO: 39, and LCDR3 according to SEQ ID NO: 40. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 38 (LCDR1), SEQ ID NO: 39 (LCDR2), and SEQ ID NO: 40 (LCDR3), as well as the sequence described in SEQ ID NO: 37.

[0127] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 42, HCDR2 according to SEQ ID NO: 43, and HCDR3 according to SEQ ID NO: 44. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 42, HCDR2 according to SEQ ID NO: 43, and HCDR3 according to SEQ ID NO: 44, as well as VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 41. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes LCDR1 according to SEQ ID NO: 46, LCDR2 according to SEQ ID NO: 47, and LCDR3 according to SEQ ID NO: 48. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 46, LCDR2, SEQ ID NO: 47, and LCDR3, as well as the sequence described in SEQ ID NO: 48.

[0128] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 50, HCDR2 according to SEQ ID NO: 51, and HCDR3 according to SEQ ID NO: 52. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 50, HCDR2 according to SEQ ID NO: 51, and HCDR3 according to SEQ ID NO: 52, as well as VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 49. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes LCDR1 according to SEQ ID NO: 54, LCDR2 according to SEQ ID NO: 55, and LCDR3 according to SEQ ID NO: 56. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, as well as the sequence described in SEQ ID NO: 53.

[0129] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 58, HCDR2 according to SEQ ID NO: 59, and HCDR3 according to SEQ ID NO: 60. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 58, HCDR2 according to SEQ ID NO: 59, and HCDR3 according to SEQ ID NO: 60, as well as VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 57. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes LCDR1 according to SEQ ID NO: 62, LCDR2 according to SEQ ID NO: 63, and LCDR3 according to SEQ ID NO: 64. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 62, LCDR2, SEQ ID NO: 63, and LCDR3, SEQ ID NO: 64, as well as the sequence described in SEQ ID NO: 61.

[0130] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 66, HCDR2 according to SEQ ID NO: 67, and HCDR3 according to SEQ ID NO: 68. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 66, HCDR2 according to SEQ ID NO: 67, and HCDR3 according to SEQ ID NO: 68, as well as VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 65. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes LCDR1 according to SEQ ID NO: 70, LCDR2 according to SEQ ID NO: 71, and LCDR3 according to SEQ ID NO: 72. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 70, LCDR2, SEQ ID NO: 71, and LCDR3, SEQ ID NO: 72, as well as the sequence described in SEQ ID NO: 69.

[0131] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 74, HCDR2 according to SEQ ID NO: 75, and HCDR3 according to SEQ ID NO: 76. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 74, HCDR2 according to SEQ ID NO: 75, and HCDR3 according to SEQ ID NO: 76, as well as VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 73. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes LCDR1 according to SEQ ID NO: 78, LCDR2 according to SEQ ID NO: 79, and LCDR3 according to SEQ ID NO: 80. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 78 (LCDR1), SEQ ID NO: 79 (LCDR2), and SEQ ID NO: 80 (LCDR3), as well as the sequence described in SEQ ID NO: 77.

[0132] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 82, HCDR2 according to SEQ ID NO: 83, and HCDR3 according to SEQ ID NO: 84. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 82, HCDR2 according to SEQ ID NO: 83, and HCDR3 according to SEQ ID NO: 84, as well as VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 81. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes LCDR1 according to SEQ ID NO: 86, LCDR2 according to SEQ ID NO: 87, and LCDR3 according to SEQ ID NO: 88. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 86, LCDR2 according to SEQ ID NO: 87, and LCDR3 according to SEQ ID NO: 88, as well as the sequence described in SEQ ID NO: 85.

[0133] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 90, HCDR2 according to SEQ ID NO: 91, and HCDR3 according to SEQ ID NO: 92. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes HCDR1 according to SEQ ID NO: 90, HCDR2 according to SEQ ID NO: 91, and HCDR3 according to SEQ ID NO: 92, as well as VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 89. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes LCDR1 according to SEQ ID NO: 94, LCDR2 according to SEQ ID NO: 95, and LCDR3 according to SEQ ID NO: 96. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment includes VL having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 94 (LCDR1), SEQ ID NO: 95 (LCDR2), SEQ ID NO: 96 (LCDR3), and SEQ ID NO: 93.

[0134] In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 according to SEQ ID NO: 98, HCDR2 according to SEQ ID NO: 99, and HCDR3 according to SEQ ID NO: 100. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment comprises HCDR1 according to SEQ ID NO: 98, HCDR2 according to SEQ ID NO: 99, and HCDR3 according to SEQ ID NO: 100, as well as VH having at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with the sequence described in SEQ ID NO: 97.

[0135] In some embodiments, the therapeutic compositions provided herein induce the expression of multiple antibodies or antigen-binding fragments in a subject. In some embodiments, the therapeutic compositions comprise one or more vectors encoding two, three, four, five, or more antibodies or antigen-binding fragments that bind to the SARS-CoV-2 protein. In some embodiments, each of the antibodies binds to the SARS-CoV-2 spike protein. In some embodiments, each of the antibodies or their antigen-binding fragments is an antibody or antigen-binding fragment provided herein.

[0136] In some embodiments, the therapeutic compositions provided herein induce the expression of multiple antibodies or antigen-binding fragments in a subject. In some embodiments, the therapeutic composition comprises one or more vectors encoding two, three, four, five, or more antibodies or antigen-binding fragments that bind to the SARS-CoV-2 protein. In some embodiments, each of the antibodies binds to the SARS-CoV-2 spike protein. In some embodiments, each of the antibodies or their antigen-binding fragments is an antibody or antigen-binding fragment provided herein. In some embodiments, the system provided herein comprises two antibodies or their antigen-binding fragments (e.g., Ab1 and Ab2). 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 can bind to the RBD of the SARS-CoV-2 spike protein simultaneously. In some embodiments, the two antibodies bind to two separate epitopes of the SARS-CoV-2 spike protein. An exemplary schematic diagram of two antibodies binding in such a manner is shown in Figure 3B. In Figure 3B, two complementary antibodies bind to the RBD at non-overlapping sites. One antibody 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 antibody that binds to the RBD core region 1. Specifically, Ab1 is a class I antibody that binds to the "receptor-binding motif" (RBM) or ACE2 region of the spike RBD and is classified as an "ACE2 blocker." Ab2 is a class IV antibody that does not overlap with the ACE2 binding site and, more precisely, binds to a conserved region (core I region) in the RBD.

[0137] In some embodiments, the expressed antibody or antigen-binding fragment comprises two or more antibodies, each containing 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. microbial infection

[0138] In some embodiments, the vectors of the systems provided herein encode antibodies or antigen-binding fragments that bind to antigens associated with infectious microorganisms. In some embodiments, the systems are effective in inducing defense against microbial infection. In some embodiments, the systems are effective in mitigating, reducing, or eliminating microbial infection. In some embodiments, the antigens associated with microorganisms are components of the microorganism. In some embodiments, the antigens associated with microorganisms are proteins, glycans, lipid membranes, cell walls, or other components. In some embodiments, the antigens associated with microorganisms are proteins. In some embodiments, the microorganisms are bacteria. In some embodiments, the bacteria are eukaryotes. In some embodiments, the bacteria are prokaryotes. In some embodiments, the microorganisms are fungi.

[0139] In some embodiments, the microorganisms targeted by the antibody or antigen-binding fragment include Bacillus anthracis, Corynebacterium diphtheria, Bordetella pertussis, Streptococcus pneumonia, Haemophilus influenzae, Salmonella typhimurium, Shigella species, and Streptococcus species, Chlamydia trachomatis, Yersinia pestis, 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, Orientia tsutsugamushi, or Bartonella species. Parasitic infection

[0140] In some embodiments, the vectors of the systems provided herein encode antibodies or antigen-binding fragments that bind to antigens associated with infectious parasites. In some embodiments, the systems are effective in inducing protection against parasitic infection. In some embodiments, the systems are effective in mitigating, reducing, or eliminating parasitic infection. In some embodiments, the parasitic antigens are components of the parasite. In some embodiments, the parasitic antigens are proteins, glycans, lipid membranes, cell walls, or other components.

[0141] In some embodiments, the parasite is 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, Schistosoma species, Cryptosporidium species, Trypanosoma brucei, Wuchereria bancrofti, Brugia malayi, Brugia timori, Entamoeba histolytica, or Onchocerca volvulus. Immunity checkpoints against infectious diseases

[0142] In some embodiments, the vector of the system provided herein encodes an antibody or antigen-binding fragment that binds to an immune checkpoint molecule. In some embodiments, such an immune checkpoint conjugate acts as an inhibitor of the immune checkpoint. In some embodiments, the immune checkpoint conjugate is useful in the treatment of infections. In some embodiments, the immune checkpoint molecule is PD-1, PD-L1, CTLA-4, TIM-3, TIGIT, 4-1BB (CD137), GITR (CD357), or a killer IgG-like receptor (KIR). In some embodiments, the immune checkpoint molecule is PD-1. In some embodiments, the immune checkpoint molecule is PD-L1. Other diseases and indications

[0143] Other indications or diseases that can be treated and / or prevented using the systems provided herein include any indications or diseases that can be treated with antibodies. Non-limiting examples of such diseases or indications include cancer, autoimmune diseases, inflammatory diseases, autoinflammatory diseases, acute toxicity due to environmental factors (e.g., environmental toxins or other toxins, such as snake venom), or allergies.

[0144] In some embodiments, the antibody or antigen-binding fragment of the system provided herein is specific to the cancer antigen. In some embodiments, cancer antigens include programmed cell death I (PD1), programmed cell death ligand 1 (PDL1), CD5, CD20, CD19, CD22, CD30, CD33, CD40, CD44, CD52, CD74, CD103, CD137, CD123, CD152, carcinoembryonic antigen (CEA), integrins, epidermal growth factor (EGF) receptor family members, vascular epidermal growth factor (VEGF), proteoglycans, disiarogangliosides, B7-H3, cancer antigen 125 (CA-125), epidermal 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 α, transmembrane glycoprotein NMB, CC chemokine receptor, prostate-specific membrane antigen (PSMA), and RON (recepteur d'origine nantais 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, beta-catenin, carboxypeptidase M, B1, CD23, CD25, CD27, CD28, CD36, CD45, CD46, CD52, CD56, CD79a / CD79b, CD317, CDK4, CO-43 (blood group Le b ), CO-514 (blood group Le a), CTLA-1, cytokeratin 8, DR5, E1 series (blood group B), 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) as seen in gastric adenocarcinoma, integrin alpha-V-beta-6, integrin β6 (ITGβ6), interleukin-13 receptor α2 (IL13Rα2), JAM-3, KID3, KID31, KS 1 / 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, MyI, MUM-1, N-acetylglucosaminyltransferase, neoglycoprotein, NS-10, OFA-1 and OFA-2, oncostatin M (oncostatin receptor beta), rho15, prostate-specific antigen (PSA), PSMA, polymorphic epithelial mucin antigen (PEMA), PIPA, prostatic acid phosphatase Phosphate), R24, ROR1, SSEA-1, SSEA-3, SSEA-4, sTn, T cell receptor-derived peptide, T5A7, tissue antigen 37, TAG-72, TL5 (blood group A), TNF-α receptor (TNFαR), TNFβR, TNFγR, TRA-1-85 (blood group H), transferrin receptor, TSTA tumor-specific transplant antigen, VEGF-R, Y hapten, Le y The group is selected from the group consisting of , and 5T4.

[0145] In some embodiments, the antibody or antigen-binding fragment of the system provided herein is specific to the allergen. In some embodiments, the allergen is derived from mites, insects, pollen, animal hides, mold, meat, fish, crustaceans, fruits, nuts, vegetables, flour or bran, milk, eggs, spices, hay, silk, cotton, latex, yeast, grass, wood, grain, or animal hair. In some embodiments, the mite allergen is Der p 1, Der f 1, or Blomia tropicalis. In some embodiments, the insect allergen is derived from cockroaches or grasshoppers. In some embodiments, the pollen allergens are derived from mugwort, birch, nettle, chrysanthemum, alder, spruce, lamb's wreath, goldenrod, Japanese hop (Humulus japonicus), pine, timothy grass, dandelion, corn, poplar, plane tree, ragweed, elm, broadleaf plantain, willow, wheat, orchard grass, oil palm, mulberry, rapeseed, or poison oats. In some embodiments, the animal epithelial allergens are derived from dog epithelium, cat epithelium, goat epithelium, duck feathers, or feathers. 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 Serpula lacrymans. 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, scallop, crab meat, shrimp, spiny lobster, or mussels.In some embodiments, fruit or nut allergens are derived from pineapple, apple, orange, banana, mango, strawberry, peanut, cashew, tangerine orange, paprika, peach, pear, tomato, walnut, grape, sunflower seed, almond, hazelnut, pistachio, pine nut, cocoa bean, chestnut, macadamia nut, Brazil nut, lupine seed, pecan nut, or pumpkin seed. In some embodiments, plant allergens are derived from potato, parsley, spinach, soybean, green onion, chives, or cabbage. In some embodiments, flour or bran allergens are derived from rice, corn flour, wheat flour, buckwheat flour, or kidney beans. In some embodiments, milk or egg allergens are derived from milk, whole egg, egg white, or egg yolk. In some embodiments, spice allergens are derived from cocoa, cinnamon, paprika, black pepper, sesame, or garlic. In some embodiments, the allergens are derived from hay, silk, cotton, latex, or yeast (e.g., baker's yeast). In some embodiments, the grass allergens are derived from white grass, timothy grass, poison ryegrass, orchard grass, long-leaved grass, or broadleaf grass. In some embodiments, the tree allergens are derived from alder, hazel, poplar, elm, willow, birch, oak, or plane tree. In some embodiments, the grass allergens are derived from mugwort, nettle, dandelion, or broadleaf plantain. In some embodiments, the grain allergens are derived from grass, barley, oat, rye, or wheat. In some embodiments, the grass allergens are derived from Australian grass. In some embodiments, the grass allergens are derived from bahia grass, halepia, gypsum, white grass, or reed. In some embodiments, the animal hair allergen is derived from hamsters, dogs, rabbits, cats, or guinea pigs.

[0146] In some embodiments, the antibody or its antigen-binding fragment specifically binds to an antigen associated with 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).

[0147] In some embodiments, the antibody or its antigen-binding fragment specifically binds to an antigen associated with an autoinflammatory disease. In some embodiments, the inflammatory disease is an autoinflammatory disease selected from familial Mediterranean fever (FMF), cryopyrin-associated periodic syndromes (CAPS), TNF receptor-associated periodic syndromes (TRAPS), IL-1 receptor antagonist deficiency (DIRA), or hyper-IgD syndrome (HIDS).

[0148] In some embodiments, the antibody or its antigen-binding fragment specifically binds to an antigen associated with 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). Lipid vesicles

[0149] In certain embodiments, the antibody or antigen-binding fragment expression systems provided herein include lipid vesicles. The lipid vesicles comprise one or more lipid components capable of encapsulating a vector provided herein (e.g., a DNA plasmid). In some embodiments, the lipid vesicles comprise one or more protein components that are in contact with or at least partially located within the lipids. In some embodiments, the lipid vesicles comprise a lipid nanoparticle (LNP) composition and a composition encapsulating a polynucleotide construct (e.g., a vector provided herein, e.g., plasmid DNA) in which the LNPs comprise the coding region of the antibody or antigen-binding fragment provided herein.

[0150] In some embodiments, compositions containing plasmid DNA encapsulated with LNPs or other lipid vesicle formulations are non-toxic and non-immunogenic in animals at doses exceeding 15 mg / kg, exhibiting efficiencies more than 80 times greater than those achievable with neutral lipid compositions and 2 to 5 times greater than those achievable with cationic lipid compositions. In some embodiments, the LNPs or other lipid vesicle cargoes deposit directly into the cytoplasm, thereby bypassing the endocytosis pathway.

[0151] In further embodiments, the Disclosure provides lipid vesicles for targeted production (and subsequently preferably efflux) of antibodies or antigen-binding fragments within target cells, the lipid vesicle composition comprising: (a) a lipid nanoparticle vector for nonspecific delivery of nucleic acids to mammalian cells, wherein the lipid nanoparticles comprise one or more lipids and one or more fusionable membrane proteins; and (b) an expression construct for preferential production of antibodies or antigen-binding fragments within target cells.

[0152] Lipid vesicle compositions according to certain embodiments of these embodiments contain one or more lipids at concentrations 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. Lipid vesicle formulations exemplified herein may contain one or more lipids at a concentration of about 20 mM.

[0153] In certain exemplary lipid vesicle compositions, one or more lipids are selected from 1,2-dioleoyl-3-dimethylammonium (dimethylammonitim)-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). LNP compositions may contain two or more lipids selected from the group consisting of DODAP, DOTAP, DOPE, cholesterol, and DMG-PEG.

[0154] 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, and 3-5 mol% DMG-PEG, or in a molar ratio of approximately 45 mol% DODAP, approximately 15 mol% DOTAP, approximately 30 mol% DOPE, approximately 6 mol% cholesterol, and approximately 4 mol% DMG-PEG, are exemplified herein. In a particular embodiment, the lipid vesicle composition comprises DODAP, DOTAP, DOPE, cholesterol, and DMG-PEG in a molar ratio of 45 mol% DODAP, 15 mol% DOTAP, 30 mol% DOPE, 6 mol% cholesterol, and 4 mol% DMG-PEG.

[0155] Lipid vesicle formulations according to other embodiments of these embodiments contain one or more fusion membrane proteins at concentrations ranging from 0.5 μM to 20 μM, or 1 μM to 10 μM, or 3 μM to 4 μM. Illustrated herein are lipid vesicle formulations in which fusion membrane proteins are present at concentrations of approximately 3.5 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, and 20 μM. Exemplary and suitable fusion membrane proteins provided herein include the p15x fusion membrane protein (SEQ ID NO: 201), the p14 fusion membrane protein (SEQ ID NO: 202), and the p14e15 fusion membrane protein (SEQ ID NO: 203).

[0156] In further embodiments of these embodiments, the lipid vesicle formulation comprises a vector containing a polynucleotide sequence encoding one or more antibodies or antigen-binding fragments.

[0157] In some embodiments, the pharmaceutical compositions provided herein include a proteolipido vehicle (PLV). In some embodiments, the proteolipido vehicle encapsulates one or more other parts of the pharmaceutical composition (e.g., a DNA vector, e.g., any DNA vector provided herein).

[0158] Lipid vesicle formulations containing vectors (e.g., DNA plasmids) in concentrations ranging from 20 μg / mL to 1.5 μg / mL, 100 μg / mL to 500 μg / mL, or approximately 250 μg / mL are exemplified herein.

[0159] Suitable exemplary lipid vesicle preparations include the following: each 1 mL lipid vesicle has a lipid concentration of approximately 20 mM, a DNA content of approximately 250 μg, and a fusion protein (e.g., p14 or p14e15) of approximately 3.5 μM; and the lipid preparation contains DODAP:DOTAP:DOPE:cholesterol:DMG-PEG in a molar ratio of approximately 45:15:30:6:4.

[0160] Lipid vesicles contain one or more lipid components. In some embodiments, the lipids of the lipid vesicles are non-immunogenic lipids. In some embodiments, the lipids of the lipid vesicles include naturally occurring lipids. In some embodiments, the lipids of the lipid vesicles include naturally occurring mammalian lipids. In some embodiments, the lipids of the lipid vesicles include naturally occurring human lipids.

[0161] In some embodiments, the lipid vesicles contain a minimum amount of cationic lipids. Cationic lipids are used in certain lipid vesicle formulations to facilitate the fusion of the lipid vesicles with another desired membrane. However, in some embodiments, the proteolipid vesicles provided herein use an alternative strategy for the fusion of the lipid vesicles with a desired cell membrane (e.g., a fusion membrane protein). Therefore, the lipid vesicles provided herein, in some examples, use less cationic lipids than other preparations, which makes the lipid vesicles provided herein less toxic. Cationic lipids have been used to concentrate negatively charged DNA molecules and to facilitate the encapsulation of DNA into liposomes due to their positive charge. In some embodiments, the lipid vesicles have a cationic lipid content (w / w relative to the total lipid content) of 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% in the proteolipid vehicle. In some embodiments, the lipid vesicles have a molar ratio 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 to the ionizable lipid vector (e.g., plasmid). In some embodiments, the molar ratio of the lipid vesicles to the ionizable lipid vector (e.g., plasmid) is between 2.5:1 and 20:1. Fusion membrane proteins

[0162] In some embodiments, the proteolipid vehicle includes a fusion membrane protein. A fusion membrane protein is a membrane-bound or membrane-type protein that facilitates lipid-lipid membrane fusion of two separate lipid membranes. Many such fusion membrane proteins are known in the art.

[0163] In some embodiments, the fusion membrane proteins are derived from viruses. Examples of such virus-derived fusion 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, Herpesviridae herpesvirus glycoprotein B, Rhabdoviridae SVS G protein, Reoviridae fusion-associated small transmembrane protein (FAST), and their derivatives.

[0164] In other embodiments of these embodiments, the lipid vesicles are fusion lipid vesicles, for example, fusion membrane proteins for catalyzing lipid mixing between the lipid vesicles and the target cell plasma membrane, such as the fusion-inducing p14 FAST membrane fusion protein derived from reptile reovirus. Suitable fusion membrane proteins are described in PCT Patent Publications WO2012 / 040825A1 and WO2002 / 044206A2, Lau, Biophys. J. :272 (2004), Nesbitt, Master of Science Thesis (2012), Zijlstra, AACR (2017), Mrlouah, PAACRAM 77 / 13Supnn:Abst 5143 (2017), Krabbe, Cancers 10:216 (2018), Sanchez-Garria, ChemComm 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, These are described in JBC 281 / 421:31778 (2006), each of which is incorporated herein by reference in the same way as when it is described in whole. Further examples of FAST membrane proteins, their fusion proteins, and exemplary formulations can be found in PCT Publication WO2022 / 067446A1, which is incorporated herein by reference in the same way as when it is described in whole.

[0165] Examples of fusionable FAST proteins include the p15 and p14e15 proteins having the amino acid sequences shown in Table 2. In some embodiments, the lipid vesicle FAST proteins provided herein include a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the p15x sequence described below. In some embodiments, the lipid vesicle FAST proteins provided herein include a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the p14 sequence described below. In some embodiments, the lipid vesicle FAST proteins provided herein include a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the p14e15 sequence shown below. Table 2 [Table 2-1] [Table 2-2]

[0166] Preferred fusion membrane proteins are non-immunogenic (e.g., they do not induce a specific immune response to the fusion membrane protein upon administration to a subject). In some cases, such fusion membrane proteins enable repeated administration of the pharmaceutical compositions provided herein and / or enhanced delivery of the inclusion material (e.g., the DNA vector provided herein) to target cells.

[0167] In some embodiments, the fusion 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 by reference as is described herein in whole.

[0168] FAST proteins are a unique family of fusion membrane proteins encoded by fusion reoviruses. FAST proteins include p10, p14, p15, and p22. FAST proteins are the smallest known viral membrane fusion proteins, ranging in size from 95 to 198 amino acids. FAST proteins are non-structural viral proteins expressed on the surface of virus-infected cells or cells transfected with the virus, rather than mediating virus-cell fusion, where they induce intercellular fusion and the formation of multinucleated syncytia. Reconstitution of purified FAST proteins on liposome membranes induces liposome-cell fusion and liposome-liposome fusion, demonstrating that FAST proteins are true membrane fusion proteins.

[0169] In contrast to most enveloped virus fusion proteins, whose cytoplasmic tails are extremely short relative to the overall size of the protein, all FAST proteins have a unique topology in which the majority of the protein is distributed to the membrane and cytoplasm, exposing only 20-43 residues of the external domain to the extracellular environment. Despite the small size of the external domain, both p14 and p10 encode hydrophobic (HP) patches that are hypothesized to induce lipid mixing, similar to the fusion peptides encoded by enveloped virus fusion proteins. The p14 HP consists of 21 residues at the N-terminus of the protein, but the peptide corresponding to this sequence requires the inclusion of 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 loop structure, which presents valine and phenylalanine residues at its apex and is linked to the rest of the protein by a flexible linker region. On the other hand, p10 HP, flanked by two cysteine ​​residues forming an intramolecular disulfide bond, shares more in common with the internal fusion peptides of Ebola virus and avian leukemia virus and avian sarcoma virus (ALSV) glycoproteins and is likely to adopt a cystine-noos structure that forces solvent exposure of conserved valine and phenylalanine residues for membrane interactions. In contrast to p14 and p10, the 20-residue external domain of p15 completely lacks hydrophobic sequences that could function as a conventional fusion peptide. In the absence of such motifs, the p15 external domain instead encodes a polyproline helix, which has been proposed to function as a membrane destabilization motif.

[0170] FAST proteins having improved properties for promoting membrane fusion in relation to synthetic lipid vesicles (e.g., proteolipid vehicles of this disclosure) have been previously described (e.g., U.S. Patent No. 10,227,386).

[0171] In some embodiments, the FAST protein comprises one or more domains derived from the FAST protein, selected from p10, p14, p15, and p22. In some embodiments, the FAST protein comprises an outer domain, a transmembrane domain, and an inner domain.

[0172] In some embodiments, the FAST protein includes an internal domain having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with an internal domain derived from p10, p14, p15, or p22. In some embodiments, the FAST protein includes an internal domain having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with an internal domain derived from p15. In some embodiments, the FAST protein includes an internal domain derived from p15.

[0173] In some embodiments, the FAST protein includes a transmembrane domain derived from the wild-type FAST protein, or a derivative thereof. In some embodiments, the FAST protein includes a transmembrane domain having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with a transmembrane domain derived from p10, p14, p15, or p22. In some embodiments, the transmembrane domain includes 23 amino acid residues, at least two hydrophobic β-branched residues adjacent to the outer domain, three consecutive serine residues directly adjacent to at least two hydrophobic β-branched residues, and glycine residues at positions 7 and 13 from the junction between the outer domain and the first hydrophobic β-branched residue. In some embodiments, the transmembrane domain includes an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with the sequence IVSSSTGIIIAVGIFAFIFSFLY (SEQ ID NO: 204).

[0174] In some embodiments, the FAST protein includes an external domain derived from the wild-type FAST protein, or a derivative thereof. In some embodiments, the FAST protein includes an external domain having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with an external domain derived from p10, p14, p15, or p22. In some embodiments, the FAST protein includes an external domain derived from p10, p14, p15, or p22. In some embodiments, the FAST protein includes an internal domain having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with an external domain derived from p14. In some embodiments, the FAST protein includes an external domain derived from p14.

[0175] In some embodiments, the FAST protein provided herein comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the sequence of the p14 FAST protein (e.g., the sequence defined by sequence MGSGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWE (SEQ ID NO: 205)), and an outer domain containing a functional myristoylation motif; 23 amino acid residues, at least two hydrophobic β-branched residues adjacent to the outer domain, three consecutive serine residues directly adjacent to the at least two hydrophobic β-branched residues, and the outer domain and a first hydrophobic β A transmembrane domain comprising glycine residues at positions 7 and 13 from the junction with a branched residue; and an internal domain comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the sequence of the p15 internal domain (e.g., the sequence defined by KLLQWYNRKSKNKKRKEQIREQIELGLLSYGAGVASLPLLNVIAHNPGS (SEQ ID NO: 206) or VISATPIYKGPCTGVPNSRLLQITSGTAEENTRILNHDGRNPDGSINV (SEQ ID NO: 207)).

[0176] In some embodiments, the FAST protein is [ka] It contains amino acids having sequence identity of at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100%.

[0177] In some embodiments, the FAST protein is supplied by a commercial vendor. In some embodiments, the FAST protein is part of the Fusogenix platform prepared by Entos Pharmaceuticals. II. Administration

[0178] This disclosure relates to the administration of the system provided herein to a subject. In some embodiments, the administration results in the transfection of one or more cells of the subject. In some embodiments, the cells transfected by the system provided herein are persistent cells (e.g., skeletal muscle cells), which results in a stable level of antibody or antigen-binding fragments in the subject, or prolonged production of antibody or antigen-binding fragments by the cells. In some embodiments, this results in the prolonged maintenance of therapeutically appropriate levels of antibody or antigen-binding fragments. Such administrations that result in the desired or optimal pharmacokinetics of antibody or antigen-binding fragments may be effective for the ongoing treatment or prevention of the associated disease. In some embodiments, the administration is carried out by injecting lipid vesicles provided herein containing the vector provided herein into the subject. dose

[0179] In some embodiments, a prescribed dose of a vector (e.g., a DNA plasmid provided herein) is administered to the subject. In some embodiments, the prescribed dose is selected to induce a desired level of antibody or antigen-binding fragment in the subject, the level depending on the clinically or therapeutically appropriate level of antibody or antigen-binding fragment.

[0180] 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. 5mg / kg~1mg / kg, 0.5mg / kg~2mg / kg, 0.5mg / kg~3mg / kg, 0.5mg / kg~4mg / kg, 0.5mg / kg~5mg / kg, 0.5mg / kg~7.5 mg / kg, 0.5mg / kg~10mg / kg, 0.5mg / kg~20mg / kg, 1mg / kg~2mg / kg, 1mg / kg~3mg / kg, 1mg / kg~4mg / kg, 1mg / kg~5 mg / kg, 1mg / kg~7.5mg / kg, 1mg / kg~10mg / kg, 1mg / kg~20mg / kg, 2mg / kg~3mg / kg, 2mg / kg~4mg / kg, 2mg / kg~5m g / kg, 2mg / kg~7.5mg / kg, 2mg / kg~10mg / kg, 2mg / kg~20mg / kg, 3mg / kg~4mg / kg, 3mg / kg~5mg / kg, 3mg / kg~7.5m The doses are g / kg, 3mg / kg~10mg / kg, 3mg / kg~20mg / kg, 4mg / kg~5mg / kg, 4mg / kg~7.5mg / kg, 4mg / kg~10mg / kg, 4mg / kg~20mg / kg, 5mg / kg~7.5mg / kg, 5mg / kg~10mg / kg, 5mg / kg~20mg / kg, 7.5mg / kg~10mg / kg, or 10mg / kg~20mg / kg. In some embodiments, the dose of vector administered to the subject is approximately 0.1mg / kg, approximately 0.5mg / kg, approximately 1mg / kg, approximately 1.5mg / kg, 2mg / kg, approximately 2.5mg / kg, approximately 3mg / kg, approximately 3.5mg / kg, approximately 4mg / kg, approximately 4.5mg / kg, approximately 5mg / kg, approximately 7.5mg / kg, approximately 10mg / kg, or approximately 20mg / 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, the subject is administered multiple doses of the same amount of vector. In some embodiments, the subject receives a first dose and (e.g., after a preferred period) a lower second dose.

[0181] In some embodiments, the dose of the vector administered to the subject is approximately 10 micrograms to approximately 5,000 micrograms. The doses are approximately 5,000 micrograms, about 100 to 250 micrograms, about 100 to 500 micrograms, about 100 to 1,000 micrograms, about 100 to 5,000 micrograms, about 250 to 500 micrograms, about 250 to 1,000 micrograms, about 250 to 5,000 micrograms, about 500 to 1,000 micrograms, about 500 to 5,000 micrograms, or about 1,000 to 5,000 micrograms. In some embodiments, the dose of vector administered to the subject is approximately 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 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 vector administered to the subject is up to 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 subject is administered multiple doses of the same amount of vector.In some embodiments, the subject receives a first dose and (for example, after a preferred period) a second, lower dose. Medication regimen

[0182] In some cases, the drug regimen is used to achieve and / or maintain a desired level of antibody in the subject. In some embodiments, the desired level and duration of antibody levels are achieved after a single dose (e.g., for the treatment of an acute infection). In some cases, repeated doses (e.g., two, three, four, or more doses) are required to achieve the initial therapeutically or clinically appropriate level of antibody or antigen-binding fragment (e.g., a lower maintenance dose following a higher single or multiple priming dose).

[0183] In some embodiments, the subject is administered once. In some embodiments, the subject is administered twice with an injection interval of two weeks. In some embodiments, the subject is administered twice with an injection interval of three weeks. In some embodiments, the subject is administered twice with an injection interval of four weeks. In some embodiments, the subject is administered twice with an injection interval of six weeks. In some embodiments, the subject is administered twice with an injection interval of eight weeks. In some embodiments, the subject is administered twice with an injection interval of twelve weeks.

[0184] In some embodiments, the subject is administered at regularly scheduled intervals (e.g., for continuous prevention against infections such as viruses). In some embodiments, the subject is administered approximately once every month, once every two months, once every three months, once every four months, once every six months, or once every year. In some embodiments, the dosing interval is selected so that a minimum level of antibody or antigen-binding fragment is consistently achieved (e.g., plasma levels above 50 ng / mL, 100 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, or 1000 ng / mL). In some embodiments, the subject is administered at regularly scheduled intervals after the initial priming phase (e.g., two or more consecutive doses at relatively short intervals, such as approximately 2-12 weeks).

[0185] When multiple doses are administered, the dose may vary as needed (e.g., a lower maintenance dose following an initial high dose).

[0186] In some embodiments, subjects receive multiple doses. Route of administration

[0187] The antibody expression systems provided herein can be administered by a wide variety of 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 injection.

[0188] In some embodiments, the systems provided herein can be administered without the need for any special equipment to achieve the desired therapeutic effect (e.g., to achieve the required antibody or antigen-binding fragment level). 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 with a standard needle and syringe setting (e.g., for intramuscular administration). Activation

[0189] In some embodiments, the administered vector can produce plasma antibody or antigen-binding fragment concentrations of 10 ng / ml to 20,000 ng / ml. In some embodiments, the administered vector can produce concentrations 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 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, and 25 ng / ml to 50 ng / ml, 25ng / ml~100ng / ml, 25ng / ml~250ng / ml, 25ng / ml~500ng / ml, 25ng / ml~1,000ng / ml, 25ng / ml~2,500ng / ml, 25ng / ml~5,000ng / ml, 2 5ng / ml~10,000ng / ml, 25ng / ml~15,000ng / ml, 25ng / ml~20,000ng / ml, 50ng / ml~100ng / ml, 50ng / ml~250ng / ml, 50ng / ml~500ng / ml, 50ng / m l~1,000ng / ml, 50ng / ml~2,500ng / ml, 50ng / ml~5,000ng / ml, 50ng / ml~10,000ng / ml, 50ng / ml~15,000ng / ml, 50ng / ml~20,000ng / ml, 100n g / ml~250ng / ml, 100ng / ml~500ng / ml, 100ng / ml~1,000ng / ml, 100ng / ml~2,500ng / ml, 100ng / ml~5,000ng / ml, 100ng / ml~10,000ng / ml, 100 ng / ml~15,000ng / ml, 100ng / ml~20,000ng / ml, 250ng / ml~500ng / ml, 250ng / ml~1,000ng / ml, 250ng / ml~2,500ng / ml, 250ng / ml~5,000ng / m l, 250ng / ml~10,000ng / ml, 250ng / ml~15,000ng / ml, 250ng / ml~20,000ng / ml, 500ng / ml~1,000ng / ml, 500ng / ml~2,500ng / ml, 500ng / ml~5,000ng / ml, 500ng / ml~10,000ng / ml, 500ng / ml~15,000ng / ml, 500ng / ml~20,000ng / ml, 1,000ng / ml~2,500ng / ml, 1,000ng / ml~5,000ng / ml, 1,000ng / ml~10,000ng / ml, 1,000ng / ml~15,000ng / ml, 1,000ng / ml~20,000ng / ml, 2,500ng / ml~5,000ng / ml, 2,500ng / ml~10,000n It is possible to produce plasma antibody or antigen-binding fragment concentrations of g / ml, 2,500 ng / ml to 15,000 ng / ml, 2,500 ng / ml to 20,000 ng / ml, 5,000 ng / ml to 10,000 ng / ml, 5,000 ng / ml to 15,000 ng / ml, 5,000 ng / ml to 20,000 ng / ml, 10,000 ng / ml to 15,000 ng / ml, 10,000 ng / ml to 20,000 ng / ml, or 15,000 ng / ml to 20,000 ng / ml. In some embodiments, the administered vector can produce plasma antibody or antigen-binding fragment concentrations 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 can produce plasma antibody or antigen-binding fragment concentrations 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.

[0190] In some embodiments, the administration results in a peak plasma level 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 of the antibody or its antigen-binding fragment. In some embodiments, the administration results in a peak plasma level of at least 1000 ng / mL of the antibody or its antigen-binding fragment. In some embodiments, the administration results in a peak plasma level of at least 1500 ng / mL of the antibody or its antigen-binding fragment. In some embodiments, the administration results in a peak plasma level of at least 2000 ng / mL of the antibody or its antigen-binding fragment. In some embodiments, the administration results in a peak plasma level of at least 2500 ng / mL of the antibody or its antigen-binding fragment. In some embodiments, administration results in a peak plasma level of at least 3000 ng / mL of the antibody or its antigen-binding fragment. In some embodiments, administration results in a peak plasma level of at least 4000 ng / mL of the antibody or its antigen-binding fragment. In some embodiments, administration results in a peak plasma level of at least 5000 ng / mL of the antibody or its antigen-binding fragment. In some embodiments, the indicated peak plasma level of the antibody or antigen-binding fragment is achieved after a single dose of the system provided herein. In some embodiments, the indicated peak plasma level of the antibody or antigen-binding fragment is achieved after a single intramuscular dose of the system. In some embodiments, the indicated peak plasma level of the antibody or antigen-binding fragment is achieved after two doses of the system provided herein. In some embodiments, the indicated peak plasma level of the antibody or antigen-binding fragment is achieved after two intramuscular doses of the system. In some embodiments, the indicated peak plasma level of the antibody or antigen-binding fragment is achieved after two intravenous doses of the system.

[0191] In some embodiments, the plasma concentration of the antibody or antigen-binding fragment is maintained over a long period at a therapeutically or clinically appropriate level (e.g., levels provided herein, e.g., 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). In some embodiments, the plasma level of the antibody or antigen-binding fragment is maintained over a period of 1 to 206 weeks. In some embodiments, the plasma antibody or antigen-binding fragment concentration is at least 1-2 weeks, 1-4 weeks, 1-8 weeks, 1-13 weeks, 1-26 weeks, 1-52 weeks, 1-104 weeks, 1-206 weeks, 2-4 weeks, 2-8 weeks, 2-13 weeks, 2-26 weeks, 2-52 weeks, 2-104 weeks, 2-206 weeks, 4-8 weeks, 4-13 weeks, 4-26 weeks, 4 weeks The concentration of the antibody or antigen-binding fragment in plasma is maintained over periods of up to 52 weeks, 4 to 104 weeks, 4 to 206 weeks, 8 to 13 weeks, 8 to 26 weeks, 8 to 52 weeks, 8 to 104 weeks, 8 to 206 weeks, 13 to 26 weeks, 13 to 52 weeks, 13 to 104 weeks, 13 to 206 weeks, 26 to 52 weeks, 26 to 104 weeks, 26 to 206 weeks, 52 to 104 weeks, 52 to 206 weeks, or 104 to 206 weeks. In some embodiments, the concentration of the antibody or antigen-binding fragment in plasma is maintained over periods of 1 week, 2 weeks, 4 weeks, 8 weeks, 13 weeks, 26 weeks, 52 weeks, 104 weeks, or 206 weeks. In some embodiments, plasma levels of antibody or antigen-binding fragment concentrations are maintained for a period of at least 1, 2, 4, 8, 13, 26, 52, or 104 weeks.

[0192] In some embodiments, the plasma concentration of the antibody or antigen-binding fragment is maintained above 50 ng / mL for at least 1, 2, 4, 8, 13, 26, 52, or 104 weeks. In some embodiments, the plasma concentration of the antibody or antigen-binding fragment is maintained above 75 ng / mL for at least 1, 2, 4, 8, 13, 26, 52, or 104 weeks. In some embodiments, the plasma concentration of the antibody or antigen-binding fragment is maintained above 100 ng / mL for at least 1, 2, 4, 8, 13, 26, 52, or 104 weeks. In some embodiments, the plasma concentration of the antibody or antigen-binding fragment is maintained above 250 ng / mL for at least 1, 2, 4, 8, 13, 26, 52, or 104 weeks. In some embodiments, the plasma concentration of the antibody or antigen-binding fragment is maintained at more than 500 ng / mL for at least 1, 2, 4, 8, 13, 26, 52, or 104 weeks. In some embodiments, the plasma concentration of the antibody or antigen-binding fragment is maintained at more than 750 ng / mL for at least 1, 2, 4, 8, 13, 26, 52, or 104 weeks. In some embodiments, the plasma concentration of the antibody or antigen-binding fragment is maintained at more than 1000 ng / mL for at least 1, 2, 4, 8, 13, 26, 52, or 104 weeks.

[0193] In some embodiments, plasma levels of the antibody or antigen-binding fragment persist 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, plasma levels of the antibody or antigen-binding fragment persist 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, plasma levels of the antibody or antigen-binding fragment persist 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.

[0194] In some embodiments, the indicated concentration of the antibody or antigen-binding fragment is achieved and maintained after a single dose of the vector. In some embodiments, the indicated concentration of the antibody is achieved and maintained after multiple doses of the vector. In some embodiments, the indicated concentration of the antibody or antigen-binding fragment is achieved and maintained after two doses of the vector. In some embodiments, the concentration of the antibody or antigen-binding fragment is maintained without any additional doses of the vector (e.g., after one or two doses of the vector, depending on the regimen described).

[0195] In some embodiments, the subject is administered two doses of the vector. In some embodiments, the second dose of the vector is administered approximately 2 to 26 weeks after the first dose. In some embodiments, the two doses are administered approximately 2 to 12 weeks apart. In some embodiments, the two doses are administered approximately 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks apart. In some embodiments, the two doses are administered approximately 4 to 12 weeks apart, approximately 6 to 12 weeks apart, approximately 8 to 12 weeks apart, approximately 4 to 10 weeks apart, approximately 6 to 10 weeks apart, or approximately 8 to 10 weeks apart. In some embodiments, the two doses are administered at least 2 weeks apart, at least 4 weeks apart, or at least 6 weeks apart. In some embodiments, the two doses are administered with an interval of up to 26 weeks, up to 20 weeks, up to 16 weeks, up to 12 weeks, or up to 10 weeks between doses. In some embodiments, the second dose is administered after a plateau period in antibody or antigen-binding fragment concentration has been achieved.

[0196] In some embodiments, the two doses are the same. In some embodiments, the first dose is higher than the second dose.

[0197] In some embodiments, the administration of a second dose achieves a peak plasma level of the antibody or antigen-binding fragment that is higher than the expected additive effect. In some embodiments, the administration of a second dose results in a peak plasma level of the antibody or antigen-binding fragment that is more than twice as high as the peak plasma level achieved after the first dose. In some embodiments, the administration of a second dose results in a peak plasma level of the antibody or antigen-binding fragment that is at least three times, at least four times, or at least five times as high as the peak plasma level achieved after the first dose. In some embodiments, the administration of a second dose results in a peak plasma level of the antibody or antigen-binding fragment that is at least three times as high as the peak plasma level achieved after the first dose. In some embodiments, the administration of a second dose results in a peak plasma level of the antibody or antigen-binding fragment that is at least four times as high as the peak plasma level achieved after the first dose. In some embodiments, the administration of a second dose results in a peak plasma level of the antibody or antigen-binding fragment that is at least five times as high as the peak plasma level achieved after the first dose. In some embodiments, each dose is administered intravenously. In some embodiments, each dose is the same amount, or the second dose is less than the first dose. subject

[0198] In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate, feline, canid, bovine, pig, sheep, goat, or rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a child or infant. In some embodiments, the subject is an adult. III. Definition

[0199] All terms are intended to be understood in the same way as they would be understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they would be generally understood by those skilled in the art to which this disclosure belongs.

[0200] The following definitions supplement those in the art and apply to this application, and should not be attributed to any related or unrelated cases, such as any jointly owned patents or applications. Any methods and materials similar or equivalent to those described herein may be used in carrying out the tests of this disclosure, but preferred materials and methods are described herein. Accordingly, the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0201] The terms used herein are for illustrative purposes only and are not intended to limit the scope of any particular case. In this application, the use of the singular form includes the use of the plural form unless otherwise specifically indicated. As used herein, the singular forms "a," "an," and "the" are intended to also include the plural form unless the context explicitly indicates otherwise.

[0202] In this application, the use of “or” means “and / or” unless otherwise specified. The terms “and / or” and “any combination thereof” as used herein, and their grammatical equivalents, can be used interchangeably. These terms can convey that any combination is specifically assumed. For illustrative purposes only, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” may mean “A individually, B individually, C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively unless the context specifically refers to disjunctive use.

[0203] The terms “about” or “approximately” can mean within an acceptable margin of error for a particular value as determined by those skilled in the art, which depends in part on how that value is measured or determined, i.e., on the limits of the measuring system. For example, “about” can mean within or above one standard deviation, according to convention in the art. Alternatively, “about” can mean within 20%, 15%, 10%, 5%, or 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within one order of magnitude, five times, or twice the value. Where a particular value is described in this application and claims, unless otherwise specified, the term “about” should be assumed to mean within an acceptable margin of error for that particular value.

[0204] As used herein and in the claims, the terms “comprising” (and any other form such as “comprise” and “comprises”), “having” (and any other form such as “have” and “has”), “including” (and any other form such as “includes” and “include”), or “containing” (and any other form such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional elements or method steps not enumerated. Any embodiment considered herein may be implemented with respect to any method or composition of the Disclosure, and vice versa. Furthermore, the compositions of the Disclosure may be used to achieve the methods of the Disclosure.

[0205] References to “some embodiments,” “a certain embodiment,” “one embodiment,” or “other embodiments” in this specification mean that certain features, structures, or characteristics described in relation to an embodiment are included in at least some embodiments of this disclosure, but not necessarily in all embodiments. To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0206] The ranges provided herein are understood to be abbreviated representations of all values ​​within that range. For example, the range 1–50 is understood to include 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, as well as any number, combination of numbers, or subrange from the group consisting of all intervening decimal values ​​between the aforementioned integers, e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from any endpoint of that range are particularly conceivable. For example, nested subranges of the exemplary range 1-50 may include 1-10, 1-20, 1-30, and 1-40 in one direction, or 50-40, 50-30, 50-20, and 50-10 in the other direction.

[0207] The term "subject" refers to an animal that is the subject of treatment, observation, or experimentation. Examples of subjects include, but are not limited to, human or non-human mammals, such as non-human primates, bovines, equids, canids, sheep, or felines.

[0208] The terms "optional" or "optionally" mean that the event or situation described thereafter may or may not occur, and that the description includes examples of when the event or situation may occur and examples of when it may not occur.

[0209] In this specification, references to the insertion and / or deletion of one or more nucleotides or amino acids from a sequence are used. When used herein in relation to a sequence, the term "ins," followed by a number, means that the listed nucleotide or amino acid sequence is inserted after the indicated residue. For example, "ins214TDR" indicates that the sequence "TDR" is inserted after residue 214 of the reference sequence. When used herein, the term "del," followed by a number or range of numbers, indicates that the nucleotide or amino acid at the indicated position number of the reference sequence is deleted from the sequence. For example, 137-145del indicates that residues 137, 138, 139, 140, 141, 142, 143, 144, and 145 are deleted from the reference sequence.

[0210] The term "V" used in this specification H The term "H" indicates that the heavy chain variable domain is obtained from, originates from, or derived from a heavy chain antibody. A heavy chain antibody is a functional antibody that has two heavy chains and no light chain. Heavy chain antibodies are found in and can be obtained from camelid animals (e.g., camels and alpacas), which are biologically members of the Camelidae family. H The H antibody was initially described as the antigen-binding immunoglobulin (variable) domain of a "heavy chain antibody" (i.e., "antibody lacking a light chain"; Hamers-Casterman et al., Nature 363: 446-448 (1993)). HThe term "H domain" was chosen to distinguish these variable domains from the heavy chain variable domains (referred to herein as "VH domains" or "VH") and the light chain variable domains (referred to herein as "VL domains" or "VL") present in conventional quadruple-chain antibodies.

[0211] "Camelization" V H The term refers to the fact that one or more amino acid residues in the amino acid sequence of the naturally occurring VH domain from conventional quadruple-chain antibodies are present in the V of heavy-chain antibodies. H This refers to an immunoglobulin single-chain variable domain that has been replaced by one or more amino acid residues located at corresponding positions within the H domain. Such “camelization” substitutions can be inserted into amino acid positions that form and / or are present at the VH-VL interface, as defined herein, and / or so-called camelid hallmark residues (see also WO9404678 and Davies and Riechmann (1994 and 1996)). See also 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). IV. Array

[0212] In some embodiments, the antibody or antigen-binding fragment in the system provided herein 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 antibodies listed in the following table. Table 3 [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0213] While the Disclosure and its merits have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the spirit and scope of the Disclosure as defined in the attached claims. The Disclosure is further illustrated in the following embodiments, which are provided for illustrative purposes only and are not intended to limit the Disclosure. [Examples]

[0214] (Example 1) Design of antibody or antigen-binding fragment expression systems Monoclonal antibody (mAb) sequences were constructed as either single transcripts (ST) or heavy / light chain (HC+LC) sequences. Antibodies in ST format were of two types: Furin T2A (T2A) was either heavy chain (HC) and light chain (LC) or V H The H-form was concatenated. The DNA sequences encoding the antibodies were codon-optimized using either Integrated DNA Technologies (IDT)'s codon optimization web tool (Strategy 1) or ThermoFisher's GeneOptimizer® web tool (Strategy 2).

[0215] 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. The DNA sequences encoding the antibodies were codon-optimized using Integrated DNA Technologies (IDT)'s codon optimization web tool (Strategy 1) or ThermoFisher's GeneOptimizer™ web tool (Strategy 2) to reduce the frequency of rare codon use, balance the GC content, and minimize the RNA secondary structure. All immunoglobulin HCs used the IHG1*01 gene sequence, and LCs used either the IGKC*01 (for mAb1) or IGLC2*01 (for mAb2) gene. A CAG promoter was placed before the complete open reading frame. Figure 5A shows an exemplary vector map of such sequences.

[0216] The HC+LC form was constructed using two plasmids (one encoding HC and the other encoding LC), with the following elements in order: Kozak sequence; signal peptide (for either HC or LC); immunoglobulin HC or LC; and stop codon. Similar to the ST form, a CAG promoter was placed before the open reading frame, followed by a BGH polyadenylation signal. In some heavy chain sequences, YTE mutations [M252Y, S254T, T256E (EU numbered)] were introduced to extend the serum / plasma half-life. Figures 5B and 5C show exemplary vector maps of such sequences.

[0217] V H The H antibody construct was designed using the following sequences in order: Kozak sequence; HC signal peptide; V HH variable domain sequence; modified human hinge region; human CH2 and CH3 domains derived from IGGH1*01; and stop codon. A CAG promoter was placed before the open reading frame, followed by a BGH polyadenylation signal. The V used in this study H The H variable domain sequence is Ty1, which was isolated and published in Hanke, et al., Nat. Comms. 2020 (doi: 10.1038 / s41467-020-18174-5) as an anti-SARS-CoV-2 V H It is H.

[0218] V H H, T2A, and HC+LC forms were constructed as circular nanoplasmids (Nature Technology Corporation). These nanoplasmids, in addition to the elements described above, include RNA-OUT selection markers and R6K origins to enable growth in bacterial hosts. These nanoplasmids are commercially available from Nature Technology Corporation under the trade name Nanoplasmid®. Table 4 [Table 4] (Example 2) In vitro testing of DNA-encoded antibodies

[0219] The DNA encoding the antibody candidate described in Example 2 was tested for in vitro expression to verify protein production. HEK293T cells were placed in 12-well plates in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and penicillin-streptomycin, with 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 cells. One day after seeding, cells were transfected with 3.75 μl of Lipofectamine 3000, 2 μl of P3000, and 1 μg of DNA per well. Plasmid DNA encoding GFP was transfected in parallel with each batch of mAb candidates, and GFP fluorescence was measured as a control 24 hours after transfection. The supernatant from mAb transfections was collected at 48 or 72 hours, and IgG titers were measured and quantified using sandwich ELISA. Plates were coated overnight with goat anti-human Fc polyclonal antibody, and human IgG in the supernatant was detected using goat anti-human H+L polyclonal antibody conjugated with horseradish peroxidase. 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 replicate tests. Samples were diluted to 1:10, 1:50, 1:250, and 1:1250 at concentrations ranging from 3 μg / ml to 1.3 ng / ml to accurately quantify titer compared to a standard curve of purified human IgG1. The data provided below were generated using a commercially available human IgG1 standard (ThermoFisher Scientific, IgG1 Human ELISA Standard (for uncoated ELISA kits), catalog no. 39-50560-65). All samples measured using this commercially available standard and described herein refer to this same standard. The standard curve was fitted to a 4-parameter sigmoid dose-response curve using nonlinear regression, and concentrations were interpolated using dilutions of cell supernatant within the linear dynamic range of the ELISA. Reported IgG expression values ​​are the mean of two biological replicate studies. Table 5: Dosage, codon optimization, antibody form, and results of in vitro expression [Table 5-1] [Table 5-2] [Table 5-3] (Example 3) In vivo expression experimental study 1

[0220] Proteolipid vesicles (PLV) containing T2A nanoplasmids were formulated at concentrations of 2.5, 2, 1, 0.6, and 0.33 mg / ml. PLV containing co-formulated HC+LC nanoplasmids were prepared at a total concentration of 1 mg / ml (0.5 mg / ml of HC nanoplasmids and 0.5 mg / ml of LC nanoplasmids).

[0221] An exemplary process for manufacturing PLV is as follows: plasmid DNA species are encapsulated within fusion-associated small transmembrane (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, San Diego, CA). Separately, PLV lipid components are dissolved in ethanol. Mixing of the DNA-protein fraction and the lipid fraction is performed on a NanoAssemblr Benchtop microfluidics instrument (Precision Nanosystems Inc, Vancouver, BC) at a ratio of 3:1 and a flow rate of 12 mL / min. The formulation is dialyzed against phosphate-buffered saline (pH 7.4) in an 8000 MWCO dialysis membrane (product code 12757486, BioDesign, Carmel, New York) for 3 hours with 3 buffer exchanges, followed by concentration using an Amicon ultracentrifugation filter (EMD Millipore, Burlington, Massachusetts), then passage through a 0.22 μm filter (GSWP04700, EMD Millipore). The resulting FAST-PLV DNA species are stored at 4° C. until use.

[0222] Rag2 knockout mice cannot mount an immune response against human antibodies, so they were used to study antibody expression and titer. To optimize in vivo antibody expression, comparisons were performed among different vector strategies (T2A versus HC+LC), doses, and administration routes (intravenous (IV) versus intramuscular (IM)). Table 6: Injection of mice with PLV, including plasmid DNA dose, vector format, administration route, and injection volume [Table 6-1] [Table 6-2]

[0223] Blood samples were collected at various time points shown herein and processed to obtain plasma. Human IgG titers in mouse plasma are measured by electrochemiluminescence assay (ECLIA) using a Meso Scale Discovery instrument. Human IgG titers in mice are quantified by measuring ECLIA signals of plasma samples diluted 1:100 and interpolating based on a standard curve of purified human IgG1 at concentrations ranging from 3.2 μg / ml to 0.78 ng / ml. The data provided in Table 7 below were generated using a commercially available human IgG1 standard. Non-linear regression is used to fit the standard curve to a 4-parameter sigmoidal dose-response curve. The reported human IgG expression values are the average for each group on day 23 post-injection. Table 7 [Table 7-1] [Table 7-2]

[0224] Figure 1A shows the IgG plasma levels in mice 9 days after administration of the illustrated construct. Figure 1B shows the IgG plasma levels in mice 16 days after administration of the illustrated construct. Figure 1C shows the IgG plasma levels in mice 23 days after administration of the illustrated construct. Figure 1D shows the IgG plasma levels in mice 30 days after administration of the illustrated construct. Figure 1E shows the IgG plasma levels in mice 37 days after administration of the illustrated construct. Figure 1F shows the IgG plasma levels in mice 44 days after administration of the illustrated construct. Figure 2 shows the IgG concentrations in mouse-derived plasma for Ab1 HC+LC and Ab2 HC+LC forms (items 4 and 7 in Table 6) administered via intravenous administration at various time points for individual animals. The data provided in Figures 1A-1F and Figure 2 were generated using commercially available human IgG1 standards.

[0225] At 44 days post-injection, mouse plasma was evaluated for binding to the SARS-CoV-2 (Wuhan) RBD protein. Binding was measured by sandwich ELISA, with ELISA plates coated overnight with commercially available SARS-CoV-2 RBD protein (SinoBiological) at a concentration of 1 μg / ml. Plasma samples (No. 11 in Tables 6 and 7) from five mice given 100 μg of Ab1 in T2A form via the intramuscular route were evaluated. 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 goat anti-human Fc polyclonal antibody conjugated with horseradish peroxidase. The results of this experiment are shown in Figure 6, where the antibody concentration on the x-axis was determined by calculating from the dilution ratio based on the initial IgG concentration using a commercially available human IgG1 standard.

[0226] Table 8 below shows the human IgG concentration in ng / mL at various time points in mice administered the system for the antibody or antigen-binding fragment described in Experiment No. 15 of Table 6 above. The data provided below was generated using a commercially available human IgG1 standard. Table 8 [Table 8]

[0227] Table 9 below shows the human IgG concentration in ng / mL at various time points in mice administered the system for the antibody or antigen-binding fragment described in Experiment No. 4 of Table 6 above. The data provided below was generated using a commercially available human IgG1 standard. Table 9 [Table 9-1] [Table 9-2]

[0228] Table 10 below shows the human IgG concentration in ng / mL at various time points in mice administered the system for the antibody or antigen-binding fragment described in Experiment No. 7 of Table 6 above. The data provided below was generated using a commercially available human IgG1 standard. Table 10 [Table 10-1] [Table 10-2]

[0229] Table 11 below shows the human IgG concentration in ng / mL at various time points in mice administered the system for the antibody or antigen-binding fragment described in Experiment No. 9 of Table 6 above. The data provided below was generated using a commercially available human IgG1 standard. Table 11 [Table 11]

[0230] Table 12 below shows the human IgG concentrations in ng / mL at various time points in mice administered with the system for the antibody or antigen-binding fragment described in Experiment No. 10 in Table 6 above. The data provided below were generated using a commercially available human IgG1 standard. Table 12 [Table 12]

[0231] Table 13 below shows the human IgG concentrations in ng / mL at various time points in mice administered with the system for the antibody or antigen-binding fragment described in Experiment No. 17 of Table 6 above. The data provided below were generated using a commercially available human IgG1 standard. Table 13 [Table 13]

[0232] Table 14 below shows the human IgG concentrations in ng / mL at various time points in mice administered with the system for the antibody or antigen-binding fragment described in Experiment No. 18 of Table 6 above. The data provided below were generated using a commercially available human IgG1 standard. Table 14 [Table 14] (Example 4) In vitro expression - effects of multiple doses

[0233] On day 60 of the study described in Example 3 above, five of the ten mice from study experiment No. 4 (Ab1 HC+LC 100ug IV), experiment No. 7 (Ab2 HC+LC 100ug IV), and experiment No. 9 (Ab1 SC 100ug IM) received a second boost dose of the same cargo previously delivered (i.e., the same vector, same administration route, same dose, etc.). Figure 1G shows the time course of antibody levels for the Ab1 HC+LC 100ug IV (experiment No. 4), Ab2 HC+LC 100ug IV (experiment No. 7), and Ab1 SC 100ug IM (experiment No. 9) forms from this experiment in single-dose and re-dose forms (second dose received on day 60 of the study). The data provided in Figure 1G were generated using a commercially available human IgG1 standard. Both IV formulations showed significantly enhanced IgG levels after boosting compared to the non-boosted control, but no substantial effect was observed for the IM formulation. Surprisingly, for both IV re-administrations, the effect of the second dose resulted in higher antibody levels than the predicted additive effect. (Example 5) High-dose substudy

[0234] To confirm the effect of higher doses administered in IM format, additional mouse groups were added to the tests described in Example 3 (Experiments No. 15, 17, and 18 in Table 6 above). Figure 1H shows the results for Ab1 HC+LC 100ug IV (Experiment No. 4), Ab1 HC+LC 250ug IV (Experiment No. 17), and Ab1 HC+LC 500ug in mice. The time course of antibody levels for single-dose formulations of IV (Experiment No. 15), Ab1 T2A 30ug IM (Experiment No. 10), Ab1 T2A 100ug IM (Experiment No. 9), and Ab1 HC+LC 250ug IM (Experiment No. 18) is shown. The data provided in Figure 1H were generated using commercially available human IgG1 standards. The results show a clear dose-response and an improved kinetic response (i.e., faster antibody production rate) with higher doses. Antibody levels also remained relatively constant until the end of the study (approximately 300 days or more). (Example 6) Comparison of commercially available standard IgG1 with IgG1 standard prepared in-house.

[0235] All results reporting the above IgG or antibody concentrations were obtained using the same commercially available human IgG1 standard (ThermoFisher IgG1 Human ELISA Standard (for uncoated ELISA kits), catalog number 39-50560-65). This commercially available standard was then compared to an in-house prepared IgG1 standard. The in-house prepared 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 suspension cell culture at a ratio of 25 μg of each plasmid using the manufacturer's recommended protocol. The supernatant was collected on day 7 post-transfection and filtered and sterilized through 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. Next, the resin was washed with 5 column volume (CV) of binding buffer, eluted with 2.5 CV of IgG elution buffer (ThermoFisher), and neutralized with 1 M Tris, pH 8.0. The buffer of the sample was changed to PBS. The sample concentration was measured using A280, the purity was measured using SDS-PAGE, and the functional activity was verified by antigen-binding ELISA.

[0236] Figure 1J shows data from experiments No. 15, 17, and 18, analyzed using in-house prepared IgG1 standards (including samples that overlap with those shown in Figure 1I, measured using commercially available standard IgG1). The data shows that the antibody concentration values ​​calculated using the in-house standards are approximately 1 / 25th lower than those of the commercially available standards used in the experiments described above. Since the antibody concentrations in the experiments provided below were calculated using these in-house standards, this approximately 25-fold correlation should be considered when comparing data generated by two different standards (commercial versus in-house). (Example 7) In vivo expression test study 2

[0237] To further optimize in vivo antibody expression, additional in vivo mouse studies were conducted using the experimental groups shown in Table 15, compared to those described in Example 3. Table 15 [Table 15-1] [Table 15-2]

[0238] Blood samples were collected and processed every 7 days after injection to produce plasma. Human IgG titers in mouse plasma were measured by electrochemiluminescence assay (ECLIA) using Meso Scale Discovery instruments. Human IgG titers in mice were quantified by measuring the ECLIA signal of plasma samples diluted 1:25 to 1:100 and interpolating it based on a standard curve of in-house purified human IgG1 at concentrations ranging from 200 ng / ml to 0.048 ng / ml. The standard curve was fitted to a 4-parameter sigmoid dose-response curve using nonlinear regression. The results from the initial point in time of this experiment are shown in Figure 7A. These results indicate that 250 micrograms and 500 micrograms of IM doses behave similarly, suggesting that the benefit of increasing the dose beyond 250 micrograms is limited. Liver preparations did not provide any significant benefit over the standard preparation.

[0239] At the initial time points of experiments No. 21, 22, and 26, antibody levels were below the lower limit of quantification for the assay described above, but an increase in levels was expected at later time points. To better evaluate these initial time points, a more sensitive assay was performed by coating the assay plate (Meso Scale Discovery) with the SARS-CoV-2 Wuhan strain receptor-binding domain (RBD) to enable better quantification. The results of this experiment are shown in Figure 7B. This experiment revealed that the HC:LC group with a molar ratio of 1.7:1 (experiment No. 23) showed a 60% increase in antibody levels on day 28 compared to the HC:LC group with a mass / mass ratio of 1:1 (experiment No. 21). This trend is expected to substantially continue at later time points, given the continued increase in antibody levels. This experiment also showed that the liver preparation performed worse than the standard preparation at this point. (Example 8) Evaluation of SV40e nuclear localization signal

[0240] Further attempts were made to increase antibody levels by using the SV50 enhancer (SV40e) in nanoplasmid expression vectors (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). See https: / / doi.org / 10.1038 / sj.gt.3301419; and Pontus Blomberg et al., "Electroporation 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 TGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACC (Sequence ID 102).This element is proposed to increase transgene expression in DNA gene therapy by providing a nuclear localization signal for targeting plasmid DNA to the cell nucleus. The SV40e element was constructed by incorporating an SV40e cassette immediately upstream of the CAG promoter. This cassette was incorporated into both heavy and light chain vectors in a split vector configuration.

[0241] Initial in vitro experiments using the SV40e element in HEK293 cells showed no substantial difference in expression compared to cells without SV40e. Subsequently, plasmids incorporating the SV40e element were administered in vivo to Rag 2 mice (e.g., those described in the examples above) in the groups shown in Table 16 below. Table 16 [Table 16]

[0242] The results from this experiment are shown in Figure 8. The results were measured using an in-house prepared IgG1 standard. Experiment No. 31 yielded similar results 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 formulations. (Example 9) V H Evaluation of H-Fc fusion format

[0243] Three Vs derived from camelid species H H-Fc fusion antibodies were also tested (see Table 17 below). To increase neutralizing efficacy and in vivo half-life, V H The H fragment was fused with the Fc domain derived from human IgG1 (V HThese constructs are N3113V-Fc and N3130V-Fc (both described in Li, et al 2022; doi 10.1016 / j.ce11.2022.03.009), and Ty1-Fc (described in Hanke, et al 2022; doi: 10.1038 / s41467-020-18174-5). H All H-Fc antibodies were designed using the following sequences in order: Kozak sequence; HC signal peptide; V H H variable domain sequence; modified human hinge region; human CH2 and CH3 domains derived from IGGH1*01; and stop codon. An open reading frame was preceded by a CAG promoter, followed by a BGH polyadenylation signal. All open reading frames were codon-optimized using commercially available software from ThermoFisher to reduce the frequency of rare codon use, balance GC content, and minimize RNA secondary structure. Table 17 [Table 17-1] [Table 17-2]

[0244] All three VHH-Fc constructs were found to be better expressed in vitro than the AB1 HC+LC form in HEK293 cells. Purified N3113V-Fc and N3130V-Fc were found to bind with high affinity to both SARS-CoV-2 Wuhan strain and Omicron RBD, while Ty-Fc did not bind substantially to Omicron RBD.

[0245] A 250ug payload of a nanoplasmid vector encoding the VHH-Fc construct (one construct / vector) was administered by IM injection to three separate groups of Rag2 mice (n=4 or 5) in the same protocol as described in Example 3. The results of this experiment are shown in Figure 9. N3130V-Fc did not produce detectable levels of antibody at any time point. Both the N3113V-Fc variant and the Ty1-Fc variant were expressed better than the Ab1 HC+LC form, with N3113V-Fc expressed approximately 3 times better than Ab1 on a molar basis, and Ty1-Fc expressed approximately 10 to 15 times better than Ab1 on a molar basis. (Example 10) WPRE vector evaluation using VHH-Fc fusion format

[0246] Introduction of woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs) into nanoplasmid expression vectors. This element has been previously reported to increase transgene expression in non-viral and viral vectors by improving the transcription, stability, transport, 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 the WPRE cassette downstream of the antibody open reading frame, prior to the BGH polyadenylation signal. This cassette was incorporated into both the heavy-chain and light-chain vectors in a split-vector configuration, as well as into the Ty1-Fc VHH construct. When a 100ug payload of the Ty1-Fc VHH construct was administered to Rag2 mice as described above, the WPRE vector showed a decrease in expression to approximately half by day 7.

[0247] While preferred embodiments of the present disclosure have been described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Those skilled in the art will likely conceive of a great many variations, modifications, and substitutions without departing from the present invention. It should be understood that various substitutes for the embodiments of the present invention described herein may be used in carrying out the present invention. The scope of the present invention is defined by the following claims, and the scope is intended to include methods and structures within these claims and their equivalents.

Claims

1. A system for expressing an antibody or its antigen-binding fragment in a target, Plasmid containing a polynucleotide sequence encoding the heavy chain variable domain of the antibody or its antigen-binding fragment Includes, The plasmid is enclosed in a lipid vesicle. A system in which, upon administration of the plasmid encapsulated in the lipid vesicles, the subject produces a peak plasma level of at least 50 ng / mL of the antibody or its antigen-binding fragment.

2. A system for expressing an antibody or its antigen-binding fragment in a target, Plasmid containing a polynucleotide sequence encoding the heavy chain variable domain of the antibody or its antigen-binding fragment Includes, A system in which the plasmid is encapsulated in a lipid vesicle.

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

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

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

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

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

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

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

10. The system according to 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 coding sequence.

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

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

1.

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

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

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

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

17. The aforementioned FAST protein has the following sequence: 【Chemistry 11】 The system according to claim 15 or 16, comprising an amino acid sequence having at least 80% sequence identity with respect to.

18. The system according to any one of the claims, wherein the plasmid comprises a promoter operably ligated to a polynucleotide sequence selected from CAG, CMV, EF1A, CBh, CBA, and SFFV.

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

20. The system according to any one of the above claims, wherein the antibody or its antigen-binding fragment comprises an IgG1, IgG2a, IgG2b, IgG3, IgG4, IgD, IgM, IgA1, IgA2, or IgE heavy chain.

21. The system according to claim 20, wherein the antibody or its antigen-binding fragment comprises the IgG1, IgG2a, IgG2b, IgG3, or IgG4 heavy chain.

22. The system according to claim 20, wherein the antibody comprises the IgG1 heavy chain.

23. The aforementioned heavy chain variable domain, 【Chemistry 12】 It includes a sequence having at least 80% sequence identity with (Sequence ID 101), The system according to any one of the above claims, wherein each X is independently either absent or any amino acid.

24. The system according to any one of the claims, wherein the antibody or antigen-binding fragment comprises an Fc domain having one or more mutations or combinations of mutations selected from Arg435His (His435), Asn434Ala (A), Met428Leu / Asn434Ser (LS), Thr252Leu / Thr253Ser / Thr254Phe (LSF), Glu294delta / Thr307Pro / Asn434Tyr (C6A-66), Thr256Asn / Ala378Val / Ser383Asn / Asn434Tyr (C6A-78), and Glu294delta (Del), and the residue position number is according to the EU numbering rules.

25. The system according to any one of the above claims, wherein the antibody or its antigen-binding fragment comprises an Fc domain having one or more mutations selected from M252Y, S254T, T256E, and any combination thereof, and the residue position numbering is in accordance with the EU numbering rules.

26. The system according to any one of the above claims, wherein the plasmid comprises an SV40e element.

27. The system according to any one of the above claims, wherein the antibody or its antigen-binding fragment specifically binds to a viral protein.

28. The system according to claim 27, wherein the viral protein is derived from a virus 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.

29. The aforementioned viral proteins include hantavirus, rabies virus, nipah virus, hendra virus, rift valley fever virus, lassa virus, Marburg virus, Crimean-Congo fever virus, hMPV, RSV, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, norovirus, monkeypox virus, cowpox virus, Japanese encephalitis virus, yellow fever virus, HSV-1, HSV-2, MERS virus, varicella virus, hand-foot-and-mouth disease virus, CMV (HHV-5), and equine encephalitis virus. The system according to claim 28, derived from a virus selected from the group consisting of Russ, EBV (HHV-4), human metapneumovirus, norovirus, enterovirus, smallpox virus, West Nile virus, paramyxovirus, rhinovirus, mononucleosis virus, coxsackievirus B, influenza virus, poliovirus, measles virus, rubella virus, HPV, Zika virus, mumps virus, herpesvirus, chikungunya virus, Haemophilus influenzae, and SARS-CoV-2 virus.

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

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

32. The system according to any one of the above claims, wherein 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 listed in Table 3.

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

34. The aforementioned cancer antigens include programmed cell death I (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 member, vascular epidermal growth factor (VEGF), proteoglycan, disiaroganglioside, B7-H3, cancer antigen 125 (CA-125), epidermal 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 α, transmembrane glycoprotein NMB, C-C chemokine receptor, prostate-specific membrane antigen (PSMA), RON (receptor) d'origin nantais) 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, beta-catenin, carboxypeptidase M, B1, CD23, CD25, CD27, CD28, CD36, CD45, CD46, CD52, CD56, CD79a / CD79b, CD317, CDK4, CO-43 (blood group Le ｂ ), CO-514 (blood group Le ａ ), CTLA-1, cytokeratin 8, DR5, E1 series (blood group B), 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) as seen in gastric adenocarcinoma, integrin alpha-V-beta-6, integrin β6 (ITGβ6), interleukin-13 receptor α2 (IL13Rα2), JAM-3, KID3, KID31, KS 1 / 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, MyI, MUM-1, N-acetylglucosaminyltransferase, neoglycoprotein, NS-10, OFA-1 and OFA-2, oncostatin M (oncostatin receptor beta), rhO15, prostate-specific antigen (PSA), PSM A, Polymorphic epithelial mucin antigen (PEMA), PIPA, Prostatic acid phosphatase, R24, ROR1, SSEA-1, SSEA-3, SSEA-4, sTn, T cell receptor-derived peptide, T5A7, Tissue antigen 37, TAG-72, TL5 (blood group A), TNF-α receptor (TNFαR), TNFβR, TNFγR, TRA-1-85 (blood group H), Transferrin receptor, TSTA tumor-specific transplant antigen, VEGF-R, Y hapten, Le ｙ The system according to claim 33, selected from the group consisting of , and 5T4.

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

36. The above bacteria include Bacillus anthracis, Corynebacterium diphtheria, Bordetella pertussis, Streptococcus pneumonia, Haemophilus influenzae, Salmonella typhimurium, Shigella species, Streptococcus species, Chlamydia trachomatis, Yersinia pestis, Methicillin-resistant Staphylococcus aureus (MRAA), Staphylococcus aureus, Clostridium tetani, Vibrio cholera, Escherichia coli, Lebsiella pneumonia, Borrelia burgdorferi, ｢orrelia ｡yonii, Clostridioides difficile, Pseudomonas aeruginosa, Helicobacter pylori, Streptococcus Genes, Francis tularensis、Acinetobacter species、Neisseria gonorrhoeae、Leptospira species、Coxiella burnetii、Clostridium botulinum、BLraholderia pseudommallei、gram-negative bacteria、Salmonella paratyphi、Mycobacterium leprae、Brucella species, Bacterium species, Listeria monocytogenes, Mycobacterium avium, Mycoplasma pneumonia, Rickettsia species, Anaplasma species, Ehrlichia species, Neorickettsia species, Neoehrlichia species, Orientia species, Mycobacterium tuberculosis, Anaplasma phagocytophilum, OrientiaThe system according to claim 35, wherein the system is tsutsugamushi or Bartonella species.

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

38. The parasite is 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 The system according to claim 37, wherein the species are Trypanosoma cruzi, Schistosoma species, Cryptosporidium species, Trypanosoma brucei, Wuchereria bankrofti, Brugia malayi, Brugia timori, Entamoeba histolytica, or Onchocerca volvulus.

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

40. The system according to claim 39, wherein the allergen is derived from dust mites, insects, pollen, animal hides, mold, meat, fish, crustaceans, fruits, nuts, vegetables, flour or bran, milk, eggs, spices, hay, silk, cotton, latex, yeast, grass, wood, grain, or animal hair.

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

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

43. The system according to any one of claims 1 to 26, wherein the antibody or its antigen-binding fragment specifically binds to an antigen associated with an inflammatory disease.

44. The system according to claim 43, wherein the inflammatory disease is selected from allergies, asthma, celiac disease, glomerulonephritis, hepatitis, and inflammatory bowel disease.

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

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

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

48. The system according to any one of the claims, wherein the administration produces a peak plasma level of the antibody or its antigen-binding fragment 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 according to any one of the above claims, wherein the administration is performed without electroporation or hydroporation.

50. The system according to any one of the above claims, wherein the plasmid is a DNA plasmid.

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

52. A method for inducing antibody production in a target, Plasmid containing a polynucleotide sequence encoding the heavy chain variable domain of the antibody or its antigen-binding fragment The step includes administering the above to the subject, The plasmid is enclosed in a lipid vesicle. A method comprising administering the plasmid encapsulated in the lipid vesicle to a subject, thereby causing a plasma level of at least 50 ng / mL of the antibody or its antigen-binding fragment.

53. The method according to claim 51 or 52, wherein the administration is performed intramuscularly, subcutaneously, intradermally, intranasally, or intrathecally.

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

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

56. The method according to any one of claims 51 to 55, wherein the administration is performed without electroporation or hydroporation.

57. The method according to any one of claims 51 to 56, wherein the administration produces a peak plasma level of the antibody or its antigen-binding fragment 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 according to any one of claims 51 to 57, wherein the administration is performed once or twice.

59. The method according to any one of claims 51 to 57, comprising the step of administering two doses of the plasmid to the subject.

60. The method according to claim 59, wherein the two doses are administered with an interval of approximately two weeks to approximately twelve weeks between them.

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

62. The method according to claim 59 or 60, wherein the administration of the second dose results in a peak plasma level of the antibody or antigen-binding fragment that is at least three times, at least four times, or at least five times higher than the peak plasma level achieved after the first dose.

63. The method according to any one of claims 51 to 62, wherein the administration comprises delivering the plasmid to the subject at a dose of approximately 0.1 mg / kg to approximately 10 mg / kg.

64. The method according to any one of claims 51 to 63, wherein the plasma level of the antibody or antigen-binding fragment persists 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 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 according to any one of claims 51 to 64, wherein the plasma level of the antibody or antigen-binding fragment persists 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 according to any one of claims 51 to 64, wherein the plasma level of the antibody or antigen-binding fragment persists 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 according to any one of claims 51 to 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 according to any one of claims 65 to 67, wherein the sustained concentration of the antibody is achieved after a single dose.

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

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