Materials, methods and systems for stimulating host immune responses

Abstract

The present disclosure relates to inter alia novel designs, compositions, systems and formulations, etc. of nucleic acid(s) (polynucleotide or oligonucleotide, etc.) products, specifically compositions and formulations of nucleic acid immunity inducing agents, such as adjuvants, immunization and or vaccine products and accessory products and nucleic acid gene therapy products, and the like. The compositions of the disclosure enhance immunity, and provide nucleic acid based medicinal and pharmaceutical products. When introduced directly into a host, such as intradermal cells and or tissues, the embodiments described herein induces production of immune responses which specifically recognize targets, such as human targets.

Description

MATERIALS, METHODSAND SYSTEMS FOR STIMULATING HOST IMMUNE RESPONSESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Serial No. 63 / 733,134 filed December12, 2024, the contents of each of which are herein incorporated by reference in its entirety.FIELD

[0002] The present disclosure relates to novel designs, systems, compositions, and formulations, etc. of nucleic acids (polynucleotides, oligonucleotides, etc.) products, specifically compositions and formulations of nucleic acid immunity inducing agents, such as adjuvants, immunization and or vaccine products and nucleic acid gene therapy products, and the like. The compositions of the disclosure enhance immunity, and provide improved nucleic acid based medicinal and pharmaceutical products. When introduced directly into a host, such as intradermally, into cells and or tissues, the invention induces production of immune responses which specifically recognize targets (e.g., antigens), such as human targets.BACKGROUND

[0003] DNA vaccines have been reported to enable humoral and cellular responses (Wolf et. al., Eur. J. Immunol., 1994, 24: pp. 759-764 ). DNA vaccines are reported to be stable, and reported to be easy to manufacture, in some instances. ZyCov-D the first DNA vaccine to be approved for human use received emergency authorization in India for Co vid in August 2021 (Lu et. al. Front. Immunol., 31 January 2024, Sec. Vaccines and Molecular Therapeutics,Volume 15, Sheridan et. al. Nat Biotechnol 39, 1481 (2021) , Li et. al., Signal Transduction and Targeted Therapy (2022) 7: 146). According to one report, despite the great potential of DNA vaccines for a broad range of applications implementation is far behind the expectations up to now (Berger et. al., Microbial Biotechnology. 2024;17:e70053).SUMMARY

[0004] Described herein are improved materials, methods and systems useful for eliciting improved host immunity. A vaccine is one of the most efficacious, safe, nontoxic and economical materials to prevent disease and to control the spread of disease and abnormal cells when used properly. Generally speaking, conventional vaccines are a form of immunoprophylaxis given before disease occurrence to afford immunoprotection by generating a strong host immunological memory against a specific antigen. The primary aim of vaccination is to effectively stimulate the immune system, immunity and, for example activate the adaptive specific immune response, primarily to generate B and T lymphocytes against specific antigen(s) associated with the disease or the disease agent.

[0005] In one aspect, a composition is provided comprising i. a synthetic polynucleotide, wherein the synthetic polynucleotide comprises a polynucleotide encoding an ovalbumin, vaccinia, diphtheria toxin and or tetanus toxin peptides or fragments thereof, or ii. a synthetic polynucleotide wherein the synthetic polynucleotide is capable of expressing ovalbumin, vaccinia, diphtheria toxin and or tetanus toxin peptides or fragments thereof when introduced into a host. In one aspect, the host is a human host, and in another aspect, the host is a nonhuman host. Non-human hosts suitable for use with the embodiments described herein include mice, rats, hamsters, simians, hominoidea, swine, lamoid, avians, llamas and the like.

[0006] In another aspect, a composition is provided, comprising i. a synthetic polynucleotide wherein the synthetic polynucleotide comprises a polynucleotide encoding an ovalbumin, vaccinia, diphtheria toxin and or tetanus toxin peptides or fragments thereof, or ii. a synthetic polynucleotide wherein the synthetic polynucleotide is capable of expressing ovalbumin, vaccinia, diphtheria toxin and or tetanus toxin peptides or fragments thereof when introduced into a host wherein the polynucleotide a. is capable of expressing ovalbumin, vaccinia, diphtheria toxin and tetanus toxin peptides, or fragments thereof when introduced into a host, and optionally, one or more target peptide(s) of fragments thereof. In some embodiments, the composition further comprises one or more target peptide(s) or immunogenic fragments thereof.

[0007] In another aspect, a composition is provided comprising i. a synthetic polynucleotide wherein the synthetic polynucleotide comprises a polynucleotide encoding an ovalbumin, vaccinia, diphtheria toxin and or tetanus toxin peptides or fragments thereof, or ii. a synthetic polynucleotide wherein the synthetic polynucleotide is capable of expressing ovalbumin, vaccinia, diphtheria toxin and or tetanus toxin peptides or fragments thereof when introduced into a host and the composition further comprising more than one target peptide.

[0008] In another aspect, a composition is provided comprising i. a synthetic polynucleotide wherein the synthetic polynucleotide comprises a polynucleotide encoding an ovalbumin, vaccinia, diphtheria toxin and or tetanus toxin peptides or fragments thereof, or ii. a synthetic polynucleotide wherein the synthetic polynucleotide is capable of expressing ovalbumin, vaccinia, diphtheria toxin and or tetanus toxin peptides or fragments thereof when introduced into a host and the composition further comprising more than one target peptidewherein the target peptide is a cancer target peptide. In some embodiments, the cancer target peptide is on or within a cancer cell.

[0009] In another aspect, a composition is provided comprising i. a synthetic polynucleotide wherein the synthetic polynucleotide comprises a polynucleotide encoding an ovalbumin, vaccinia, diphtheria toxin and or tetanus toxin peptides or fragments thereof, or ii. a synthetic polynucleotide wherein the synthetic polynucleotide is capable of expressing ovalbumin, vaccinia, diphtheria toxin and or tetanus toxin peptides or fragments thereof when introduced into a host and the composition further comprising more than one target peptide wherein the target peptide is a cancer cell target peptide and wherein the target peptide comprises angiopoietin, BCMA, MAGE, CD19, CD20, CD22, CD25, CD33, CD37, CD38, mTOR, EGFR, ALK, VEGF, VEGFR, PDGF / PDGFR, PD1 / PD-E1, ROS1, HER2, EMP3, ENPP3, DEE3, and androgen receptor or a fragmen t(s) thereof.

[0010] In one aspect a composition is provided comprising the DNA design of FIG. 1, or equivalents thereof. In one aspect, the DNA of FIG. 1 is provided, or fragments thereof. In some embodiments, the DNA sequence comprises a origin of replication. In some embodiments, the DNA sequences comprises an enhancer. In some embodiments, the DNA sequences comprises a promoter. In some embodiments, the DNA sequences comprises a T- Helper Cassette. In some embodiments, the DNA sequences comprises a sequence encoding an immunogen. In some embodiments, the DNA sequences comprises a bGH poly (A) signal. In some embodiments, the DNA sequences comprises an antibody resistance cassette.

[0011] In one aspect, a system for immunization is provided, comprising the compositions of the embodiments disclosed herein, wherein the system produces or is capable of producing immune molecules. In some embodiments, the immune molecules have binding in the rangeof 0. InM to 5nM, serological data indicating high affinity binding with a Kd less than or equal to InM, screening data of at least IpM binding, an antibody yield equal to or greater than 5ug of antibody from of about 2 ml of expression media, sera or blood, and / or wherein the antibody sequences have an antibody sequence diversity. In some embodiments, the antibodies have the sequence similarity and or epitope group identity from a diversity analysis, wherein clustering over CDR sequences reveals DNA and protein immunogens provide comparable coverage of target epitopes as measured by epitope binning.

[0012] In one aspect, a system is provided. An embodiment of this aspect is a system for immunization comprising a composition as described herein, wherein the system produces immune molecules that are polyclonal or clonal molecules.

[0013] In one aspect, a composition is provided wherein the composition is as described herein and is administrable or is administered to a host. In one aspect, a composition is provided wherein administration of the composition is oral, rectal, inhalation, nebulization, dermal, transdermal, injection (intrathecal, subcutaneous, intravenous, and or intramuscular), intraocular, intraotic, nasal, sublingual, or buccal, or any combination thereof. In another aspect, an administrable composition is provided wherein administration is intradermal. In one aspect, a composition is provided comprising an antibody or fragment thereof that specifically binds a target peptide, said antibody or fragment thereof obtained following administration of the composition. In another aspect, compositions are provided and administrated in accordance with the information available to a person of ordinary skill in the art (Kutzler, M.A. et. al., Nature Reviews Genetics volume 9, pages776-788 (2008)).

[0014] In another aspect, an antibody or fragment thereof hat specifically binds a target peptide following administration of the composition of any one of the embodiments herein is provided.

[0015] In another aspect, a method for immunizing against a tumor-associated antigen, suppressing or attenuating tumor growth, and treating cancer, is provided. In some embodiments, the method comprises a step for inducing an antibody response using the composition of any one of the embodiments herein, and / or a step for inducing a cell mediated immune response through CD4, CD8 or other lymphocyte subsets.

[0016] In another aspect, a method of preventing disease recurrence is provided. The method comprises a step for inducing an antibody response using the composition of any one of the embodiments herein, and / or a step for inducing a cell mediated immune response through CD4, CD8 or other lymphocyte subsets.

[0017] In another aspect, a composition is provided. In some embodiments, the composition comprises a means for encoding an ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof, or a means for expressing ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof when introduced into a host. In some embodiments, the means is a synthetic polynucleotide encoding an ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof. In some embodiments, the means is capable of expressing ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof when introduced into the host.

[0018] In another aspect, a means for producing the composition of any of the compositions provided herein is provided.

[0019] In another aspect, a system for performing a method of immunization is provided. The system comprises a means for encoding an ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof, or a means for expressing ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof when introduced into a host. In some embodiments, the means comprises the composition of any one of the embodiments described herein. In some embodiments, the system further comprises a means for administration.

[0020] In another aspect a method for immunizing against a tumor-associated antigen, suppressing or attenuating tumor growth, and treating cancer is provided. The method comprises a means for administering the composition of any one of the embodiments described herein into a host and / or a means for inducing a cell mediated immune response in the host.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows an overview of an exemplary DNA immunogen design of the present invention, a circular polynucleotide for the expression of an immunogen(s). The DNA map of the circular DNA is illustrated linearly, and is circular. The design comprises a origin of replication (ori), CMV Enhancer, CMV Promoter, T-Helper Cassette, sequence encoding the immunogen(s), bGH poly (A) signal and a NeoR / KanR cassette. The DNA polynucleotide is about 4000 to 6000 kb. Equivalents of boxed elements are known in the art.

[0022] FIG. 2 shows exemplary peptide immunogens and their binding partners which have their T helper cell epitopes.

[0023] FIG. 3 shows expression of a representative target or tumor associated antigen (TAA) in HEK293. Cell surface expression of a representative target TAA (ENPP3) was detected using anti-human 203c antibody (clone NP4D6). Immunization strategy with DNA immunogen is described in the examples herein, wherein mice are immunized in accordance with embodiments described herein, and a response is determined. As shown, a pVAX DNA sequence encoding the TAA had a THC (right panel) or no THC (left panel). Surface expression of the protein was measured using the anti-human 203c antibody.

[0024] FIG. 4 shows binding data illustrative of the immune response obtained by the embodiments herein. The serum was used to evaluate binding of serum from each individual mouse to KU812 cells (top left) , HepG2 cells (top right) and HEK293 cells (bottom).

[0025] FIG. 5 shows serology data from mice that were immunized in accordance with the present invention. Serum from individual mice were collected and evaluated for binding to cell lines expressing a TAA (ENPP3), KU- 182 and HepG2. Mouse identifier number as 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90.

[0026] FIG. 6 shows primary screening data on KU812 plotted against IgG titers from sera.

[0027] FIG. 7A, FIG. 7B and FIG. 7C shows CDRH3 length distribution and VH allelic utilization. FIG. 7A) illustrates antibody yields from 2mL transient expression system comparing antibodies recovered from both DNA and protein immunogen; and FIG. 7B) illustrates antibody purity measured by aSEC post protein A purification; and FIG. 7C) illustrates hydrophobicity of kappa and lambda light chain as measured by interaction by Hydrophobic interaction chromatography (HIC) column.

[0028] FIG. 8A, FIG. 8B, and FIG 8C shows sequence diversity of antibodies generated with protein and DNA immunization. FIG. 8A shows TAA is used for analyzing for a unique sequence cluster and shows sequence diversity for DNA and Protein Immunogens, FIG. 8B shows the distribution of CDRH3 length, and FIG. 8C shows the VH / VL allele distribution, such as the distribution of VH and VK / L in DNA and protein immunization. The top row on FIG 8C shows the DNA distribution of VH alleles (left) and Distribution of VK / VL alleles (right). The bottom row on FIG. 8C shows the data for protein the left panel showing the distribution of VH alleles and the right panel showing the distribution of VK / VL alleles.

[0029] FIG. 9 shows an exemplary dendrogram depicting sequence similarity and epitope group determined by cross-competition assay.DETAILED DESCRIPTION

[0030] The disclosed methods can be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed methods are not limited to the specific methods described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods. All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth fully herein.

[0031] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0032] In an attempt to help the reader of the present application, the description has been separated in various paragraphs or sections. These separations should not be considered as disconnecting the substance of a paragraph or section from the substance of another paragraph or section. To the contrary, the present description encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated.

[0033] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0034] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.”

[0035] Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0036] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to thespecific embodiments of the application described herein. Such equivalents are intended to be encompassed by the application.

[0037] The transitional terms “comprising,” “consisting essentially of,” and “consisting of’ are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) “consisting of’ excludes any element, step, or ingredient not specified in the claim; and (iii) “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claims. Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of’ and “consisting essentially of.”

[0038] It should also be understood that the terms “about,” “approximately,” “generally,” “substantially” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred embodiment, indicate that the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

[0039] “Antigen” refers to any molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) capable ofbeing bound by an antigen binding domain or a T-cell receptor capable of mediating an immune response. Exemplary immune responses include antibody production and activation of immune cells, such as T cells, B cells or NK cells. Antigens may be expressed by genes, synthetized, or purified from biological samples such as a tissue sample, a tumor sample, a cell or a fluid with other biological components, organisms, subunits of proteins / antigens, killed or inactivated whole cells or lysates.

[0040] “Antibodies” is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies (mAbs) including murine, human, humanized and chimeric monoclonal antibodies, antigen binding fragments, multispecific antibodies, such as bispecific, trispecific, tetraspecific, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity. “Full length antibodies” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g. IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CHI, hinge, CH2 and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and the VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4.Antibody light chains of any vertebrate species may be assigned to one of two clearly distinct types, namely kappa (K) and lambda (X), based on the amino acid sequences of their constant domains.

[0041] “Fab” or “Fab fragment” refers to an antibody fragment composed of VH, CHI, VL and CL domains.

[0042] “Fv” or “Fv fragment” refers to an antibody fragment composed of the VH and the VL domains from a single arm of the antibody.

[0043] “Single chain Fv” or “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region (VL) and at least one antibody fragment comprising a heavy chain variable region (VH), wherein the VL and the VH are contiguously linked via a polypeptide linker, and capable of being expressed as a single chain polypeptide. Unless specified, as used herein, a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N- terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL. As used herein “scFv” includes stapled single chain Fv (spFv).

[0044] The term “polypeptide” is used interchangeably with the term “protein” and in its broadest sense refers to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or aprotein. “Peptide” can be used to describe a compound of two or more amino acids linked in a chain.

[0045] “Specifically binds” or “binds specifically” or derivatives thereof when used in the context of antibodies, or antibody fragments, represents binding via domains encoded by immunoglobulin genes or fragments of immunoglobulin genes to one or more epitopes of a protein of interest, without preferentially binding other molecules in a sample containing a mixed population of molecules.

[0046] As used herein, the terms nucleic acid, polynucleotide and nucleotide are interchangeable and refer to any nucleic acid, whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphoro thioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sultone linkages, and combinations of such linkages.

[0047] The terms nucleic acid, polynucleotide and nucleotide also specifically include nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

[0048] As used herein, a nucleic acid molecule is said to be “isolated” when the nucleic acid molecule is substantially separated from contaminant nucleic acid molecules encoding other polypeptides.

[0049] As used herein, the term “host” “individual,” “subject,” or “patient,” can be used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans. As used herein, the term “mammal” means a rodent (i.e., a mouse, a rat, or a guinea pig), a monkey, a cat, a dog, a cow, a llama, a horse, a pig, or a human. In some embodiments, the mammal is a human. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a rat.

[0050] “Enhancer” as described herein, is a DNA sequence that increases the transcription of a gene.

[0051] “Immnogen” as described herein is any substance that can cause an immune response, such as an antigen in a vaccine, that may trigger a host to produce antibodies.

[0052] DNA vaccines have generated significant interest due to the ability to generate both a humoral and cellular response. DNA vaccines are stable and easy to manufacture making them a cost effective and attractive therapeutic platform (Lu, B. et. al. Front. Immunol., 31 January 2024, Sec. Vaccines and Molecular Therapeutics, Volume 15 - 2024). However, despite the advantages of DNA vaccines, their effectiveness has been limited by low or lower immunogenicity and thus is not yet widely adopted as a clinically viable therapeutic modality (Khobragade et. al., The Lancet 399, 1313-1321 (2022)). Similarly, for in vivo discovery of therapeutic monoclonal antibodies immunization strategies often utilize proteins and cells as immunogen of choice successfully. However, this process can be labor and resource intensive, often with extended project timelines ultimately affecting time to impact hosts, including patients. As taught and disclosed herein are how utilizing DNA immunization can shorten timelines to generate hits, for evaluating therapeutic candidates for numerous human diseases if immunogenicity can be addressed either through use of adjuvants or route of administration.

[0053] DNA immunizations are often administered intramuscularly where most of the antigen expression occurs in muscle but where professional antigen presenting cells (APCs) such as dendritic cells (DCs) and macrophages are not present. Thus, it is thought the efficacy of DNA vaccines could be limited by the lack of sufficient APCs at the site of immunization. This is addressed by changing the route of administration to an intradermal injection site, where there is evidence of higher IFN-gamma and IL-2 production and an increase in cellular responses.

[0054] To further enhance immunogenicity, co-immunization of adjuvant is commonly utilized to recruit additional APCs to the injection site. Common adjuvant for vaccines used in human therapeutic products are aluminum-based salt, such as aluminum hydroxide and aluminum phosphate, whereas for in vivo drug discovery, adjuvants GM-CSF, Zinc Chitosan, and nanoparticles are commonly utilized. Additionally, CD4+ T helper epitopes have been explored in the vaccine field to enhance immune response and generate immunity for infectious disease and cancer.

[0055] In one such approach, “string of beads” of linking multiple epitope peptides has been reported in literature, with optimized spacers for proper processing by both HLA class I and class II receptors. In yet another approach, using CD4+ helper epitopes have been utilized to generate immune response for prophylactic protection in human diseases. However, there are limited reported uses of CD4 helper epitopes for generating therapeutic antibodies in vivo. Given DNA immunization’s durability of response, including enhanced T-cell immunity, stability, and relative ease of production of plasmid DNA, the invention optimized an in vivo platform that utilizes DNA immunization to generate diverse repertoire of antibodies with a wide range of affinities for therapeutic usage.

[0056] Numerous antigens, also called tumor associated antigen (TAA), in some instances, referring to cancer antigens, are suitable for use with the embodiments described herein, including, for example ectonucleotide pyrophosphatase / phosphodiesterase 3, part of a family of membrane proteins consisting of seven structurally related molecules. This antigen is a target in the field of oncology and is a 150 kDa, single pass Type II transmembrane protein, with an extracellular region containing nuclease-like domain, a catalytic domain, and a somatomedin B like domain. Functionally, it is known to hydrolyze ATP to AMP and isdifferentially upregulated in renal cell carcinoma making it an attractive therapeutic intervention target.

[0057] As a non-limiting example, as described herein, are screening and selection of a vaccine vector, with the usage of T-Helper epitopes called T-Helper Cassettes (THC), as a built- in adjuvant, to boost immune response and application through an intradermal delivery system to generate therapeutic antibodies against tumor associated antigen (TAA) (e.g., exemplary ENPP3 (Ecto-Nucleotide pyrophosphatase / phosphodiesterase3). The embodiments described herein includes an optimized DNA vaccine methodology, useful for generating a diverse antibody repertoire with sub nanomolar affinity and favorable developability ready for therapeutic usage similar to antibodies generated by protein immunogen.

[0058] Accordingly, the present disclosure provides novel, more efficient and improved approaches for using DNA based systems and reagents to deliver antigenic stimulation in vivo to cause immunity and or immunize the host. The embodiments disclosed herein avoids many processes involved in vaccine preparation, driving a more efficient, cost effective and sustainable strategy. Quality control and purity of plasmid DNA (or RNA) can be more easily monitored in accordance with the present invention. Various modes or formulations of delivery are included, including the use of liposomes, which provide an efficient vehicle to package, deliver and direct nucleic acid to specific targets and at the same time protect against nucleic acid degrading enzymes in body fluids and cytoplasmic organelles. In one aspect, tumor antigen is expressed together with immunostimulatory proteins on a background of normal cell(s) and tumor cells, for example. In one aspect, nucleic acid vaccination provides an opportunity for molecular immuno-physiologic manipulation of antigen expression that can be a useful tool in, for example, tailored cancer vaccine design or anti-cancer immune responses.In one aspect, the composition provided by the invention does not necessarily incorporate into hemopoietic-derived cells, as do some liposome delivery systems.

[0059] In one aspect, the embodiments described herein provides methods and compositions for immunizing against a tumor-associated antigen, suppressing or attenuating tumor growth, and treating cancer. The methods and compositions provided may induce an antibody response (IgG, IgM, IgA) or a cell-mediated immune response through CD4, CD8 or other lymphocyte subsets.

[0060] The compositions provided herein are comprised of a nucleic acid encoding immunostimulatory polypeptides and a selected antigen. In additional aspects, a method of producing the DNA is to use anionic or cationic liposomes with an inactive virus, preferably HVJ (Dzau, et al., Proc. Natl. Acad. Sci USA, vol. 93 (Oct. 1996); pp. 11421-11425 and U.S. Pat. No. 5,631,237).

[0061] Any number and combination of nucleic acid sequences encoding antigen(s), including TAAs may be included. For example, in addition to TAA ENPP3, the MAGE gene family members are examples of TAAs that would be useful in the embodiments described herein. MAGE-1, MAGE-2, and MAGE-3 antigens were first described in melanoma and subsequently demonstrated in various other cancers. MAGE-1 and MAGE-3 genes are expressed in greater than 30 percent of melanomas and carcinomas such as lung, breast, liver and gastrointestinal cancers, but not in normal tissues except testes. The MAGE-1 and MAGE- 3 antigens have been shown to be immunogenic, expressed by a wide variety of human cancers and not expressed by normal tissues. Nucleic acid sequences encoding many other TAAs will be useful in the compositions and vaccines provided by the embodiments described herein. B- catenin, TRP-2, TRP-1, gpl00 / pmell7, MART-1, GAGE-1, BAGE-1, HSP-70, gp43, HCG,Ras mutation, MUC-1, 2, and 3, PSA, p53 mutation, HMW melanoma antigen, MUC-18, HOJ- 1, tyrosinase, and carcinoembryonic antigen (CEA) are examples. In general, any antigen that is found to be associated with cancer tumors may be used (Gomella, et. al., Gerhard, et al., Zhang, et al., Nollau, et al., Mivechi, et al., Ralhan, et al., Yoshino, et al, Shirasawa, et al., Cheung, et al., Sarantou, et al., Cancer Research, 57, pp. 1371-1376, Apr. 1, 1997, Doi, et aL, Hoon, et al., Abstract 69, 3rd Annual Meeting of The Japan Society of Gene Therapy (1997), Eynde, et al., Hoon, et al. (1996), Takahashi, et al., Kawakami, et al., Proc. Nat'l. Acad. Sci. USA, vol. 91, pp. 3515-3519, Apr. 1994, Wolfel, et al., Vijayasaradhi, et al., Yokoyama, et al., Kwon, and Sensi, et al.). Multiple genes can be incorporated into the vaccine to produce a polyvalent antigen DNA adjuvant or vaccine. In one aspect, effective vaccination requires a polyvalent antigen vaccine to control human tumor progression effectively. Nucleic acids encoding these antigens can be incorporated into the vaccine provided by the embodiments described herein .

[0062] In one aspect, a drug sensitive gene can be incorporated into the DNA to turn off protein expression at any time in vivo. Examples of drugs to which genes can be sensitive are Tetracycline, Ampicillin, Gentamycin, etc.

[0063] In one aspect, additional immunogenic polypeptides or determinants, are provided, such as alternative Diphtheria toxin immunogens also may be included as additional "helper" antigen with the TAA to improve efficacy. Diphtheria toxin B fragment COOH-terminal region has been shown to be immunogenic in mice (Autran, B., et al., Immunology. 1987 Apr;60(4):531-8). HSP70, in part or in whole, as well as other immunogenic peptides, such as influenza viral or immunogenic sequences peptide with an anchoring motif to HLA class I and class II molecules, also may be included in the vaccines of the embodiments described herein.

[0064] In some aspects, the compositions may include other components to serve certain functions, for example, directing the nucleic acid to a certain location in the cell or directing transcription of the tumor-associated antigen. Compositions for transport to the nucleus may be included, particularly members of the high mobility group (HMG), more particularly HMG- 1, which is a non-histone DNA-binding protein. In combination with antisense molecules, RNAses such as RNAseH, may be used, which degrade DNA-RNA hybrids. Other proteins which will aid or enhance the function of the TAA may be included, such as peptide sequences that direct antigen processing, particularly HLA presentation, or movement in the cytoplasm.

[0065] The vaccine provided by the embodiments described herein may be administered subcutaneously, intramuscularly, intradermally, or into an organ. Administration may be directly or indirectly. The vaccine also may be injected directly into tissues. In another aspect, a tumor is administered the vaccine to enhance or induce immunity. Intramuscular injection has been shown in the past to a delivery route for induction of immunity. Skeletal muscle has properties such as high vascularization and multi-nucleation. In addition, it is nonreplicating and capable of expressing recombinant proteins. These properties are advantageous for gene therapy.

[0066] In one aspect, muscle tissue and or muscle cell(s) presents the proteins and induce immune responses such that recombinant protein is produced and released into the vascular network of the muscle and eventually presented by professional antigen-presenting cells such as dendritic cells, myoblasts, or macrophages infiltrating the muscle. In another aspect, the injection site in muscle injury induces myoblast proliferation and activation of infiltrating macrophages or dendritic-like cells, which present antigens through MHC class II antigen.Thus, other tissues which have similar qualities also would be good delivery sites for the vaccine.

[0067] In one aspect, administration depends on the vaccine composition and the disease status of hosts or patients. Considerations include the types of immune cells to be activated, the time which the antigen is exposed to the immune system and the immunization schedule. Although many vaccines are administered consecutively within a short period, spreading the immunizations over a longer time may maintain effective clinical and immunological responses.

[0068] In one aspect, a process of administration by a single injection, and in another aspect a process of administration by more than one injection, are provided. For example, in a human host injection at weeks 0, 2, 4, 8, 12, 16, and every fourth week successively for 1 year. After that, hosts are laced on a 3- to 6-month vaccine schedule for one or more years, optionally for several years. In another aspect, a preventative immunization schedule may consist of three immunizations, one every three to four weeks. Treatment after removal of a tumor may consist of immunization every week for one month. In some aspects, the method is used for prophylactic vaccination. This helps in preventing disease recurrence in hosts to improve survival and control tumor progression. The DNA compositions, and vaccination can be applied in this situation. In some embodiments, the DNA comprises a CMV Enhancer, a CMV promoter, A T-Helper Cassette, and a sequence encoding an immunogen. In some embodiments, the DNA comprises a promoter, an enhancer and a sequence encoding an immunogen. Vaccination has no major deleterious side effects corresponding to those of chemotherapy or radiation. Vaccines also may be given to high risk individuals likely to have cancer (e.g., based on congenital, family history of cancer, high frequency of nevi on the body,or other known indicators). In one aspect, a system for immunization is provided that comprises the composition of any one of the embodiments herein, wherein the system produces immune molecules having binding data according FIG. 4, the serological data according to FIG. 5, the screening data according to FIG. 6, the antibody yield according to FIG. 7 and the antibody sequence diversity according to FIG. 8. In some embodiments, the system comprises a sequence similarity and / or epitope group identity in accordance with FIG. 9.

[0069] In some embodiments, a system for immunization is provided wherein the system produces immune molecules having a binding data of about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1.0 nM, 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, about 2.0 nM, 2.1 nM, about 2.2 nM, about2.3 nM, about 2.4 nM, about 2.5 nM, about 2.6 nM, about 2.7 nM, about 2.8 nM, about 2.9 nM, about 3.0 nM, 3.1 nM, about 3.2 nM, about 3.3 nM, about 3.4 nM, about 3.5 nM, about3.6 nM, about 3.7 nM, about 3.8 nM, about 3.9 nM, about 4.0 nM, 4.1 nM, about 4.2 nM, about4.3 nM, about 4.4 nM, about 4.5 nM, about 4.6 nM, about 4.7 nM, about 4.8 nM, about 4.9 nM, about 5.0 nM, 5.1 nM, about 5.2 nM, about 5.3 nM, about 5.4 nM, about 5.5 nM, about5.6 nM, about 5.7 nM, about 5.8 nM, about 5.9 nM, about 6.0 nM 6.1 nM, about 6.2 nM, about6.3 nM, about 6.4 nM, about 6.5 nM, about 6.6 nM, about 6.7 nM, about 6.8 nM, about 6.9 nM, about 7.0 nM, 7.1 nM, about 7.2 nM, about 7.3 nM, about 7.4 nM, about 7.5 nM, about7.6 nM, about 7.7 nM, about 7.8 nM, about 7.9 nM, about 8.0 nM, 8.1 nM, about 8.2 nM, about8.3 nM, about 8.4 nM, about 8.5 nM, about 8.6 nM, about 8.7 nM, about 8.8 nM, about 8.9 nM, about 9.0 nM, 9.1 nM, about 9.2 nM, about 9.3 nM, about 9.4 nM, about 9.5 nM, about9.6 nM, about 9.7 nM, about 9.8 nM, about 9.9 nM, about 10.0 nM, 10.1 nM, about 10.2 nM,about 10.3 nM, about 10.4 nM, about 10.5 nM, about 10.6 nM, about 10.7 nM, about 10.8 nM, about 10.9 nM, about 11.0 nM, 11.1 nM, about 11.2 nM, about 11.3 nM, about 11.4 nM, about11.5 nM, about 11.6 nM, about 11.7 nM, about 11.8 nM, about 11.9 nM, about 12.0 nM, 12.1 nM, about 12.2 nM, about 12.3 nM, about 12.4 nM, about 12.5 nM, about 12.6 nM, about 12.7 nM, about 12.8 nM, about 12.9 nM, about 13.0 nM, 13.1 nM, about 13.2 nM, about 13.3 nM, about 13.4 nM, about 13.5 nM, about 13.6 nM, about 13.7 nM, about 13.8 nM, about 13.9 nM, about 14.0 nM, 14.1 nM, about 14.2 nM, about 14.3 nM, about 14.4 nM, about 14.5 nM, about14.6 nM, about 14.7 nM, about 14.8 nM, about 14.9 nM, or about 15.0 nM.

[0070] In some embodiments, the serological data indicates a high binding affinity with a Kd of about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1.0 nM, 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, about 2.0 nM, 2.1 nM, about 2.2 nM, about 2.3 nM, about 2.4 nM, about 2.5 nM, about 2.6 nM, about 2.7 nM, about 2.8 nM, about 2.9 nM, about 3.0 nM, 3.1 nM, about 3.2 nM, about3.3 nM, about 3.4 nM, about 3.5 nM, about 3.6 nM, about 3.7 nM, about 3.8 nM, about 3.9 nM, about 4.0 nM, 4.1 nM, about 4.2 nM, about 4.3 nM, about 4.4 nM, about 4.5 nM, about4.6 nM, about 4.7 nM, about 4.8 nM, about 4.9 nM, about 5.0 nM, 5.1 nM, about 5.2 nM, about5.3 nM, about 5.4 nM, about 5.5 nM, about 5.6 nM, about 5.7 nM, about 5.8 nM, about 5.9 nM, about 6.0 nM 6.1 nM, about 6.2 nM, about 6.3 nM, about 6.4 nM, about 6.5 nM, about6.6 nM, about 6.7 nM, about 6.8 nM, about 6.9 nM, about 7.0 nM, 7.1 nM, about 7.2 nM, about7.3 nM, about 7.4 nM, about 7.5 nM, about 7.6 nM, about 7.7 nM, about 7.8 nM, about 7.9 nM, about 8.0 nM, 8.1 nM, about 8.2 nM, about 8.3 nM, about 8.4 nM, about 8.5 nM, about8.6 nM, about 8.7 nM, about 8.8 nM, about 8.9 nM, about 9.0 nM, 9.1 nM, about 9.2 nM, about9.3 nM, about 9.4 nM, about 9.5 nM, about 9.6 nM, about 9.7 nM, about 9.8 nM, about 9.9 nM, about 10.0 nM, 10.1 nM, about 10.2 nM, about 10.3 nM, about 10.4 nM, about 10.5 nM, about 10.6 nM, about 10.7 nM, about 10.8 nM, about 10.9 nM, about 11.0 nM, 11.1 nM, about11.2 nM, about 11.3 nM, about 11.4 nM, about 11.5 nM, about 11.6 nM, about 11.7 nM, about11.8 nM, about 11.9 nM, about 12.0 nM, 12.1 nM, about 12.2 nM, about 12.3 nM, about 12.4 nM, about 12.5 nM, about 12.6 nM, about 12.7 nM, about 12.8 nM, about 12.9 nM, about 13.0 nM, 13.1 nM, about 13.2 nM, about 13.3 nM, about 13.4 nM, about 13.5 nM, about 13.6 nM, about 13.7 nM, about 13.8 nM, about 13.9 nM, about 14.0 nM, 14.1 nM, about 14.2 nM, about14.3 nM, about 14.4 nM, about 14.5 nM, about 14.6 nM, about 14.7 nM, about 14.8 nM, about14.9 nM, or about 15.0 nM.

[0071] In some embodiments, the screening data comprises a Signal to Background noise of about 2, equal to 2, or greater.

[0072] In some embodiments, the antibody yield is at least about 4 ug. In some embodiments, the antibody yield is about 5 ug, about 6 ug, about 7 ug, about 8 ug , about 9 ug, about 10 ug, about 11 ug , about 12 ug, about 13 ug, about 14ug , or about 15ug from about 2 ml or blood and / or sera or from expression.

[0073] In some embodiments, the system produces immune molecules having an antibody sequence diversity. In some embodiments, the system produces immune molecules having an antibody sequence diversity as shown in FIG. 8A , 8B and 8C. In some embodiments, the system produces immune molecules having the sequence similarity and or epitope group identity in accordance with FIG. 9.

[0074] Unlike most non-neoplastic diseases treated with vaccines, an actively growing cancer is in some instances viewed as a dynamic biological entity that is genetically andphenotypically continuously evolving. A tumor that is allowed to evolve genetically and phenotypically will eventually become more difficult for the host immune system to control. However, cancer vaccines may be more effective when combined with other adjuvant therapies such as chemotherapies, or other immunotherapies such as monoclonal antibodies and cytokines. A more aggressive treatment regimen approach at early stages of tumor development may be more effective in preventing the evolution of escape mechanisms. A more aggressive treatment also may be necessary for cancers that are highly malignant versus relatively benign cancers with a low risk of recurrence. In general, if the host has a weak immune response to the vaccination, then a larger dose or a more frequent vaccination should be given or alternatively immunomodulatory reagents obtained and designed based on robust immune responses are used to provide new approaches to address cancers. The embodiments described herein are suitable for use with various targets, and particularly suitable for challenging to drug targets. Included are various targets relevant to various types of cells and tissues and organs, including those associated with normal cells, undesired cells, pathological cells, tissues, etc. See for example, Y. Zhou, Y. T. Zhang, D. H. Zhao, X. Y. Yu, X. Y. Shen, Y. Zhou, S. S. Wang , Y. Q. Qiu*, Y. Z. Chen* & F. Zhu* . TTD: Therapeutic Target Database describing target druggability information. Nucleic Acids Research. 52(D1): 1465-1477 (2024). PMID: 37713619 and https: / / ttd.idrblab.cn / (database of therapeutic protein and nucleic acid targets, the targeted disease, pathway information and the corresponding drugs directed at each of these targets). Such targets are, for example highly restricted to normal, autoreactive, and or malignant tissues, making them valuable cell, tissue and organ specific markers and such targets may be in or associated with one or more plasma membrane(s) and or cytoplasm(s) of cells, such as neurons, epithelial cells, cardiac cells and the like.Furthermore, target expression can be regulated, such as hormonally regulated or otherwise, and such targets suitable for use with the present invention include targets having limited expression in certain cells, tissues and organs where such targets helps regulate biological functions, such as osmolarity, brain function, metabolism, such as lipid metabolism, and myelin regulation, for example.

[0075] In one aspect of the embodiments described herein, adjuvants are included, such as chemical adjuvants and or molecular adjuvants including nucleic acids, proteins directly applied or nucleic acid encoded. Such adjuvants include, for example aluminum salt, manganese, NPs, Oil-in-water emulsions (e.g., MF59), Poly(I:C), CpG ODNs, IL- 12, TNF-a, IFN-y, GM-CSF, RANTES (CCL5), NF-KB, MyD88, CD40, shRNA, siRNA, and the like.

[0076] All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein.EXAMPLES

[0077] The embodiments of the present disclosure is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the described embodiments are not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.

[0078] Example 1. DNA Immunogens and Immunizations.

[0079] Four epitopes to skew T helper cells, hereby called T-helper cell cassette (THC) were created. The four epitopes were comprised of vaccinia virus, tetanus toxin, diphtheria, and ovalbumin with a cathepsin cleavage site in between each epitope for enhanced antigen processing (FIG. 2). The THC was cloned upstream of gene of interest (GOI) into a pVAXl plasmid from Thermo Scientific (FIG. 1). Early immediate CMV enhancer upstream of CMV promoter were introduced to increase expression level of THC and antigen. Expression of GOI in the optimized vector was designed by transiently expressing pVAXl in HEK-293 cell line and testing for target (ENPP3) expression by flow cytometry (FIG. 3) in accordance with known protocols. As shown in FIG. 3, the overlays of the data include the transfected cells, transfected cells plus the Isotype Ab control, untransfected cells plus the primary antibody and transfected cells with the primary antibody. The transfected cells with the primary antibody shows that there is an increase of the primary antibody binding. In some examples, antibodies bound TAA (e.g., ENPP3) with high affinity (less than 1 mM) as measured by SPR (data not shown).

[0080] Example 2. Immunizations.

[0081] Mice were immunized intradermally to enhance antigen processing by antigen presenting cells (APC). Ablexis transgenic mice, for example, have been designed to produce antibodies with human variable regions and mouse constant regions, allowing bypass of time consuming humanization of therapeutic antibodies to reduce immunogenicity. Immunization was conducted with an electroporator needle into the intradermal layer of the skin, where it is processed by APCs and presented to immune cells, the B and T cells. The immunization isperformed with one or more electroporator needles. The mice were immunized and boosted every two weeks with 40ug of DNA.

[0082] Example 3. Early responses: DNA immunogen and Screening.

[0083] Mice were injected on Days 0, 14, 28, 35, 42, and 56, and immune responses were assessed by collecting serum from individual mice on Day 35 post-immunization. Immune responses to immunization were evaluated by collecting serum from 16 individual mice on Day 35 post immunization. The presence and binding of circulating antibodies were tested on cell lines expressing antigens of interest via flow cytometry in accordance with known methodologies. As shown, there was binding to KU812 and HepG2 cells. Antigen specific titer was observed in all mice immunized, indicating a robust immune response from DNA immunization (FIG. 4). Lymphocytes from immunized mice were harvested on day 60 and fused with NO mouse myeloma cells creating immortalized B -lymphocytes for in vitro production of antibodies. The supernatants from the hybridoma were screened for binding to cells with Meso Scale Discovery platform (MSD). Hybridoma that displayed >lpM binding with signal over background noise ratio were selected for variable region gene recovery (FIG. 6) in accordance with known methods.

[0084] Example 4. High throughput production of antibody from V-Gene Recovery.

[0085] The variable region (V-gene) of antibody was recovered and cloned into an expression plasmid containing the human IgGl constant domains. From DNA immunized mice 114 unique variable regions were identified, compared to 413 unique variable regions identified from mice immunized with protein (FIG. 6)

[0086] The antibodies were expressed transiently where >90% of antibodies yielded >5ug of protein from 2mL expression, with >80% purity as measured by analytical size exclusionchromatography (aSEC) (FIG. 7A and FIG. 7B). Antibodies with <5ug of yield were omitted from further characterizations.

[0087] Example 5. Characterization of antibody binders.

[0088] The antibodies recovered from both DNA and protein immunized mice were assayed for sequence diversity in silico and in vitro (FIG. 8A). More sequences were recovered from protein immunization (413 total) than DNA immunization (114 total). Surprisingly and unexpectedly, the diversity of the recoveries in terms of sequence diversity, CDRH3 length distribution and VH allelic utilization were comparable between the two immunogens (FIG. 8B and FIG. 8C).

[0089] Most of the mice immunized with protein had greater percentage of lambda (lambda light chain constant region (-57%), whereas majority of the sequences from the DNA immunization were kappa (kappa light chain (-91%), which was surprising. Interestingly, K light chain represents -90% of light chains found in clinical therapeutic antibodies.

[0090] A subset of sequences was characterized by cross-competition assay and grouped by sequence similarity. Total of eight groups were identified by cross-competition assay which correlated to sequence (CDR) similarity (see FIG. 9). From the eight groups, six of the groups included sequences generated by both protein and DNA immunogen; the remaining two groups (Group 5 and 8) included sequences from protein immunogen (Table 1). However, Group 5 only included two sequences, and Group 8 included 17 sequences, representing a small subset of epitope coverage not generated by DNA immunogen. Table 2 Table outlining representation of sequence group and type of immunogen used to generate sequence.

[0091] Table 2

[0092] To test the hydrophobicity of the antibodies from the immunization campaigns, analysis using hydrophobic interaction chromatography (HIC) columns was performed and lambda light chains were indeed found to be more hydrophobic compared to kappa light chain (Fig 7C).

[0093] Example 6. Immunization prime boosting strategy.

[0094] Sprague Dawley rats were immunized using a heterologous prime -boost strategy incorporating DNA, nanodiscs, and micelles. The regimen began with multiple DNA immunizations, followed by subsequent immunization (nanodisc boosting and / or micelle boosting). Blood samples were collected for serological analysis, and the resulting sera demonstrated a positive response in nanodisc-based MSD assays. To enhance immune cell recovery, an optional DNA boost was administered three days prior to harvesting spleen, lymph nodes, and bone marrow tissues for single B cell sorting. Thus, this technology is suitable for use with various targets, and particularly suitable for challenging drug targets.

[0095] The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

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Claims

Claims1. A composition, comprising: i. a synthetic polynucleotide wherein the synthetic polynucleotide comprises a polynucleotide encoding an ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof, or ii. a synthetic polynucleotide wherein the synthetic polynucleotide is capable of expressing ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof when introduced into a host.

2. The composition of claim 1, wherein the polynucleotide: a. is capable of expressing ovalbumin, vaccinia, diphtheria toxin and tetanus toxin peptides, or fragments thereof when introduced into a host, and b. optionally, one or more target peptide(s) or fragments thereof.

3. The composition of claim 1 or 2, further comprising one or more target peptide(s) or immunogenic fragments thereof.

4. The composition of claim 3, wherein the target peptide is a cancer target peptide, wherein optionally the cancer target peptide is on or within a cancer cell.

5. The composition of claim 4, wherein the target peptide comprises angiopoietin, BCMA, MAGE, CD19, CD20, CD22, CD25, CD33, CD37, CD38, mTOR, EGFR, ALK, VEGF, VEGFR, PDGF / PDGFR, PD1 / PD-E1, ROS1, HER2, EMP3, ENPP3, DEE3, and androgen receptor or a fragment(s) thereof.

6. A composition comprising the DNA design of FIG. 1 or equivalents thereof.

7. A system for immunization, comprising the composition of any one of claims 1-6, wherein the system produces or is capable of producing immune molecules having: i. binding in the range of O.lnM to 5nM; ii. serological data indicating high affinity binding with a Kd less than or equal to InM; iii. screening data of at least IpM binding, iv. an antibody yield equal to or greater than 5ug of antibody from of about 2 ml of expression media, sera or blood; and / or v. wherein the antibody sequences have an antibody sequence diversity.

8. The system of claim 7, having the sequence similarity and or epitope group identity from a diversity analysis, wherein clustering over CDR sequences reveals DNA and protein immunogens provide comparable coverage of target epitopes as measured by epitope binning9. The composition of claim 1 or the system of claim 7, wherein the composition or system is administered to a host.

10. The composition of claim 9, wherein the administration is oral, rectal, inhalation, nebulization, dermal, transdermal, injection (intrathecal, subcutaneous, intravenous, and or intramuscular), intraocular, intraotic, nasal, sublingual, or buccal, or any combination thereof.

11. The composition according to claim 9, wherein the administration is intradermal.

12. An antibody or fragment thereof that specifically binds a target peptide following administration of the composition of any one of claims 1 to 6 and 11.

13. A method for immunizing against a tumor-associated antigen, suppressing or attenuating tumor growth, and treating cancer, the method comprising: a. a step for inducing an antibody response using the composition of claim 1; and / or b. a step for inducing a cell mediated immune response through CD4, CD8 or other lymphocyte subsets.

14. A method of preventing disease recurrence, the method comprising: a. a step for inducing an antibody response using the composition of claim 1; and / orb. a step for inducing a cell mediated immune response through CD4, CD8 or other lymphocyte subsets.

15. A composition comprising: a. a means for encoding an ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof, or b. a means for expressing ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof when introduced into a host.

16. The composition of claim 15, wherein the means is a synthetic polynucleotide encoding an ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof.

17. The composition of claim 15, wherein the means is capable of expressing ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof when introduced into the host.

18. A means for producing the composition of any one of claims 1-6, 15 or 16.

19. A system for performing a method of immunization, the system comprising a means for encoding an ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof, or b. a means for expressing ovalbumin, vaccinia, diphtheria toxin and / or tetanus toxin peptides or fragments thereof when introduced into a host.

20. The system of claim 19, wherein the means comprises the composition of any one of claims 1-6.

21. The system of claim 19 or 20 wherein the system further comprises a means for administration.

22. A method for immunizing against a tumor-associated antigen, suppressing or attenuating tumor growth, and treating cancer; the method comprising a. a means for administering the composition of claim 1 into a host and / or b. a means for inducing a cell mediated immune response in the host.

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