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WO2017075615A1 - Virus-like particle intermediates, agents attached thereto, methods for making and uses thereof - Google Patents

Virus-like particle intermediates, agents attached thereto, methods for making and uses thereof Download PDF

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Publication number
WO2017075615A1
WO2017075615A1 PCT/US2016/059786 US2016059786W WO2017075615A1 WO 2017075615 A1 WO2017075615 A1 WO 2017075615A1 US 2016059786 W US2016059786 W US 2016059786W WO 2017075615 A1 WO2017075615 A1 WO 2017075615A1
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Prior art keywords
vlp
inad
amino acid
antibody
cell
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PCT/US2016/059786
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French (fr)
Inventor
Thomas Theriault
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Bullet Biotechnology, Inc.
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Publication of WO2017075615A1 publication Critical patent/WO2017075615A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10123—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • VLPs Virus-like particles attached or joined to therapeutics agents in order to induce an immune response sufficient for use against diseases such as cancer are known.
  • VLPs Virus-like particles attached or joined to therapeutics agents in order to induce an immune response sufficient for use against diseases such as cancer are known.
  • VLPs Virus-like particles attached or joined to therapeutics agents in order to induce an immune response sufficient for use against diseases such as cancer are known.
  • VLPs Virus-like particles attached or joined to therapeutics agents in order to induce an immune response sufficient for use against diseases such as cancer are known.
  • VLPs Virus-like particles attached or joined to therapeutics agents in order to induce an immune response sufficient for use against diseases such as cancer are known.
  • novel VLPs having particular therapeutic agents to provide better agents against disease.
  • the invention solves the problem of the art by providing novel virus-like particles (VLPs) attached or joined to agents of interest and novel methods for making them.
  • VLPs virus-like particles
  • the invention provides methods for making a population of VLP intermediates to which one or more agents of interest may be attached when desired.
  • the method comprises synthesizing virus coat or capsid proteins having at least one non-naturally occurring amino acid to which one of two binding/interacting partners of an InaD (Inactivation no afterpotential D) domain complex can attach.
  • the virus coat or capsid proteins may then be assembled into a population of VLPs. After assembly, one of two binding/interacting partners of the InaD domain complex is attached to the VLP thereby producing a population of VLP intermediates which may attach to one or more agents of interest at the site of the binding/interacting partner.
  • the invention further provides VLPs produced by the methods of the invention.
  • the invention additionally provides methods for making VLPs attached to one or more agents of interest comprising making the population of VLP intermediates by the method above and attaching to the VLP intermediates one or more agents of interest.
  • the agents of interest attaches or is fused to one of two binding/interacting partner of the InaD domain complex that recognizes and binds the other remaining binding/interacting partner on the VLP thereby resulting in VLPs attached to one or more agents of interest.
  • VLPs attached to one or more agents of interest produced by the methods of the invention.
  • compositions for treatment of cancer comprising the VLPs of the invention including pharmaceutical compositions admixed with a therapeutic agent and kits.
  • Figure 1 shows the amino acid and nucleotide sequences of a Hepatitis B core antigen (HBc) as well as nucleotide sequence of an expression plasmid, pET21 -Flep B Core 319.44.1.4, used to produce the HBc protein.
  • HBc Hepatitis B core antigen
  • FIG. 2 shows the amino acid and nucleotide sequences of the stabilized Hepatitis B core antigen (sHBc)
  • Figure 3 is a table providing exemplary molarities and ratios of components, total HepB monomer (HepB), approximate total molarity of azido groups (Azide), Pra-PEG6-TEFCA (PEG) and CpG- alkyne (CpG), in Click conjugation reaction.
  • HepB total HepB monomer
  • Azide approximate total molarity of azido groups
  • PEG Pra-PEG6-TEFCA
  • CpG- alkyne CpG
  • Figure 4 is a reducing SDS-PAGE gel analysis of adapter-CpG-VLP intermediate, obtained from reacting VLP-azide with varying amounts of CpG-alkyne and Pra-PEG6-TEFCA adapter peptide in the presence or absence of Cu(I) as catalyst.
  • Evidence of conjugation can be seen by a shift in migration to higher apparent molecular weight consistent with the combined HBc monomer+CpG oligonucleotide or FIBc monomer+adapter peptide molecular weights.
  • Figure 5 are two graphs showing dose response curve for adapter-CpG-VLP intermediates in a mTLR HEK-Blue I M assay for CpG activity. Note a general correlation between a higher CpG activity and a higher dose of adapter-CpG-VLP intermediate as well as a higher CpG activity and a greater CpG ratio or concentration used in the preparation of the adapter-CpG-VLP intermediate.
  • Figure 6 shows and SDS-PAGE gel analysis of Adapter-sVLP intermediates and subsequent docking of an InaD-scFv Id antigen to the intermediates.
  • Evidence of correct intermediate formation is seen for two different adapter-sVLPs based on the shift in migration of conjugated sI IBc monomer in lanes 6 and 7.
  • Evidence of docking of InaD-scFv Id is seen by retention of the InaD-scFv Id after docking with the sVLP.
  • Figure 7 shows the sequence of eight fusion proteins comprising a portion of an antibody of interest and a binding/interacting partner of the InaD domain complex.
  • Figure 8 shows the sequence of six fusion proteins comprising a portion of a protein antigen of interest and a binding/interacting partner of the InaD domain complex.
  • Figure 9 shows flow cytometry results for the staining of PBMCs with biotin labelled antiCD3- antiCD19-sVLP (MS-VLP). Show in clockwise order from the top left are MS-VLP stained (MS- VLP+) vs side scatter, MS-VLP co-stained with antiCD3 antibody (antiCD3+) and MS-VLP co- stained with antiCD19 antibody (antiCD19+) along with tabulation of the percentage of cells that are stained. In total 88.6 percent of all lymphocytes were stained by the MS-VLP. In the subset of either CD3 or CD19 positive cells, 91.8 percent were also stained with the MS-VLP. As expected cells that stain with the labeled MS-VLP also stain with antiCD3 and antiCD19 antibodies.
  • Figure 10 shows flow cytometry results for the staining of PBMCs with biotin labelled antiCD3- antiCD19-sVLP (MS-VLP). Show in clockwise order from the top left are MS-VLP stained (MS- VLP+) vs side scatter, MS-VLP co-stained with antiCD4 antibody (antiCD4+) and MS-VLP co- stained with antiCD20 antibody (antiCD20+) along with tabulation of the percentage of cells that are stained. In total 91.5 percent of all lymphocytes were stained by the MS-VLP. In the subset of either CD4 or CD20 positive cells, 96.2 percent were also stained with the MS-VLP. As expected cells that stain with the labeled MS-VLP also stain with antiCD4 and antiCD20 antibodies.
  • Figure 1 1 shows flow cytometry results for the staining of PBMCs with antiCD19-sVLP (aCD19- VLP+) vs side scatter, antiCD19-sVLP co-stained with antiCD4 antibody (antiCD4+) and antiCD19-sVLP co-stained with antiCD20 antibody (antiCD20+). As expected, cells that are stained by the antiCD19-sVLP are co-stained by antiCD20 but not antiCD4.
  • Figure 12 shows the amino acid and nucleotide sequences of a wild-type Hepatitis B core antigen (HBc).
  • Figure 13 shows the amino acid and nucleotide sequences of an InaD template.
  • Vaccine as used herein, is a preparation comprising a virus-like particle (VLP) of the invention that when administered stimulates an immune response in a subject.
  • VLP virus-like particle
  • a therapeutic vaccine may be administered during or after onset of a disease or disorder.
  • a prophylactic treatment vaccine may be administered prior to onset of the disease such as a cancer and is intended to prevent, inhibit or delay onset of the disease.
  • VLP as used herein is a virus-like particle made from non-infectious subunits of a virus that form a structure, commonly in the form of an icosahedral matrix. VLP lacks a viral genome required for producing more infectious particles.
  • the VLP may contain an assemblage of capsid protein monomers/subunits, for example, about a multiple of 60 coat or capsid protein monomers/subunits.
  • HBV core protein monomers also referred to herein as viral coat polypeptides
  • the invention provides in one embodiment a HBV coat protein truncated at the C-terminus leaving intact the first 149 amino acid at the N-terminus (aa 1 -149), and the HepB Core VLP is formed by the assembly of, e.g., 180 or 240 C-terminally truncated HepB core proteins.
  • Capsid protein participates in the formation of a capsid, which generally form a helical or icosahedral structure. Capsid proteins participate in forming a protective shell around the genome of a virus particle. Throughout the application, a “capsid protein” may be used interchangeably with a “coat protein.” In the case of hepatitis B virus (HBV), its “capsid protein” is often referred to as HBV core (HBc) protein. As used herein, HBc, HBC, HepB core, and HBV core are used interchangeably to refer to the Hepatitis B virus core protein monomer or variations thereof.
  • VLP-azide refers to the presence of at least one azide functional group in VLP, such as through the incorporation of a non-natural amino acid with an azide functional group, e.g., azidohomoalanine.
  • Azidohomoalanine may be used to substitute for methionine in a polypeptide chain in vivo by supplying azidohomoalanine to a methionine auxotroph grown in methionine- deficient medium.
  • azidohomoalanine may be introduced in vitro synthesis using a cell-free protein synthesis (CFPS) system.
  • CFPS cell-free protein synthesis
  • an azide functional group permits participation in copper-catalyzed [3+2] cycloaddition or "click chemistry" with an alkyne function group.
  • Other non-natural amino acids with an azide function group including -azido-L- phenylalanine, are known in the art and are available and may be introduced into a polypeptide including a capsid protein, either intact or truncated, which can form a VLP.
  • VLP-alkyne refers to the presence of at least one alkyne functional group in VLP, such as through the incorporation of a non-natural amino acid with an alkyne functional group, e.g., by supplying homopropargylglycine as a partial or complete substitute for methionine while expressing VLP in a methionine auxotroph strain, thus replacing methionine with the alkyne- containing non-natural amino acid.
  • a non-natural amino acid may also be incorporated into a polypeptide at a desired site through the introduction of stop codon, e.g., amber stop codon UAG, and use of a suppressor tRNA charged with the desired non-natural amino acid, e.g., ?-propargyloxyphenylalanine, permitting site-specific incorporation of a non-natural amino acid through suppression of an engineered stop codon in a RNA transcript encoding a specific polypeptide (Bundy and Swartz, Bioconjugate Chem. 21 :255-263 (2010)). In either case, presence of an alkyne functional group permits participation in copper-catalyzed [3+2] cycloaddition or "click chemistry" with an azide functional group.
  • stop codon e.g., amber stop codon UAG
  • a suppressor tRNA charged with the desired non-natural amino acid e.g., ?-propargyloxyphenylalanine
  • Immune checkpoint inhibitors refers to agents that block immune checkpoint proteins.
  • Immune checkpoint proteins involve or are associated with inhibitory pathways present in immune cells important for maintaining self-tolerance and controlling the degree of an immune response. Blocking these pathways may lead to reduced modulation of immune cells, or increased activation of immune cells.
  • vector refers to a recombinant nucleic acid molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the coding sequence in a particular host organism.
  • Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences.
  • Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • a "vector,” “construct” or “plasmid” may also be used outside the context of a particular host organism, such as in a cell free protein synthesis system following production RNA transcripts or in an in vitro transcription- translation system.
  • an "active ingredient” includes any compound or composition of matter which, when administered to an organism (human or animal subject) induces a desired pharmacologic and/or physiologic effect by local and/or systemic action.
  • a "subject” means a mammal.
  • the mammal can be a human or an animal such as a non-human primate, mouse, rat, dog, cat, horse, monkey, ape, rabbit or cow, but are not limited to these examples.
  • Mammals, other than humans can be advantageously used as subjects that represent animal models of disorders associated with, e.g., cancer.
  • the methods and compositions described herein can be used to treat domesticated animals and/or pets.
  • the terms, "patient” and “subject” are used interchangeably.
  • a subject can be male or female.
  • the VLP vaccines of the invention may be administered in the form of a pharmaceutical composition comprising the active ingredient in a pharmaceutically acceptable dosage form.
  • the compositions may be administered at varying doses. Administration may be by methods including, but not limited to, intratumoral delivery, peritumoral delivery, intraperitoneal delivery, intrathecal delivery, intramuscular injection, subcutaneous injection, intravenous delivery, nasal spray and other mucosal delivery (e.g. transmucosal delivery), intra-arterial delivery, intraventricular delivery, intrasternal delivery, intracranial delivery, intradermal injection, electroincorporation (e.g., with electroporation), ultrasound, jet injector, and topical patches.
  • intratumoral delivery peritumoral delivery, intraperitoneal delivery, intrathecal delivery, intramuscular injection, subcutaneous injection, intravenous delivery, nasal spray and other mucosal delivery (e.g. transmucosal delivery), intra-arterial delivery, intraventricular delivery, intrasternal delivery, intracranial delivery, intradermal
  • Formulations suitable for administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • an administration protocol useful for the invention comprises multiple administrations of the multivalent VLP vaccine of the invention during an initial period (such as, for example, a six week period, with, for example, administration every two weeks).
  • an administration protocol may also include multiple administrations of the multivalent VLP vaccine of the invention at first administration (such as at multiple sites within a tumor at first administration of the multivalent VLP vaccine).
  • an effective amount as used herein with respect to a VLP vaccine of the invention, is meant an amount of the multivalent VLP, administered to a subject that results in an immune response by the mammal so as to inhibit the disease such as cancer. Further, an effective amount may include any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.
  • inhibiting a tumor may be measured in any way as is known and accepted in the art, including complete regression of the tumor(s) (complete response); reduction in size or volume of the tumor(s) or even a slowing in a previously observed growth of a tumor(s), e.g., at least about a 10-30% decrease in the sum of the longest diameter (LD) of a tumor, taking as reference the baseline sum LD (partial response); mixed response (regression or stabilization of some tumors but not others); or no apparent growth or progression of tumor(s) or neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease, taking as reference the smallest sum LD since the treatment started (stable disease).
  • complete regression of the tumor(s) complete response
  • reduction in size or volume of the tumor(s) or even a slowing in a previously observed growth of a tumor(s) e.g., at least about a 10-30% decrease in the sum of the longest diameter (LD) of a tumor, taking as reference the baseline sum LD (par
  • Tumor or cancer status may also be assessed by sampling for the number, concentration or density of tumor or cancer cells, alone or with respect to a reference. Tumor or cancer status may also be assessed through the use of surrogate marker(s), such as Her-2 in breast cancer or PSA in prostate cancer.
  • surrogate marker(s) such as Her-2 in breast cancer or PSA in prostate cancer.
  • treating means using a therapy to ameliorate a disease or disorder or one or more of the biological manifestations of the disease or disorder; to directly or indirectly interfere with (a) one or more points in the biological cascade that leads to, or is responsible for, the disease or disorder or (b) one or more of the biological manifestations of the disease or disorder; to alleviate one or more of the symptoms, effects or side effects associated with the disease or disorder or one or more of the symptoms or disorder or treatment thereof; or to slow the progression of the disease or disorder or one or more of the biological manifestations of the disease or disorder.
  • Treatment includes eliciting a clinically significant response.
  • Treatment may also include improving quality of life for a subject afflicted with the disease or disorder (e.g., a subject afflicted with a cancer may receive a lower dose of an anti-cancer drug that cause side-effects when the subject is immunized with a composition of the invention described herein).
  • a subject afflicted with a cancer may receive a lower dose of an anti-cancer drug that cause side-effects when the subject is immunized with a composition of the invention described herein.
  • compositions of the invention and methods for the use thereof are provided and are chosen to provide suitable treatment for subjects in need thereof.
  • treatment with a composition of the invention described herein induces and/or sustains an immune response in a subject.
  • Immune responses include innate immune response, adaptive immune response, or both.
  • Innate immune response may be mediated by neutrophils, macrophages, natural killer cells (NK cells), and/or dendritic cells.
  • Adaptive immune response includes humoral responses (i.e., the production of antibodies), cellular responses (i.e., proliferation and stimulation of T-lymphocytes), or both.
  • Measurement of activation and duration of cellular response may be by any known methods including, for example, cytotoxic T- lymphocyte (CTL) assays.
  • Humoral responses may be also measured by known methods including isolation and quantitation of antibody titers specific to the compositions of the invention (e.g., vaccines) such as IgG or IgM antibody fractions.
  • the methods of treatment e.g., immunotherapy
  • the methods of treatment is used as a stand-alone therapy without combining with any other therapy.
  • the methods of treatment (e.g., immunotherapy) described herein provide adjunct therapy to other therapies, e.g., cancer therapy, prescribed for a subject.
  • the methods of treatment (e.g., immunotherapy) described herein may be administered in combination with radiotherapy, chemotherapy, gene therapy or surgery. The combination is such that the method of treatment (e.g., immunotherapy) described herein may be administered prior to, with or following adjunct therapy.
  • the effect of anti-disease or disorder treatment may be assessed by monitoring the patient, e.g., by measuring and comparing survival time or time to disease progression (disease-free survival). Any assessment of response may be compared to individuals who did not receive the treatment or were treated with a placebo, or to individuals who received an alternative treatment.
  • preventing is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation.
  • prevention is not an absolute term.
  • Prophylactic therapy is appropriate, for example, when a subject is considered at high risk for developing a particular disease or disorder (e.g., cancer), such as when a subject has a strong family history of a disease or disorder or when a subject has been exposed to e.g., a disease causing agent, e.g., a carcinogen.
  • the invention provides methods for making a population of VLP intermediates to which one or more agents of interest may attach when desired.
  • the method comprises synthesizing virus coat or capsid proteins having at least one non-naturally occurring amino acid to which one of two binding/interacting partners of an InaD domain complex or equivalent can attach.
  • the virus coat or capsid proteins may then be assembled into a population of VLPs.
  • one of two binding/interacting partners of the InaD domain complex or equivalent e.g. any PDZ domain complex (any PDZ domain with its binding partner) including those described herein
  • the invention further provides VLPs produced by the methods of the invention.
  • the InaD domain complex or equivalent contains two binding/interacting partners.
  • One binding/interacting partner may be an InaD domain or fragment thereof comprising a domain of InaD protein.
  • the second remaining binding/interacting partner may be a polypeptide or a peptide comprising a sequence TEFCA.
  • the domain of InaD protein may be PDZ1 (PSD-95, DiscsLarge, ZO l ) domain or fragment, mutant, variant or derivative thereof.
  • one of the two binding/interacting partners of the the InaD domain complex or equivalent may comprise a non-naturally occurring amino acid.
  • an InaD domain may comprise a non-naturally occurring amino acid which can be used to attach to the assembled VLP.
  • the polypeptide or peptide comprising a TEFCA sequence may comprise a non-naturally occurring amino acid which can be used to attach the InaD domain to the assembled VLP.
  • the unnatural amino acid may include an alkyne or azide reactant group for, e.g., Click chemistry reactions.
  • the unnatural amino acid having an azide reactant group may be a L-azidohomoalanine or £>-azido-L-phenylalanine.
  • the unnatural amino acid having an alkyne reactant group may be a L-homopropargylglycine or p- propargyloxyphenylalanine.
  • the unnatural amino acid may be a N6- ((2-propynyloxy)carbonyl)-L-lysine or /?-acetyl-L-phenylalanine.
  • the virus capsid polypeptides of the VLP may be modified before assembly to comprise at least one first unnatural amino acid (also referred to herein as non- natural amino acid or non-canonical amino acid (nnAA)) at a site of interest, such as the incorporation of azidohomoalanme during virus capsid polypeptide synthesis in the place of methionine, and the partner attached to an alkyne functional group, such as L-propargylglycine, L-propargylphenylalanine or L-propoargylalanine at the N-terminus of the partner, e.g., adapter peptide terminating with TEFCA sequence, or alternatively N- or C-terminus or surface exposed positions of a PDZ domain so as not to interfere with peptide binding, such as N-terminal PDZ domain from Drosophila inactivation no after
  • first unnatural amino acid also referred to herein as non- natural amino acid or non-canonical amino acid (nnAA)
  • the azide functional group of azidohomoalanine incorporated into a capsid protein of a VLP may participate in a (3+2) cycloaddition click reaction with an alkyne functional group of partner-X, resulting in VLP crosslinked to the binding/interacting partner.
  • Other unnatural amino acid-containing capsid proteins within the same VLP may similarly participate in the (3+2) cycloaddition click reaction to produce a VLP attached or joined to a partner, producing a VLP with two or more partners.
  • the VLP contains at least one or at least two unnatural amino acid per capsid monomer subunit (also referred to herein as a virus coat protein).
  • a virus coat protein also referred to herein as a virus coat protein.
  • at least one-fiftieth of the total number of unnatural amino acids in a VLP may be used to attach a partner.
  • one-twentieth of the total number of unnatural amino acids in a VLP may be used to attach a partner.
  • one-tenth of the total number of unnatural amino acids in a VLP may be used to attach a partner.
  • about one fourth of the total number of unnatural amino acids in a VLP may be used to attach a partner.
  • about one-third of the total number of unnatural amino acids in a VLP may be used to attach a partner.
  • about one half of the total number of unnatural amino acids in a VLP may be used to attach a partner.
  • about two-thirds of the total number of unnatural amino acids in a VLP may be used to attach a partner.
  • about four-fifths of the total number of unnatural amino acids in a VLP may be used to attach a partner.
  • At least one-twenty fifth of the viral coat proteins may display a partner attached thereto.
  • at least one-tenth of the viral coat proteins may display a partner.
  • at least one-fifth of the viral coat proteins may display a partner.
  • about half of the viral coat proteins may display a partner.
  • about two-thirds of the viral coat proteins may display a partner.
  • nearly all of the viral coat proteins may display a partner.
  • the invention additionally provides methods for making VLPs attached to one or more agents of interest comprising making the population of VLP intermediates by the method above and attaching to the VLP intermediates one or more agents of interest.
  • the agents of interest may attach or may fuse to one of two binding/interacting partner of the InaD domain complex or equivalent.
  • an agent of interest may be attached or may be fused to a binding/interacting partner that recognizes and binds the other remaining binding/interacting partner which is on the VLP thereby resulting in a complex that generates VLPs attached to one or more agents of interest.
  • VLPs which are attached to one or more agents of interest and produced by the methods of the invention.
  • one binding/interacting partner of the InaD domain complex or equivalent binds the other remaining binding/interacting partner on the VLP intermediates so that an InaD domain complex or equivalent is formed.
  • an agent of interest may bind an InaD domain of the InaD protein.
  • an agent of interest may bind a polypeptide or peptide comprising the TEFCA sequence. Either configuration is contemplated. In either configuration, an InaD domain complex or equivalent is formed which permits the agent of interest to attach to the VLP, either directly or indirectly.
  • a InaD domain complex or equivalent is formed when the InaD domain of InaD protein binds the carboxyl terminus of a polypeptide or peptide comprising a TEFCA sequence.
  • the polypeptide or peptide TEFCA sequence of the InaD domain complex may be directly attached to the VLP.
  • the InaD domain of the InaD domain complex or equivalent may be directly attached to the VLP.
  • either one of the binding/interacting partners of the InaD domain complex or equivalent may be attached to a spike region of the VLP.
  • the spike region of the VLP may be the spike tip. But other portions of the spike region may be the attachment site.
  • the InaD domain complex or equivalent may additionally comprise a covalent bond formed between the binding/interacting partners of the InaD domain complex or equivalent.
  • the covalent bond may be a disulfide bond formed between two cysteine residues.
  • the disulfide bond may be formed under oxidizing condition permissive for the formation of a disulfide bond between two nearby cysteine residues.
  • the agent of interest may be joined to a binding/interacting partner as a fusion protein.
  • the agent of interest may be chemically joined to a binding/interacting partner of the InaD domain complex or equivalent.
  • Examples of an average amount of the agents of interest attached to the VLP is in an amount equivalent to about 10 to 50 copies per VLP, about 40 to 80 copies per VLP, about 70 to 170 copies per VLP, about 100 copies per VLP or about 160 to 240 copies per VLP.
  • the agent of interest may an immunostimulatory oligonucleotide comprising an unmethylated cytosine.
  • the immunostimulatory oligonucleotide comprising an unmethylated cytosines e.g., CpG oligonucleotide comprising a CpG dinucleotide with an unmethylated cytosine
  • the immunostimulatory oligonucleotide comprising an unmethylated cytosines may be attached to the VLP using the InaD domain complex or equivalent, described herein.
  • the immunostimulatory oligonucleotide comprising an unmethylated cytosines may be attached to the VLP using, other means, e.g., as described herein in Example 2, where, for example, an alkyne reactive group of 5-octadiynyl dU attached to an end of a CpG oligonucleotide and an azide reactive group of a non-naturally occurring amino acid of the VLP participate in a Click chemistry reaction to yield a VLP attached to an immunostimulatory oligonucleotide.
  • CpG oligonucleotide unmethylated cytosines
  • the agents of interest on the VLP may be a tumor associated antigen and/or an immunostimulatory oligonucleotide comprising an unmethylated cytosine.
  • the agents of interest on the VLP are an antibody or fragment or derivative therewith which binds a checkpoint protein and/or an immunostimulatory oligonucleotide comprising an unmethylated cytosine.
  • the agents of interest on the VLP are an Id antigen and/or an immunostimulatory oligonucleotide comprising an unmethylated cytosine.
  • the unmethylated cytosine may be a CpG oligonucleotide attached to a VLP intermediate in an amount (molar) such that the CpG oligonucleotide to VLP monomer ratio is equivalent to 1 :24 to 1 : 12, 1 : 12 to 1 :6, 1 :6 to 1 :3, 1 :3 to 2:3 or 1 :2 to 1 : 1 .
  • the CpG oligonucleotide so attached to the VLP may be in an average amount equivalent to 10 to 50 copies per VLP, 40 to 80 copies per VLP, 70 to 170 copies per VLP, or 160 to 240 copies per VLP.
  • the CpG oligonucleotide comprises a sequence, 5' - TGACTGTGAACGTTCGAGATGA-3 ' .
  • the sequence has phosphorothioate bonds as shown in 5 > T*G*A*C*T*G*T*G*A*A*C*G*T*T*C*G*A*T*G*A 3 ', where * represents replacement of a phosphodiester bond with a phosphorothioate bond.
  • the CpG oligonucleotide further comprises a 5-octadiynyl deoxyuridine or a modified deoxyuridine or a linker at the 3 ' end of the sequence.
  • the binding/interactive partner of an InaD domain complex or equivalent attached to VLP may be in an amount such that the partner to VLP weight ratio is equivalent to 1 : 1000 to 1 : 100, 1 : 100 to 1 : 10, 1 : 10 to 1 :4, 1 :4 to 1 :2 or 1 :2 to 1 : 1 .
  • the partner attached to a VLP is in an amount (molar) such that the partner to VLP ratio is equivalent to 1 :24 to 1 : 12, 1 : 12 to 1 :6, 1 :6 to 1 :3, 1 :3 to 2:3 or 1 :2 to 1 : 1.
  • agents of interest include, but are not limited to, an isolated antibody that specifically recognizes and binds a leucocyte antigen; an isolated antibody that specifically recognizes and binds a cell adhesion molecule; an isolated antibody that specifically recognizes and binds a prostate specific membrane antigen; or an isolated antibody that specifically recognizes and binds an oncogene.
  • the isolated antibody may be an isolated or purified monoclonal antibody.
  • the antibody or antigen-binding fragment is a labeled antibody, a bivalent antibody, a polyclonal antibody, a bispecific antibody, a chimeric antibody, a recombinant antibody, an anti-idiotypic antibody, a humanized antibody, or an affinity matured antibody.
  • the antigen-binding fragment is a camelized single domain antibody, a diabody, an scfv, an scfv dimer, a dsfv, a (dsfv) 2 , a dsFv-dsfv', a bispecific ds diabody, a Fv, a Fab, a Fab', a F(ab') 2 , or a domain antibody.
  • the antigen-binding fragment is operably attached to a constant region, wherein the constant region is a kappa light chain, gamma- 1 heavy chain, gamma-2 heavy chain, gamma- 3 heavy chain or gamma-4 heavy chain.
  • An example of a leucocyte antigen includes, but is not limited to, an immune checkpoint protein.
  • immune checkpoint proteins include, but are not limited to, a PD-1 , a PDL1 , a PDL2, a B7-H3, a B7-H4, a CTLA-4, a LAG3, a KIR, a TIM3, a TIGIT, a BTLA, a CD 160, an A2aR, and/or a VISTA protein(s).
  • leucocyte antigens include, but are not limited to, CD1 , CD2, CD3, CD4, CD5, CD6, CD6L, CD7, CD8, CD9, CD10, CD1 1/CD18, CDl l a, CDl l b, CDl lc, CDl ld, CDwl 2, CD13, CD14, CD15, CD15s, CD16, CDwl 7, CD18, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD26, CD27, CD27L, CD28, CD29, CD30, CD30L, CD31 , CD32, Fc Gamma Receptor, CD33, CD34, CD35, CD36, CD37, CD38, CD40, CD40L, CD41 , CD42, CD42a, CD43b, CD43c, CD43, CD44, CD45, CD45-AP, CD46, CD48, CD49, CD49a, CD49b, CD49c, CD49d, CD49e,
  • An example of a prostate specific membrane antigen includes, but is not limited to, a FOHL1.
  • oncogenes include, but are not limited to, HER2/ErbB-2/Neu, HER3/ErbB-3, HER4/ErbB-4, and EGFR/ErbB-1 or portion thereof.
  • An example of a cell adhesion molecule includes, but is not limited to, an EPCAM.
  • the invention provides specific embodiments such as a VLP which includes antibodies or portion thereof that bind HER2 and antibodies or portion thereof that bind CD3; a VLP which includes antibodies or portion thereof that bind CD 19 and antibodies or portion thereof that bindsCD3; a VLP which includes antibodies or portion thereof that bind EPCAM and antibodies or portion thereof that bind CD3; a VLP which includes antibodies or portion thereof that bind FOLHl and antibodies or portion thereof that bind CD3; a VLP which includes antibodies or portion thereof that bind FIER2 and antibodies or portion thereof that bind FcGR; and a VLP which includes antibodies or portion thereof that binds CD20 and a second antibody or portion thereof that binds CD3.
  • a VLP which includes antibodies or portion thereof that bind HER2 and antibodies or portion thereof that bind CD3
  • a VLP which includes antibodies or portion thereof that bind CD 19 and antibodies or portion thereof that bindsCD3
  • a VLP which includes antibodies or portion thereof that bind EPCAM and antibodies or portion thereof that
  • the VLP may further comprise an additional agent of interest, e.g., CpG, antibodies or portion thereof that binds a checkpoint inhibitor, or both.
  • additional agent of interest e.g., CpG
  • Suitable average amounts of an antibody attached to a VLP may be an average amount equivalent to about 10 to 50 copies of an antibody per VLP, about 40 to 80 copies per VLP, about 70 to 170 copies per VLP, about 100 copies per VLP or about 160 to 240 copies per VLP.
  • the agent of interest so attached to the VLP may be a tumor associated antigen.
  • tumor-associated antigens include, but are not limited to, 17- 1 A, 707-AP, AFP, Annexin II, ART-4, BAGE, BAGE- 1 , b- catenin, BCG, bcr/abl, Bcr/abl el4a2 fusion junction, bcr-abl (polypeptide from translation of b3a2 transcript), bcr-abl (polypeptide from translation of b2a2 transcript), bcr-abl p210 (polypeptide from translation of b2a2 transcript), bcr-abl p210 (polypeptide from translation of b3a2 transcript), bullous pemphigoid antigen- 1 , CA 19-9, CA 125, CA21 5, CAG-3 cancer peptide, CAMEL tumor antigen, Cancer-testis antigen, Caspase-8, CCL3, CCL4,
  • the antibodies against immune checkpoint inhibitors may be modified to comprise at least one second unnatural amino acid, wherein the first unnatural amino acid is different from, and reactive with the second unnatural amino acid.
  • An example of one first unnatural amino acid is azidohomoalanine.
  • An example of a second unnatural amino acid is propargyloxyphenylalanine.
  • the azide functional group of azidohomoalanine incorporated into a capsid protein of a VLP may participate in a (3+2) cycloaddition click reaction with an alkyne functional group of propargyloxyphenylalanine incorporated into a polypeptide agent, such as a polypeptide-based antibody against immune checkpoint inhibitor, resulting in VLP crosslinked to a polypeptide agent.
  • a polypeptide agent such as a polypeptide-based antibody against immune checkpoint inhibitor
  • Other unnatural amino acid-containing capsid proteins within the same VLP may similarly participate in the (3+2) cycloaddition click reaction to produce a VLP attached or joined to an antibody against immune check point inhibitors, producing A VLP with two or more antibodies against immune check point inhibitors.
  • a therapeutic agent includes but is not limited to an agent that suppresses Treg activity.
  • a therapeutic agent is an agent of interest as used herein.
  • An example of a Treg suppressor includes agents that stimulate GITR (e.g., a GITR agonist), or a ligand, or a mimic of a ligand thereof.
  • the agent may be an isolated antibody or fragment or derivative thereof that stimulates the target receptor (e.g., GITR).
  • An example antibody is TRX-518.
  • Another example protein is GITR-L.
  • the agent may be a small molecule that stimulates the target receptor.
  • Treg depleting agents include agents that induce cell death in Treg cells (e.g., binding to a surface antigen on Treg cells (e.g., FR4, CD4, CD25 (IL-2Ra), CD 127 (IL7Ra), CD45RA, CD45RO, CD39, CD73, GITR, CD101 , GARP)) causing ADCC cytotoxicity (e.g., antibodies that mediate ADCC (antibody-dependent cell-mediated cytotoxicity)), CDC (complement-dependent cytotoxicity), or mediate cell death through other effector functions.
  • examples of Treg depleting agents include agents that induce PCD (programmed cell-death).
  • the agent may be an antibody or fragment or derivative thereof that induces cell death. Further, the agent may be a small molecule that induces cell death.
  • a therapeutic agent includes but is not limited to an agent (such as an antibody or small molecule) that binds to a tumor necrosis factor superfamily receptor (TNFRSFR) or ligand (TNFRSFRL).
  • agents such as an antibody or fragment or derivative thereof or small molecule
  • TNFRSFR or a ligand e.g., CD 137 agonist, an NGFR agonist, a BAFFR agonist, an Osteoprotegerin agonist, a BCMA agonist, a OX40 agonist, a CD27 agonist, a RANK agonist, a CD30 agonist, a RELT agonist, a CD40 agonist, a TACI agonist, a DcR3 agonist, a TNF RI agonist, a DcTRAIL Rl agonist, a TNF agonist, a DcTRAIL R2 agonist, a TRAIL Rl agonist, a DR3 agonist, a TRACI agonist, a
  • Examples also include inhibitors of a TNFRSFR or ligand thereof (e.g., CD137 antagonist, an NGFR antagonist, a BAFFR antagonist, an Osteoprotegerin antagonist, a BCMA antagonist, an OX40 antagonist, a CD27 antagonist, a RANK antagonist, a CD30 antagonist, a RELT antagonist, a CD40 antagonist, a TACI antagonist, a DcR3 antagonist, a TNF RI antagonist, a DcTRAIL Rl antagonist, a TNF antagonist, a DcTRAIL R2 antagonist, a TRAIL Rl antagonist, a DR3 antagonist, a TRAIL R2 antagonist, a DR6 antagonist, a TRAIL R3 antagonist, a EDAR antagonist, a TRAIL R4 antagonist, a Fas antagonist, a TROY antagonist, a GITR antagonist, a TWEAK R antagonist, a HVEM antagonist, a XEDAR antagonist, a Lymphotoxin beta receptor antagonist,
  • the therapeutic agent may be an anti-cancer agent that inhibits cell proliferation or induces apoptosis.
  • therapeutic agents include, but are not limited to, lenalidomide; ipilimumab; rituximab; alemtuzumab; ofatumumab; flavopiridol; Adriamycin; Dactinomycin; Bleomycin; Vinblastine; Cisplatin; ABT-199; acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; amino glutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride;
  • the therapeutic agent may be an alkylating agent which may be nitrogen mustards, ethylenimine and methylmelamines, alkyl sulfonates, nitrosoureas, or triazenes.
  • alkylating agent may be nitrogen mustards, ethylenimine and methylmelamines, alkyl sulfonates, nitrosoureas, or triazenes.
  • VLP with reactive azide functional groups could be attached (e.g., coupled) to other non-proteinaceous or non-nucleic acid-based therapeutic agents, such as antagonist ligands or inhibitors, including small molecule inhibitors, of immune checkpoint proteins which are not protein or nucleic acid, through functionalizing these agents with an alkyne functional group.
  • Such non-proteinaceous or non-nucleic acid-based agents may be attached to a VLP through the (3+2) cycloaddition click reaction to produce a VLP attached or joined to non- proteinaceous or non-nucleic acid-based agents.
  • the VLPs of the invention are VLPs free of a viral genome which may comprise virus capsid polypeptides derived from any of an Adenoviridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papillomaviridae, Rhabdoviridae, Togaviridae or Paroviridae families.
  • the VLP is a stable icosahedral VLP free of a viral genome.
  • viruses from which the virus coat or capsid proteins may be derived include but are not limited to any of a bacteriophage, adenovirus, coxsackievirus, Hepatitis A virus, poliovirus, Rhinovirus, Herpes simplex virus, Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, Human herpes virus, Hepatitis B virus, Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, HIV, Influenza virus, Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human metapneumovirus, Human papillomavirus, Rabies virus, Rubella virus, Human bocavirus or Parvovirus, and Norovirus.
  • the bacteriophage may be a MS2 bacteriophage, PI like viruses, P2 like viruses, T4 like viruses, P22 like viruses, and lambda-like viruses.
  • a VLP derived from Hepatitis B virus is preferred.
  • the invention additionally provides pharmaceutical composition for treatment of cancer comprising the VLPs of the invention, e.g., the VLP intermediates (not yet attached to an agent of interest) or a VLP attached to one or more agents of interest.
  • the pharmaceutical composition further comprises a therapeutic agent admixed therein.
  • the invention additionally provides for a pharmaceutical composition for treatment of a solid tumor or cancer comprising any of the VLP compositions of the invention and one or more therapeutic agents that may be admixed with the VLP compositions or, alternatively, the therapeutic agent(s) may administered before or after the VLP compositions of the invention.
  • a pharmaceutical composition for treatment of a solid tumor or cancer comprising any of the VLP compositions of the invention and one or more therapeutic agents that may be admixed with the VLP compositions or, alternatively, the therapeutic agent(s) may administered before or after the VLP compositions of the invention.
  • the second therapeutic agent may be the same as the therapeutic agent admixed or sequentially administered or the second therapeutic agent may be a different therapeutic agent.
  • VLP compositions of the invention and “VLPs of the invention” include a VLP intermediate (including a VLP intermediate attached to one or more agents of interest but free of an InaD complex or equivalent, such as a VLP intermediate attached to a CpG oligonucleotide free of an InaD complex or a VLP intermediate attached to a TLR agonist free of an InaD complex), a VLP free of a viral genome comprising capsid proteins or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, compositions containing them (e.g., pharmaceutical compositions, dosage forms, immunostimulatory compositions, immunomodulatory compositions,, vaccines); and VLPs having two of more agents of interest attached thereto through means other than by the InaD domain complex or equivalent thereof (free of InaD domain complex or equivalent).
  • VLP compositions of the invention and “VLPs of the invention” may be used interchangeably.
  • therapeutic agents include, but are not limited to, an agent that inhibits an immune checkpoint protein (also referred to herein as an immune checkpoint inhibitor).
  • immune checkpoint inhibitors include agents that inhibit PD- 1 (e.g., a PD-1 inhibitor or an anti- PD- 1 agent); CTLA-4 (e.g., a CTLA-4 inhibitor or an anti-CTLA-4 agent); LAG3 (e.g., a LAG 3 inhibitor or an anti-LAG3 agent); KIR (e.g., a KIR inhibitor or an anti-KIR agent); TIM3 (e.g., an ⁇ 3 inhibitor or an anti-TIM3 agent); TIGIT (e.g., a TIGIT inhibitor or an anti-TIGIT agent); BTLA (e.g., a BTLA inhibitor or an anti-BTLA agent); CD160 (e.g., a CD160 inhibitor or an anti- CD 160 agent); VISTA (e.g.
  • the immune checkpoint inhibitor may inhibit a ligand of a checkpoint receptor, examples of which would include PDL 1 (e.g., a PDL 1 inhibitor or an anti-PDLl agent), PDL2 (e.g., a PDL2 inhibitor or an anti-PDL2 agent), B7-IT3 (e.g., a B7- H3 inhibitor or an anti-B7I I3 agent); B7-LI4 (e.g., a B7-H4 inhibitor or an anti-B7-H4 agent).
  • PDL 1 e.g., a PDL 1 inhibitor or an anti-PDLl agent
  • PDL2 e.g., a PDL2 inhibitor or an anti-PDL2 agent
  • B7-IT3 e.g., a B7- H3 inhibitor or an anti-B7I I3 agent
  • B7-LI4 e.g., a B7-H4 inhibitor or an anti-B7-H4 agent.
  • the agent may be an isolated antibody or fragment or derivative thereof that blocks the target receptor (e.g., PD- 1 , B7-H3, B7-H4, CTLA-4, LAG3, KIR, TIM3, TIGIT, BTLA, CD160, or A2aR) or a ligand.
  • the agent may be a small molecule that blocks activity of an immune checkpoint protein or a ligand.
  • the ligand may be an antagonist or selective modulator of an immune checkpoint protein, such as a target receptor in an immune checkpoint pathway.
  • therapeutic agents include, but are not limited to, an agent that is a co- stimulatory molecule.
  • co-stimulatory agents include HVEM; ICOSL; 4- 1 BBL; OX40L; GITRL; CD40L; and agents that stimulate CD28 (e.g., a CD28 agonist); 1COS (e.g., an ICOS agonist); CD137 (e.g., a CD 137 agonist); OX40 (e.g., an OX40 agonist); CD27 (e.g., an CD27 agonist); CD40 (e.g., a CD40 agonist); CD40L (also known as gp-39) (e.g., an CD40L agonist); LIGHT (e.g., a LIGHT agonist); LT-alpha (e.g., an LT-alpha agonist); GITR (e.g., a G1TR agonist); and a mimic of a ligand of the aforementioned.
  • the agent may be an isolated antibody or fragment or derivative thereof that stimulates the target receptor (e.g., CD28, ICOS, CD 137, OX40, CD27, CD40, CD40L, LIGHT, LT-alpha, and/or GITR also known as TNFRSF 18) such as an anti-CD28 antibody, anti-ICOS antibody, anti-CD 137 antibody, anti-OX40 antibody, anti-CD27 antibody, anti-CD40 antibody, anti-CD40L antibody, anti-LIGHT antibody, anti-LT- alpha antibody, and anti-GITR antibody.
  • the agent may be a small molecule that stimulates the target receptor.
  • the invention provides VLP intermediate to which one or more agents of interest may attach when desired comprising a VLP comprising capsid protein or viral coat proteins having at least one non-naturally occurring amino acid to which one of two binding/interacting partners of an InaD domain complex or equivalent is attached and the VLP is free of a viral genome.
  • the VLP may be a stabilized VLP.
  • the capsid protein or viral coat protein is a monomer.
  • the capsid protein or viral coat protein is a dimer.
  • the capsid protein or viral coat protein is assembled into a VLP.
  • the VLP is formed by the assembly of HBc polypeptides, corresponding to the first 149 amino acids of a Hepatitis B capsid protein or coat protein.
  • the VLP is formed by the HBc polypeptide with the amino acid sequence provided in Figure 1 with the nucleic acid encoding this polypeptide provided below. This nucleic acid may be used to produce the HBc polypeptide with an amino acid sequence provided in Figure 1.
  • the VLP is formed by the HBc polypeptide with the amino acid sequence provided in Figure 2.
  • a VLP may contain one type of HBc polypeptide or alternatively a VLP may be formed by a mixture of more than one HBc polypeptides such as for example the two peptides provided in Figure 2.
  • a VLP intermediate may be attached to one or more agents of interest (not through the InaD domain complex), wherein the VLP intermediate attached to such agents can be later used to attach additional agents of interest when so desired (though the InaD domain complex or other means, e.g. cross linking).
  • the VLP intermediate may be stored or stock piled.
  • One preferred embodiment provides a VLP intermediate attached to a CpG oligonucleotide but not through the InaD domain complex.
  • the InaD domain complex or equivalent may contain two binding/interacting partners, wherein one binding/interacting partner is an InaD domain or fragment thereof or equivalent comprising a domain of InaD protein or equivalent and wherein a second binding/interacting partner is a peptide with at least four amino acids to which the InaD domain or fragment or equivalent binds.
  • the peptide with at least four amino acids may be a binding/interacting partner of PDZl domain of the InaD protein.
  • the peptide is 5 amino acid long.
  • the peptide may comprise a sequence TEFCA.
  • the peptide with at least four amino acids additionally comprises a non-naturally occurring amino acid and optionally a linker separating the peptide from the non-naturally occurring amino acid which is or can be used to attach the peptide to a VLP.
  • the domain of an InaD protein may be a PDZl domain or a first PDZ domain of a five PDZ domain-containing InaD protein comprising an amino acid sequence from position 1 1 to 107 of an InaD protein (e.g., having a GenBank Accession Number AAF81203.1 or a closely corresponding sequence in Figure 13 (beginning at amino acid position 2 (alanine) and ending at amino acid position 98 (lysine)).
  • the PDZl domain may have a mutation such as a substitution mutation.
  • substitution mutation include an amino acid change at position 62 of GenBank Accession Number AAF81203.1 from a cysteine residue to an alanine residue, such that the resulting amino acid sequence is AGELIHMVTLDKTGKKSFGICIVRGEVKDSPNTKTTGIFIKGIVPDSPAHLAGRLKVGDRI LSLNGKDVRNSTEQAVIDLIKEADFKIELEIQTFDK. or as provided in Figure 13.
  • Figure 13 also provides a nucleic acid sequence which may be used to produce an InaD domain used in the invention.
  • the peptide binding/interacting partner of PDZl domain comprises a PDZl -binding consensus sequence, X-F/W/Y-C-F/A-COOH, where X denotes any amino acid and COOFI, F/W/Y are amino acid residues at the 3 rd amino acid from the C-terminus, C denotes cysteine at the 2" d amino acid from the C-terminus, and -COOH denotes a non-side chain carboxylic acid/carboxylate group of the last amino acid at the C-terminus of the peptide.
  • the peptide binding/interacting partner of PDZl domain includes any of XFCF-COOH, XWCF-COOFI, and XYCF-COOH, where X denotes any amino acid, F denotes phenylalanine, W denotes tryptophan, Y denotes tyrosine, and -COOH denotes a non-side chain carboxylic acid/carboxylate group of phenylalanine at the C-terminus of the peptide.
  • the peptide binding/interacting partner of PDZl domain includes any of VFCF-COOH, KYCF-COOH, LYCF-COOH, QYCF-COOH, EYCF-COOH, QWCF-COOH, RFCF-COOH, SYCF-COOH, GYCF-COOH, KWCF-COOH and PYCF-COOH, where X denotes any amino acid and -COOH denotes a non-side chain carboxylic acid/carboxylate group of phenylalanine at the C-terminus of the peptide.
  • the peptide binding/interacting partner of PDZl domain comprises a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide.
  • the peptide comprises a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide may be one not found in nature, may be from a peptide library or may be derived from C-terminal end of naturally occurring proteins identifiable in publically available databases.
  • Examples of peptides having a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide includes, but are not limited to, peptides having as their last three amino acids, FCA-COOH, TCL-COOH, VCV-COOH, TCA-COOH, SCV-COOH, ACA- COOH, ICA-COOH, LCL-COOH, FCL-COOH, YCA-COOH, SCA-COOH, LCT-COOH, SCV- COOH, ACL-COOH, TCF-COOH, SCL-COOII, TCV-COOH, ACV-COOH, FCL-COOH, XCY- COOH and FCF-COOH, wherein the 4 lh amino acid from the C-terminus may be any amino acid and -COOFI denotes a non-side chain carboxylic acid/carboxylate group of the last amino acid at the C-terminus of the peptide.
  • the peptide having a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide may be the sequence TEFCA or TEFCF.
  • the peptide with at least four amino acids may be 4, 5, 6, 7, 8 or 9 amino acid in length.
  • the cysteine amino acid as a penultimate amino acid from C-terminus of the peptide may participate in intermolecular disulfide bond formation with the PDZ1 domain of the InaD protein.
  • the intermolecular disulfide bond formation may occur with cysteine-31 of the PDZ1 domain of the InaD protein (GenBank Accession Number AAF81203.1 ), corresponding to cysteine at position 22 in the amino acid sequence of the InaD domain of Figure 13).
  • the InaD complex may additionally comprise an intermolecular disulfide bond between a cysteine of the InaD domain and a cysteine of the peptide binding/interacting partner.
  • the disulfide bond formation between the two binding/interacting partners may further stabilizes the InaD domain complex.
  • the peptide binding/interacting partner of PDZ1 domain may be free of a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide.
  • the peptide free of a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide includes any of the peptides having as their last three amino acids, YSF-COOH, WSF- COOH, YEF-COOH, FMF-COOH, YYF-COOH, YMF-COOH and WTF-COOH, wherein the 4 lh amino acid from the C-terminus may be any amino acid and -COOH denotes a non-side chain carboxylic acid/carboxylate group of phenylalanine at the C-terminus of the peptide.
  • the peptide free of a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide may be any of the peptides having as their last four amino acids, SYSF- COOH, CYSF-COOH, DWSF-COOH, VYEF-COOH, VFMF-COOH, HYSF-COOH, EYYF- COOH, NYMF-COOI I and DWTF-COOH, wherein -COOH denotes a non-side chain carboxylic acid/carboxylate group of phenylalanine at the C-terminus of the peptide.
  • the peptide binding/interacting partner within the InaD domain complex adopts a ⁇ -strand structure or an extended conformation, wherein presence of a penultimate cysteine residue from the C-terminus of the peptide may permit formation of an intermolecular disulfide bond with the InaD domain and wherein presence of a phenylalanine 3 amino acids from the C-terminus of peptide may induce an abrupt turn to the ⁇ -strand or extended conformation.
  • the equivalent of a domain of InaD protein may be any PDZ domain which forms a ⁇ -barrel structure comprising 6 ⁇ -strands (B l to B6), a short a-helix (Al ) and a long ⁇ -helix (A2) and wherein the ⁇ -strands and a-helices are arranged in the order, B 1 -B2- B3-A 1 -B4-B5-A2-B6, or a circular permutation with a carboxylate-binding loop between ⁇ - strands, B l and B2, found at one end of a groove formed by a-helix A2 and ⁇ -strand B2, wherein a peptide binding/interacting partner of 4 to 9 amino acid in length can fit and form hydrogen bonds, ionic interactions and hydrophobic interactions, and optionally a covalent bond between binding partner cysteine residues, in the InaD domain complex equivalent.
  • the peptide binding/interacting partner may be any peptide sequence which adopts a ⁇ -strand, extended conformation or a ⁇ -hairpin conformation and permits binding to a PDZ domain binding pocket comprising a-helix A2 and ⁇ -strand B2, wherein the peptide may comprise a non-side chain carboxylic acid/carboxylate group normally present at a C-terminus of a peptide, free of a non-side chain carboxylic acid/carboxylate group normally present at a C-terminus of a peptide, a cysteine residue as a penultimate amino acid from the C-terminus or be free of said cysteine, a peptide with no free ends so as to lack non-side chain amino or carboxyl groups normally associated with ends of a peptide, and 4 to 9 amino acids or a combination thereof.
  • the equivalent of a domain of InaD protein may be a variant of a ⁇ -barrel structure of the PDZ domain, wherein the variant can function to bind a peptide binding/interacting partner of 4 to 9 amino acid in length.
  • the equivalent of a domain of InaD protein or PDZ domain or its variant, or the peptide binding/interacting partner of the domain of InaD protein or PDZ domain or its variant comprises a primary sequence or structure, secondary structure or tertiary structure as provided in RCSB Protein Data Bank for structure of a PDZ domain or a PDZ-domain variant in a complex with a peptide binding/interacting partner.
  • Examples of the structure of PDZ domain or its variant or PDZ domain or its variant in a complex with a peptide binding/interacting partner may be found in RCSB Protein Data Bank with Accession Numbers 1IIIJ, 1N7T, 2101, 2EXG, 2H3L.1W9E, 1W90, 1W9Q, 1Z86, 2FNE, 2FCF, 1 YBO, 2FE5, 2F5Y, 2CSS, 1 Y7N, 2F0A, 2CSJ, 2CS5, 1X5N, 1X5Q, 1X5R, 1XZ9, 1X45, 1VJ6, 1TP3, 1TP5, 1TQ3, lZUB, 2BYG, 1U37, 1U38, 1U39, 1U3B, 1V1T, 1 WF8, 1 WI4, 1ZOK, 1VA8, 1T2M, 1X8S, 1WJL, 1WH1, 1WHA, 1WHD, 1WI2, 1WIF, 1WFV,
  • sequences of the equivalent of a domain of InaD protein or PDZ domain or its variant, or the peptide binding/interacting partner of the domain of InaD protein or PDZ domain or its variant include sequences for the group of domain of InaD protein or PDZ domain or its variant and respective peptide binding/interacting partners as provided in RCSB Protein Data Bank with Accession Numbers 1IHJ, 1N7T, 2101, 2EXG, 2H3L,1 W9E, 1 W90, 1W9Q, 1Z86, 2FNE, 2FCF, 1YBO, 2FE5, 2F5Y, 2CSS, 1Y7N, 2F0A, 2CSJ, 2CS5, 1X5N, 1X5Q, 1X5R, 1XZ9, 1X45, 1VJ6, 1TP3, 1TP5, 1TQ3, 1ZUB,2BYG, 1U37, 1U38, 1U39, 1U3B, 1V1T, 1WF8, 1WI4, 1ZOK
  • the peptide binding partner with at least four amino acids to which the InaD domain or fragment or equivalent binds may have a free amino terminus, a free carboxyl terminus or lack both free amino and carboxyl ends.
  • the peptide binding partner with at least four amino acids to which the InaD domain or fragment or equivalent binds comprises a free carboxyl terminus with a non-side chain carboxylate or carboxylic acid group.
  • the non-naturally occurring amino acid and optionally a linker separating the peptide from the non-naturally occurring amino acid is attached N-terminal to the peptide.
  • linkers include, but are not limited to, a polyethylene glycol (PEG) or one or more amino acids.
  • PEGs include PEGs comprising more than 3 ethylene glycol repeats and less than 50 ethylene glycol repeats. Suitable examples of PEG include any of PEG&, PEG i2 and PEG24.
  • non-naturally occurring amino acid provides an alkyne or azide reactant group for Click chemistry reactions.
  • non-naturally occurring amino acids having an azide reactant group include L-azidohomoalanine, /7-azido-L-phenylalanine or N6-((2-azidoethoxy)carbonyl)-L-lysine.
  • non-naturally occurring amino acids having an alkyne reactant group include L-homopropargylglycine, L- propargylglycine, / propargyloxyphenylalanine or N6-((2-propynyloxy)carbonyl)-L-lysine.
  • the non-naturally occurring amino acid is N6-((2-propynyloxy)carbonyl)-L- lysine, N6-((2-azidoethoxy)carbonyl)-L-lysine or /?-acetyl-L-phenylalanine.
  • a VLP of the invention e.g., a VLP intermediates or a VLP intermediate attached to one or more agents of interest
  • a VLP of the invention is formed by a hepatitis B core protein (HBc) or its variant or a portion thereof.
  • HBc hepatitis B core protein
  • other viral species are contemplated and encompassed herein.
  • the HBc polypeptide is 149 amino acid in length and is derived from the first 149 amino acids at the N-terminus of a hepatitis B virus (HBV) coat protein of HBV subtype adyw (UniProt accession number P03147) .
  • the HBc polypeptide variant from HBV subtype adyw has a methionine-to-serine substitution at amino acid 66 (M66S) and a leucine-to-methionine substitution at amino acid 76 (L76M), wherein the variant comprises an amino acid sequence as shown in Figure 1.
  • the VLP comprises an HBc polypeptide comprising the amino acid sequence of Figure 1 or portion thereof.
  • the VLP is a stabilized VLP comprising hepatitis B core proteins (HBc) or its variant and wherein at least two amino acids of a HBc polypeptide are substituted with cysteine residues that can form intermolecular disulfide bonds when the HBc is assembled into a virus like particle (VLP), thereby stabilizing the VLP structure.
  • the stabilized VLP comprises an HBc polypeptide comprising the amino acid sequence of Figure 2 or portion thereof.
  • the amino acid substitutions for stabilizing a VLP are selected from any of [D29C, R127C]; [T109C, V120C]; [Y132C, N136C]; [Y132C, A137C]; [R133C, N136C]; [R133C, A137C]; [P134C, P135C]; [P134C, N136CJ; [P134C, A137C]; and [P135C, N136C].
  • the amino acid substitutions for stabilizing a VLP include [D29C, R127C]; [P134C, N136C]; or [D29C, R127C, P134C, N 136C].
  • the amino acid substitutions for stabilizing a VLP include [D29C, R127C].
  • one of two binding/interacting partners of the InaD domain complex or equivalent is attached to a spike region of the VLP.
  • the spike region of the VLP is a spike tip.
  • the amino acids of the spike region at residues 73-81 are substituted to reduce the negative charge, relative to an amino acid sequence as shown in Figure 1.
  • the spike region comprises a hydrophobic pocket and wherein the amino acids of the hydrophobic pocket at residues 57-81 are substituted relative to an amino acid sequence as shown in Figure 1.
  • amino acid substitutions include any one of [159V, L60S, G63R, D64E, L65V, M66T, T67D, L68F, A69G, T70D, T74N, L76M, E77Q, P79Q, S81 A, S87N, T91A, V93I, F97I] or [ ⁇ 74 ⁇ , L76M, E77Q, P79Q, S81A],
  • the VLP may comprise an amino acid sequence NxMQxQxA, where x may be any amino acid. Additionally, in yet another embodiment, the VLP may comprise an amino acid sequence VSxxREVTDFGDxxxNxMQxQxAxxxxxNxxxAxIxxxI, where x may be any amino acid. In a further embodiment, the VLP comprises an amino acid sequence NxMQxQxA, where x may be any amino acid so long as the amino acid does not disrupt HBc dimer formation, formation of a HBc hydrophobic pocket and formation of HBc dimer spike.
  • the VLP comprises an amino acid sequence VSxxREVTDFGDxxxNxMQxQxAxxxxxNxxxAxIxxxl, where x may be any amino acid so long as the amino acid does not disrupt HBc dimer formation, formation of a HBc hydrophobic pocket and formation of HBc dimer spike.
  • the invention further provides an embodiment, wherein the VLP further comprises at least one pair of amino acid substitutions selected from [D29C, R127C]; [T109C, V 120C]; [Y132C, N 136C]; [Y132C, A 137C]; [R133C, N136C]; [R133C, A137C]; [P 134C, P 135C]; [P 134C, N 136C]; [P 134C, A 137C] ; and [P 135C, N 136CJ .
  • the VLP further comprises amino acid substitutions any of [D29C, R 127C]; [P 134C, N 136C]; and [D29C, R127C, P134C, N 136C].
  • the VLP further comprising amino acid substitution [D29C, R127C].
  • the VLP comprises a hepatitis B core protein (HBc) which has an amino acid other than a methionine at amino acid 66 of wild-type HBc ( Figure 12).
  • the amino acid other than a methionine at amino acid 66 is a serine, threonine or an isoleucine.
  • the amino acid other than a methionine at amino acid 66 is a serine. In a further example, the amino acid other than a methionine at amino acid 66 is a threonine.
  • the hepatitis B core protein (HBc) has an amino acid substitution M66S.
  • the invention further provides a VLP free of a viral genome comprising capsid proteins or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, wherein the capsid proteins or viral coat proteins have at least one non-naturally occurring amino acid to which one of two binding/interacting partners of the InaD domain complex or equivalent is attached, wherein an agent of interest is attached to a second binding/interacting partner of an InaD domain complex or equivalent, and wherein the two binding/interacting partners form the InaD domain complex or equivalent, such that the VLP displays one or more agents of interest.
  • the VLP free of a viral genome additionally comprises a CpG oligonucleotide.
  • the VLP free of a viral genome is produced from a VLP intermediate previously attached to one or more agents of interest free of an InaD domain complex or equivalent. In one embodiment, the VLP free of a viral genome is produced from a VLP intermediate previously attached to a CpG oligonucleotide or a TLR agonist free of an InaD domain complex or equivalent.
  • agents of interest include, but are not limited to, a peptide, polypeptide, nucleic acid molecule, polymer of a nucleic acid molecule, lipopolysaccharide, lipopeptide, peptidoglycan, small molecule, antibody, Id antigen, tumor-associated antigen, TLR agonist and immunostimulatory oligonucleotide.
  • agents of interest include, but are not limited to, a peptide, polypeptide, nucleic acid molecule, polymer of a nucleic acid molecule, lipopolysaccharide, lipopeptide, peptidoglycan, small molecule, antibody, Id antigen, tumor-associated antigen, TLR agonist and immunostimulatory oligonucleotide.
  • tumor-associated antigens are described hereinabove and contemplated herein but not repeated to avoid duplication.
  • leucocyte antigens are described hereinabove and contemplated herein but not repeated to avoid duplication.
  • the VLP (also referred to herein as a VLP intermediate or a VLP free of a viral genome) may contain at least one non-naturally occurring amino acid or unnatural amino acid per capsid protein or viral coat protein.
  • at least one-fourth of the total number of non-naturally occurring amino acids in a VLP is used to attach a an agent of interest (examples include, but are not limited to, a peptide, polypeptide, nucleic acid molecule, polymer of nucleic acid molecules, lipopolysaccharide, lipopeptide, peptidoglycan, small molecule, antibody, Id antigen, tumor-associated antigen, TLR agonist and/or immunostimulatory oligonucleotides).
  • at least one-fourth of the total number of non-naturally occurring amino acids in a VLP is used to attach one of two binding/interacting partners of an InaD domain complex or equivalent.
  • At least one-third of the total number of non-naturally occurring amino acids in a VLP is used to attach an agent of interest. In yet another embodiment, at least one-third of the total number of non-naturally occurring amino acids in a VLP may be used to attach one of two binding/interacting partners of an InaD domain complex or equivalent.
  • At most 120 of the 240 capsid protein or viral coat proteins display an agent of interest.
  • One or more types of agents of interest may be attached.
  • at most 120 of the 240 capsid proteins or viral coat proteins display one of two binding/interacting partners of an InaD domain complex or equivalent.
  • the VLP may display only one type or kind of agent of interest.
  • the VLP may display a combination of two or more different types or kinds of agents of interest.
  • the VLP displays agents of interest using the INAD domain complex described herein.
  • the VLP may display agents of interest using the INAD domain complex and other agents through other attachment means, for example, via crosslinking as described herein, such as crosslinking of CpG to the VLP as described in Example 2.
  • the VLP comprises one or more additional moieties conjugated to the non-naturally occurring amino acid.
  • the one or more additional moieties may be a peptide binding partner of the InaD domain complex or equivalent.
  • the one or more additional moieties is an InaD domain or equivalent.
  • the VLP may be an isolated VLP or purified VLP.
  • the VLP 10 further comprising two or more types of agents of interest.
  • the second agent of interest is attached to a second binding/interacting partner of an InaD domain complex.
  • one agent of interest comprises a reactive functional group, which can participate in a crosslinking reaction with the non-naturally occurring amino acid.
  • the crosslinking reaction may be an alkyne-azide cycloaddition or click chemistry between an alkyne or azide functional group of the agent of interest and respective alkyne or azide functional group of the non-naturally occurring amino acid of the capsid protein or viral coat protein.
  • the agent of interest is an antibody that recognizes and binds a leucocyte antigen, cell adhesion molecule, prostate specific membrane antigen or oncoprotein.
  • the leucocyte antigen is an immune checkpoint protein.
  • immune checkpoint protein include, but are not limited to, a PD-1 , a PDL l , a PDL2, a B7-H3, a B7-H4, a CTLA-4, a LAG3, a KIR, a TIM3, a TIGIT, a BTLA, a CDl 60, an A2aR, and a VISTA protein.
  • the antibody that recognizes and binds an immune checkpoint is an antibody that recognizes and binds any of a PD-1, a PDL1, a PDL2, a B7-I I3, a B7-H4, a CTLA- 4, a LAG3, a KIR, a TIM3, a TIGIT, a BTLA, a CD 160, an A2aR, and a VISTA protein.
  • an agent of interest include, but are not limited to, an antibody specifically directed against any of CDl, CD2, CD3, CD4, CD5, CD6, CD6L, CD7, CD8, CD9, CDIO, CD11/CD18, CDlla, CDl lb, CDllc, CDl Id, CDwl2, CD13, CD14, CD15, CD15s, CD16, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD27L, CD28, CD29, CD30, CD30L, CD31, CD32, Fc Gamma Receptor, CD33, CD34, CD35, CD36, CD37, CD38, CD40, CD40L, CD41, CD42, CD42a, CD43b, CD43c, CD43, CD44, CD45, CD45-AP, CD46, CD48, CD49, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50,
  • the invention further provides a VLP free of a viral genome comprising two or more antibodies selected from the group of antibodies for CDl, CD2, CD3, CD4, CD5, CD6, CD6L, CD7, CD8, CD9, CDIO, CDl 1/CD18, CDl la, CDl lb, CDl lc, CDl Id, CDwl2, CD13, CD14, CD15, CD15s, CD16, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD27L, CD28, CD29, CD30, CD30L, CD31, CD32, Fc Gamma Receptor, CD33, CD34, CD35, CD36, CD37, CD38, CD40, CD40L, CD41 , CD42, CD42a, CD43b, CD43c, CD43, CD44, CD45, CD45- AP, CD46, CD48, CD49, CD49a, CD49b, CD49c, CD49d,
  • two antibodies displayed (attached) on a VLP of the invention include any of (a) a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds CD3; (b) a first antibody or portion thereof that binds CD 19 and a second antibody or portion thereof that binds CD3; (c) a first antibody or portion thereof that binds EPCAM and a second antibody or portion thereof that binds CD3; (d) a first antibody or portion thereof that binds FOLHl and a second antibody or portion thereof that binds CD3; (e) a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds FcGR; and (f) a first antibody or portion thereof that binds CD20 and a second antibody or portion thereof that binds CD3.
  • the Id antigen is derived from a T cell receptor (TCR).
  • TCR T cell receptor
  • An embodiment of the invention includes an Id antigen which comprises an immunoglobulin variable heavy (VTI) chain domain or sequence having an amino acid motif Q-(A or P)-(P or L)- G-(Q or K)-G-L-E-W-(M or V or I) immediately preceding a tri-peptide motif, (G or A or S)-(X)- I, wherein X is any amino acid.
  • VTI immunoglobulin variable heavy
  • Id antigens include, but are not limited to, an Id antigen designated N2517-H comprises an amino acid sequence, EVQLVESGGALVQPGGSLRLSCAASGFTFRDFWMSWVRQAPGKGLEWVANIREDGND YYVGAVRGRFTVSRDNARNSLYLQMNSLRAEDTAVYYCARDLGGYCSSTNCEGYFD YWGQGTLVTVSS or a portion thereof; an Id antigen designated N2517-K comprises an amino acid sequence, DIQMTQSPSTLSASVGDRVTITCRASQSISTWLAWYQQKPGRAPKLLIYKASTLESGVPS RFSGSGSGTEFTLTISGLQPDDFATYYCQQYADYRTFGQGTKVEIK or a portion thereof ; an Id antigen designated N3803-H comprises an amino acid sequence, EVQLVESGGGVVRPGGSRRLSCAASGFSFDDYGMSWVRQAPG GLEWVSGIKWNGGS RGYLDSVKGRFTI
  • VLP compositions of the invention where an agent of interest is attached through a InaD domain complex or equivalent thereof provides specific VLPs having two of more agents of interest attached thereto through means other than by the InaD domain complex or equivalent thereof (free of InaD domain complex or equivalent).
  • these alternative attachment means are described in PCT/US2014/030788 or PCT/US 14/069406 which are incorporated by reference and in Example 2 herein.
  • VLPs free of a viral genome comprising two or more display polypeptides comprising two or more antibodies selected from the group of antibodies for CD1 , CD2, CD4, CD5, CD6, CD6L, CD7, CD8, CD9, CD10, CD1 1/CD18, GDI l a, CD1 l b, CD1 lc, CD1 I d, CDwl 2, CD13, CD14, CD15, CD15s, CDwl 7, CD18, CD19, CD21 , CD22, CD23, CD24, CD25, CD26, CD27, CD27L, CD28, CD29, CD30L, CD31 , CD32, Fc Gamma Receptor, CD33, CD34, CD35, CD36, CD37, CD38, CD40, CD40L, CD41 , CD42, CD42a, CD43b, CD43c, CD43, CD44, CD45, CD45-AP, CD46, CD48, CD49, CD49a, CD49b, CD
  • the invention provides a VLP which comprises any of two antibodies displayed (attached, for example, not through an InaD domain complex but by, e.g., crosslinking) thereon including any of (a) a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds CD3; (b) a first antibody or portion thereof that binds CD 19 and a second antibody or portion thereof that binds CD3; (c) a first antibody or portion thereof that binds EPCAM and a second antibody or portion thereof that binds CD3; (d) a first antibody or portion thereof that binds FOLH1 and a second antibody or portion thereof that binds CD3; (e) a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds FcGR; and (f) a first antibody or portion thereof that binds CD20 and a second antibody or portion thereof that binds CD3.
  • the invention further provides a nucleic acid molecules encoding the VLP and sVLP of the invention, e.g., as shown in Figure 1 and Figure 2.
  • the nucleic acids of the invention may comprise nucleotide sequences and encode polypeptides (amino acid sequences) which are at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the reference nucleotide and amino acid sequences of the present invention (i.e., see examples herein, e.g., the sequences in Figures 1 and 2) when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences.
  • amino acid sequences amino acid sequences
  • Polypeptides comprising amino acid sequences which are at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%o) to the reference amino acid sequences of the present invention when the comparison is performed with a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences, are also included in the present invention.
  • the nucleic acid molecule may be a DNA molecule (e.g., an isolated cDNA) encoding the VLP of the invention. Additionally, the nucleic acid molecule may be a RNA (e.g., an isolated RNA such as isolated mRNA). Alternatively, the nucleic acid molecule may be a hybrid of cDNA and mRNA. For example, the invention provides for a DNA construct comprising a vector that expresses the VLP free of a viral genome of the invention.
  • the nucleic acid molecules of the invention also include derivative nucleic acid molecules which differ from DNA or RNA molecules.
  • Derivative molecules include peptide nucleic acids (PNAs), and non-nucleic acid molecules including phosphorothioate, phosphotriester, phosphoramidate, and methylphosphonate molecules, that bind to single-stranded DNA or RNA in a base pair- dependent manner (Zamecnik, P. C, et al., 1978 Proc. Natl. Acad. Sci. 75 :280284; Goodchild, P. C, et al., 1986 Proc. Natl. Acad. Sci. 83 :4143-4146).
  • the invention provides a vector which comprises the nucleic acid molecule of the invention.
  • the term vector includes, but is not limited to, plasmids, cosmids, and phagemids.
  • the host vector system comprises the vector of the invention in a suitable host cell. Examples of suitable host cells include but are not limited to bacterial cell and eukaryotic cells.
  • the invention provides a process comprising recovering a VLP of the invention and/or VLP monomers from a culture medium and from cultured cells. In the case of VLP monomers from a culture medium or cultured cells, such monomers may be first isolated and then allowed to form VLPs.
  • the invention also provides for a method for producing a VLP free of a viral genome protein comprising culturing the host vector system of the invention under suitable culture conditions so as to produce the VLP free of a viral genome in the host and recovering the VLP free of a viral genome so produced.
  • the method comprises culturing the host vector system of the invention under suitable culture condition so as to produce VLP coat protein in the host, assembling VLP from VLP coat protein isolated from the host in the absence of a viral genome, and recovering the VLP free of a viral genome so produced.
  • VLP may also be produced from assembly of VLP capsid monomers following isolation from a host cell.
  • the VLP may be assembled from capsid proteins outside of the host cell.
  • the VLP of the invention may be produced following synthesis and assembly of its capsid protein or coat proteins in a cell free in vitro transcription and/or translation system.
  • the invention provides methods for producing, in a cell-free in vitro reaction, a VLP free of a viral genome.
  • the VLP is a population of icosahedral virus like particles free of a viral genome.
  • This method may comprise synthesizing virus coat proteins in a prokaryotic cell-free in vitro translation reaction (e.g. substantially free of polyethylene glycol).
  • the prokaryotic cell-free in vitro translation reaction may contain a bacterial cell extract, components of polypeptide and/or mRNA synthesis machinery; a template for transcription for the translation of the polypeptide; monomers for synthesis of the polypeptide; and co-factors, enzymes and other reagents necessary for translation to produce the virus coat proteins (e.g., at least about 250 ug/ml of the virus coat proteins) under conditions permissive for the virus coat proteins to self-assemble into a stable icosahedral virus like particle free of a viral genome which comprises at least 60 separate proteins.
  • virus coat proteins e.g., at least about 250 ug/ml of the virus coat proteins
  • the degeneracy of the genetic code provides a predictable number of nucleic acid sequences encoding the VLP of the invention, the codons of which may be selected to optimally express the isolated nucleic acid in a host organism (including without limitation, bacteria, yeast, mammalian cells cultured in vitro, and cells of a mammal (including a human). Such expression is useful for production of the nucleic acid or the polypeptide in a host organism for subsequent isolation and use according to the invention or in cell free in vitro transcription and/or translation system.
  • a host organism including without limitation, bacteria, yeast, mammalian cells cultured in vitro, and cells of a mammal (including a human).
  • Such expression is useful for production of the nucleic acid or the polypeptide in a host organism for subsequent isolation and use according to the invention or in cell free in vitro transcription and/or translation system.
  • compositions pharmaceutically acceptable compositions
  • dosage forms refer to a composition containing the active ingredient(s) of the invention in a form suitable for administration to a subject.
  • compositions of the present invention may be mixed with one or more pharmaceutically acceptable carriers, binders, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, polymers, disintegrating agents, glidants, wetting agents, emulsifying agents, suspending agents, lubricating agents, acidifying agents, dyes, preservatives and dispensing agents, or compounds of a similar nature depending on the nature of the mode of administration and dosage forms.
  • pharmaceutically acceptable carriers and excipients that may be used to formulate dosage forms, are described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association ( 1986), incorporated herein by reference in its entirety.
  • Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation.
  • examples of pharmaceutically acceptable carriers include water, saline, Ringer's solution, dextrose solution, ethanol, polyols, vegetable oils, fats, ethyl oleate, liposomes, waxes polymers, including gel forming and non-gel forming polymers, and suitable mixtures thereof.
  • the carrier may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability.
  • Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
  • the carrier may be a parenteral carrier
  • binders include, but are not limited to, microcrystalline cellulose and cellulose derivatives, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polvinylpyrrolidine, povidone, crospovidones, sucrose and starch paste.
  • diluents examples include salt.
  • excipients include, but are not limited to, surfactants, lipophilic vehicles, hydrophobic vehicles, sodium citrate, calcium carbonate, and dicalcium phosphate.
  • wetting agents include, but are not limited to, propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene laural ether.
  • the agents of the invention can be formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier(s) described above.
  • Any dosage form used for therapeutic administration should be sterile. Sterility can readily be accomplished by filtration through sterile filtration membranes. Therapeutics generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. KITS OF THE INVENTION
  • kits are provided.
  • Kits according to the invention include package(s) comprising a composition of the invention or VLP compositions of the invention.
  • packaging means any vessel containing compositions presented herein.
  • the package can be a box or wrapping.
  • Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes (including pre-filled syringes), bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
  • the kit can also contain items that are not contained within the package but are attached to the outside of the package, for example, pipettes.
  • Kits may optionally contain instructions for administering compositions of the present invention to a subject having a condition in need of treatment. Kits may also comprise instructions for approved uses of components of the composition herein by regulatory agencies, such as the United States Food and Drug Administration. Kits may optionally contain labeling or product inserts for the present compositions. The package(s) and/or any product insert(s) may themselves be approved by regulatory agencies.
  • the kits can include compositions in the solid phase or in a liquid phase (such as buffers provided) in a package.
  • the kits also can include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.
  • the kit may optionally also contain one or more other compositions for use in combination therapies as described herein.
  • the package(s) is a container for intravenous administration.
  • compositions are provided in an inhaler.
  • compositions are provided in a polymeric matrix or in the form of a liposome.
  • the invention also provides methods of stimulating an immune response, e.g., in a subject.
  • the method comprises contacting immune cells with an effective amount of a VLP intermediate free of a viral genome, comprising a VLP having capsid proteins or viral coat proteins comprising at least one non-naturally occurring amino acid to which one of two binding/interacting partners of an InaD domain complex or equivalent is attached thereby stimulating an immune response.
  • the method comprises contacting immune cells with an effective amount of a VLP free of a viral genome comprising capsids or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, wherein the capsid proteins or viral coat proteins have at least one non-naturally occurring amino acid to which one of two binding/interacting partners of the InaD domain complex or equivalent is attached, and wherein an agent of interest is attached to a second binding/interacting partner of an InaD domain complex or equivalent, thereby stimulating an immune response.
  • the VLP intermediate further comprises an immunostimulatory oligonucleotide.
  • the VLP intermediate comprising an immunostimulatory oligonucleotide remains as a VLP intermediate so long as additional agents of interest may be attached to the VLP through the non-naturally occurring amino acid on the VLP or through one of the binding/interacting partner of the InaD complex or equivalent attached to the VLP.
  • the VLP intermediate further comprises additional agents of interest and wherein the VLP intermediate so further attached to additional agents of interest comprises unreacted non-naturally occurring amino acid and/or unoccupied binding/interacting partner of the InaD complex or equivalent.
  • the invention provides methods of inhibiting an immune response, e.g., in a subject.
  • the method comprises contacting immune cells with an effective amount of a VLP free of a viral genome comprising capsid proteins or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, wherein the capsid proteins or viral coat proteins have at least one non-naturally occurring amino acid to which one of two binding/interacting partners of the InaD domain complex or equivalent is attached, and wherein an agent of interest is attached to a second binding/interacting partner of an InaD domain complex or equivalent, thereby inhibiting an immune response.
  • the invention provides method of inhibiting tumor cells.
  • the method comprises contacting the tumor cells with an effective amount of a VLP intermediate free of a viral genome, comprising a VLP having capsid proteins or viral coat proteins comprising at least one non-naturally occurring amino acid to which one of two binding/interacting partners of an InaD domain complex or equivalent is attached thereby inhibiting the tumor cells.
  • the method comprises contacting the tumor cells with an effective amount of a VLP free of a viral genome comprising capsid proteins or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, wherein the capsid proteins or viral coat proteins have at least one non-naturally occurring amino acid to which one of two binding/interacting partners of the InaD domain complex or equivalent is attached, wherein an agent of interest is attached to a second binding/interacting partner of an InaD domain complex or equivalent, and wherein the two binding/interacting partners form the InaD domain complex or equivalent, such that the VLP displays one or more agents of interest thereby inhibiting the tumor cells.
  • the method comprises contacting the tumor cells with an effective amount of the VLP of the invention thereby inhibiting the tumor cells.
  • the invention also provides methods of treating, inhibiting or preventing the progression of a tumor in a subject, which comprises administering to said subject an effective amount of a VLP compositions of the invention thereby treating, inhibiting or preventing the progression of a tumor in the subject.
  • the invention further provides for a method of treating, inhibiting or preventing the progression of a disease or disorder comprising administering to said subject an effective amount of a VLP composition of the invention.
  • the disorder is an autoimmune disorder and may be a myasthenia gravis, chronic active hepatitis, primary biliary cirrhosis, dilated cardiomyopathy, myocarditis, dilated cardiomyopathy, autoimmune polyendocrine syndrome type I (APS- 1 ), autoimmune hepatitis, cystic fibrosis vasculitidis, acquired hypoparathyroidism, Goodpasture syndrome, Crohn's disease, coronary artery disease, pemphigus foliaceus, pemphigus vulgaris, Guillain-Barr syndrome, type 1 diabetes, stiff man syndrome, Rasmussen encephalitis, autoimmune gastritis, Addison disease, insulin hypoglycemic syndrome (Hi rata disease), type B insulin resistance, acanthosis, systemic lupus erythematosus (SLE), pernicious anemia, treatment- resistant Lyme arthritis, polyneuropathy, multiple sclerosis, demyelinating disease,
  • the disorder may be a systemic autoimmune disorder and may include ACTH deficiency, myositis, dermatomyositis, polymyositis, dermatomyositis, SLE, Sjogren syndrome, systemic sclerosis, rheumatoid arthritis (RA), progressive systemic sclerosis, systemic sclerosis, deimatomyositis, scleroderma, morphea, primary antiphospholipid syndrome, bullous pemphigoid, herpes gestationis, cicatricial pemphigoid, chronic idiopathic urticaria, necrotizing and cescentic glomerulonephritis (NCGN), system vasculitis, Wegener granulomatosis, Churg-Strauss syndrome, polymyositis, scleroderma, Raynaud syndrome, chronic liver disease, visceral leishmaniasis, and systemic autoimmune disease.
  • ACTH deficiency myositis,
  • the disorder may be a cancer or a paraneoplastic autoimmune disorder which may include neuropathy, small lung cell cancer, hepatocellular carcinoma, liver cancer, paraneoplastic pemphigus, paraneoplastic stiff man syndrome, paraneoplastic encephalomyelitis, sub-acute autonomic neuropathy, cancer, SLE, hepatocellular carcinoma, cancer-associated retinopathy, paraneoplastic opsoclonus myoclonus ataxia, lower motor neuron syndrome, Lambert-Eaton myasthenic syndrome, and paraneoplastic cerebellar degeneration.
  • a paraneoplastic autoimmune disorder which may include neuropathy, small lung cell cancer, hepatocellular carcinoma, liver cancer, paraneoplastic pemphigus, paraneoplastic stiff man syndrome, paraneoplastic encephalomyelitis, sub-acute autonomic neuropathy, cancer, SLE, hepatocellular carcinoma, cancer-associated retinopathy, paraneoplastic
  • the disorder may be a solid tumor cancer which may be a adrenal cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, cancer of unknown primary origin, Castieman Disease, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Leukemia, Liver Cancer, Lung Cancer, Lymphoma, Malignant Mesothelioma, Multiple Myeloma, Myelodysplasia Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non- Hodgkin Lymphoma, Oral Cavity and Or
  • the cancer may be any of head and neck cancer, breast, salivary gland, thyroid, pancreas, stomach, bladder, endometrial or uterine carcinoma, cervical cancer, ovarian, vulvar cancer, prostate, colon, rectal, colorectal, lung, non-small cell lung cancer, osteosarcoma, glioblastoma, kidney, liver, metastatic cancer.
  • the cancer is a B-cel l lymphoma (such as CLL).
  • the cancer is a T-cell lymphoma.
  • the cancer is prostate cancer.
  • the subject is a human, a farm animal, a horse, a dog, or a cat.
  • the disorder may be a plasma protein autoimmune disorder or cytokine autoimmune disorder.
  • plasma protein autoimmune disorder or cytokine autoimmune disorder include but not limited to autoimmune CI deficiency, SLE membrane proliferative glomerulonephritis, RA, systemic sclerosis, autoimmune thrombocytopenia purpura, immunodeficiency disorder, and atherosclerosis.
  • the disorder may be a B-cel l malignancy.
  • B-cell malignancy include but not limited to non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), mantle cell lymphoma and multiple myeloma, B-cell prolymphocyte leukemia, lymphoplasmocytic leukemia, splenic marginal zone lymphoma, marginal zone lymphoma (extra-nodal or nodal), plasma cell neoplasms (e.g., plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases), and follicular lymphoma (e.g., Grades 1 , II, III or IV).
  • the disorder may be a T-cell malignancy.
  • T-cell malignancy include but not limited to chronic lymphocytic leukemia (CLL), large granular lymphocyte leukemia (T gamma lymphoproliferative disease, mycosis fungoides/Sezary syndrome, diffuse aggressive lymphomas of adults, peripheral T-cell lymphomas (mixed cell type and large cell, immunoblastic), adult T-cell leukemia/lymphoma, angiocentric lymphomas (lymphomatoid granulomatosis polymorphic reticulosis, acute lymphocytic leukemia, or lymphoblastic lymphoma.
  • CLL chronic lymphocytic leukemia
  • T gamma lymphoproliferative disease mycosis fungoides/Sezary syndrome
  • diffuse aggressive lymphomas of adults include peripheral T-cell lymphomas (mixed cell type and large cell, immunoblastic), adult T-cell leukemia/lymphoma, angiocentric lymphomas
  • the VLP is produced by a method for producing a population of icosahedral virus like particles free of a viral genome in a cell-free in vitro reaction.
  • the method for producing a population of icosahedral virus like particles free of a viral genome in a cell-free in vitro reaction comprise synthesizing virus coat proteins in a prokaryotic cell-free in vitro translation reaction substantially free of polyethylene glycol and comprising a bacterial cell extract, components of polypeptide and/or mRNA synthesis machinery; a template for transcription for the translation of the polypeptide; monomers for synthesis of the polypeptide; and co-factors, enzymes and other reagents necessary for translation to produce at least about 250 ug/ml of the virus coat proteins-under conditions permissive for the virus coat proteins to self- assemble into a stable icosahedral virus like particle free of a viral genome, and comprising at least 60 separate proteins.
  • the invention provides a method of treating a cancer in a subject further comprising administering to the subject a therapeutically effective amount of one or more chemotherapeutic agents (which are included herein as an agent of interest), wherein the chemotherapeutic agents are one or more of the following: alkylating agents; thiotepa; cyclosphosphamide; alkyl sulfonates; busulfan; improsulfan; piposulfan; aziridines; benzodopa; carboquone; meturedopa; uredopa; ethylenimines; methylamelamines; altretamine; triethylenemelamine; trietylenephosphoramide; triethylenethiophosphaoramide; trimethylolomelamine; nitrogen mustards; chlorambucil; chlornaphazine; cholophosphamide; estramustine; ifosfamide; mechlorethamine; mechlorethamine oxide
  • the disorder is an infectious disease and may be polio, respiratory syncytial virus (RSV) infection AIDS, hepatitis B, hepatitis C, hepatitis E, rabies, herpes, HSV, EBV, influenza, smallpox, myxoma infection, rhinovirus infection, coronavirus infection, whooping cough (rubella virus infection), adenovirus infection, papilloma virus infection or human T-cell leukemia virus (HTLV) infection.
  • the infectious disease is HIV.
  • the infectious disease is influenza.
  • the infectious disease is RSV infection.
  • the invention provides for a method for treating, inhibiting, or preventing the progression of a solid tumor cancer, in a subject.
  • the method comprises administering to the subject, in need thereof, an effective amount of VLP vaccine or pharmaceutical composition of the invention so as to inhibit tumor growth or metastasis, kill tumor cells or reduce tumor burden.
  • the invention provides for a method of inhibiting tumor cells for a solid tumor which comprises contacting the tumor cells with an effective amount of a vaccine or composition of the invention.
  • the VLP compositions of the invention may be administered, e.g., in the case for cancer, by directly injection into or near a solid tumor.
  • the administration may be intratumoral.
  • the administration may be made directly into or around the lymph node, spleen, thyroid, bone marrow, or other organ of the body with a high concentration of tumor cells.
  • the administration may be intramuscular, intraperitoneal, intranasal, intradermal, or transmucosal.
  • the VLP compositions of the invention may be admixed with the therapeutic agent (included herein as an agent of interest) just prior to administration of the composition to the subject.
  • the composition may be available premixed so as to contain both the VLP vaccine and the therapeutic agent.
  • the solid tumor cancer may be an adrenal cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, cancer of unknown primary origin, Castleman Disease, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Liver Cancer, Lung Cancer, Lymphoma, Malignant Mesothelioma, Multiple Myeloma, Myelodysplastic Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer
  • the cancer may be any of head and neck cancer, breast, salivary gland, thyroid, pancreas, stomach, bladder, endometrial or uterine carcinoma, cervical cancer, ovarian, vulvar cancer, prostate, colon, rectal, colorectal, lung, non-small cell lung cancer, osteosarcoma, glioblastoma, kidney, liver, melanoma or metastatic cancer.
  • the invention also provides a method for regulating functional CD 19 positive B cell and CD3 positive T cells interactions comprising contacting CD 19 positive B cells and CD3 positive T cells with a VLP composition of the invention that comprises a first antibody or portion thereof that binds CD 19 and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous CD19 positive B cells with CD3 positive T cells.
  • a VLP composition of the invention that comprises a first antibody or portion thereof that binds CD 19 and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous CD19 positive B cells with CD3 positive T cells.
  • the interaction of said CD 19 positive B cell and CD3 positive T cells stimulates an immune response (e.g., activating cytotoxic T cells).
  • the invention additionally provides a method for regulating functional HER2 positive B cell and CD3 positive T cells interactions comprising contacting HER2 positive B cells and CD3 positive T cells with a VLP composition of the invention that comprises a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous HER2 positive B cells with CD3 positive T cells.
  • a VLP composition of the invention that comprises a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous HER2 positive B cells with CD3 positive T cells.
  • the interaction of said HER2 positive B cell and CD3 positive T cells stimulates an immune response (e.g., activating cytotoxic T cells).
  • the invention provides a method for regulating functional EPCAM positive B cell and CD3 positive T cells comprising contacting EPCAM positive B cells and CD3 positive T cells with a VLP composition of the invention that comprises a first antibody or portion thereof that binds EPCAM and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous EPCAM positive B cells with CD3 positive T cells.
  • a VLP composition of the invention that comprises a first antibody or portion thereof that binds EPCAM and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous EPCAM positive B cells with CD3 positive T cells.
  • the interaction of said EPCAM positive B cell and CD3 positive T cells stimulates an immune response (e.g., activating cytotoxic T cells).
  • the invention further provides a method for regulating functional FOLHl positive B cell and CD3 positive T cells comprising contacting FOLHl positive B cells and CD3 positive T cells with a VLP composition of the invention that comprises a first antibody or portion thereof that binds FOLHl and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous FOLHl positive B cells with CD3 positive T cells.
  • a VLP composition of the invention that comprises a first antibody or portion thereof that binds FOLHl and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous FOLHl positive B cells with CD3 positive T cells.
  • An embodiment of this invention provides that the interaction of said FOLH 1 positive B cell and CD3 positive T cells stimulates an immune response (e.g., activating cytotoxic T cells).
  • the invention provides a method for regulating functional CD20 positive B cell and CD3 positive T cells comprising contacting CD20 positive B cells and CD3 positive T cells with a VLP composition of the invention that comprises a first antibody or portion thereof that binds CD20 and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous CD20 positive B cells with CD3 positive T cells.
  • a VLP composition of the invention that comprises a first antibody or portion thereof that binds CD20 and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous CD20 positive B cells with CD3 positive T cells.
  • the interaction of said CD20 positive B cell and CD3 positive T cells stimulates an immune response (e.g., activating cytotoxic T cells).
  • any of the methods that involve increased activation of immune cells may further comprise contact or administration of one or more of an immune checkpoint inhibitor, examples which are provided herein, i.e., agents that block immune checkpoint proteins.
  • an immune checkpoint inhibitor examples which are provided herein, i.e., agents that block immune checkpoint proteins.
  • Contact or administration of one or more of immune checkpoint inhibitors may be effected before, during or after contact or administration of one or more VLP compositions of the invention.
  • Plasmid construction and host cell bank preparation The sequence encoding the human Hepatitis B core (HBc) monomer of subtype adyw (Pasek et al., 1979) with the C-terminus truncated at amino acid 149 ( Figure 1 ) was codon optimized for E. coli expression, produced by overlapping oligonucleotide gene synthesis and cloned into a pET21a plasmid. The plasmid was used to transform T7 Express Crystal Competent E. coli (High Efficiency) bacterial cells along with a second plasmid containing the pLysS gene. Cells were selected for growth using both ampicillin and chloramphenicol. Several colonies were selected for production of the HBc protein and a high-producing line was identified and stored in a Research Cell Bank.
  • HBc-producing E. coli from the Research Cell Bank were expanded in shake flasks in minimal media, M9 (Sigma) supplemented with glucose, magnesium sulfate, calcium chloride ferric ammonium citrate, riboflavin, niacinamide, pyridoxic hydrochloride, thiamine, biotin, 13 amino acids mixture (arginine, glutamine, lysine, histidine, glycine, isoleucine, phenylalanine, leucine, cysteine, aspartic acid, valine, serine and threonine), ampicillin, chloramphenicol and methionine at 37 degrees Centigrade for approximately 8 hours at approximately 175 rpm.
  • HBc protein was purified by multiple rounds of precipitation in 30 percent ammonium sulfate/re- suspension in 50 mM Tris / 500 mM sodium chloride / pH7.5 to form HBc azido-VLP intermediates. sHBc Azido-sVLP intermediate production
  • Hepatitis B core (HBc) virus-like particles (VLPs) were described by Lu, W. Chan, B. Y. Ko, C. C. VanLang and J. R. Swartz (2015) Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery. Proc Natl Acad Sci U S A Vol 1 12, Num 40, pp 12360. Plasmid construction and host cell bank preparation. The sequence encoding the stabilized human Hepatitis B core (sHBc) monomer of subtype adyw (Pasek et al., 1979) with the C-terminus truncated at amino acid 149 ( Figure 2) was codon optimized for E.
  • sHBc stabilized human Hepatitis B core
  • coli expression produced by overlapping oligonucleotide gene synthesis and cloned into a pET24a plasmid.
  • the plasmid was used to transform T7 Express Crystal Competent E. coli (High Efficiency) bacterial cells. Cells were selected for growth using kanamycin. Several colonies were selected for production of the sHBc protein and a high-producing line was identified and stored in a Research Cell Bank. sFIBc-producing E.
  • coli from the Research Cell Bank were expanded in shake flasks in minimal media, M9 (Sigma) supplemented with glucose, magnesium sulfate, calcium chloride, ferric ammonium citrate, riboflavin, niacinamide, pyridoxic hydrochloride, thiamine, biotin, 13 amino acids mixture (arginine, glutamine, lysine, histidine, glycine, isoleucine, phenylalanine, leucine, cysteine, aspartic acid, valine, serine and threonine), kanamycin and methionine at 37 degrees Centigrade for approximately 8 hours at approximately 175 rpm.
  • Lysis Buffer 50 mM Tris / 25 mM Imidazole / 5 mM DTT / pl l 7.5
  • the supernatant of the cell lysis was collected by centrifugation at 15,000g and azido-sHBc protein was purified by Nickel Column affinity chromatography. Formation of azido-sVLP intermediates was accomplished by dialysis into 1.5 M sodium chloride followed by dialysis into PBS and treatment with diamide to induce disulfide bond formation. Azido-sVLP was then dialysed into conjugation buffer for click reactions.
  • Lysis Buffer 50 mM Tris / 25 mM Imidazole / 5 mM DTT / pl l 7.5
  • CpG-X A CpG-containing oligonucleotide with a cross-linkable functional group (CpG-X) was synthesized and purified by Nitto Avecia Oligrow ⁇ custom services group (http://www.avecia.com/avecia/nitto-avecia-oligrow.html).
  • the sequence used is 5' TsGsAsCsTsGsTsGsAsAsCGsTsTsCsGsAsGsAsTsGsA- ⁇ 5-0ct-dU ⁇ 3', where 's' denotes a phosphorothioate linkage in the sequence and 5-Oct-dU is 5-octadiynyl dU at the 3 ' end of the oligonucleotide. Presence of a 5-Oct-dU moiety introduces an alkyne functional group to the CpG oligonucleotide, and the resulting CpG-X oligonucleotide is also referred to as CpG-alkyne. 5 octadiynyl dU attached at the 3' end of the oligo formed the basis of alkyne-azide conjugation to the VLP.
  • a peptide, PEG6-TEFCA with C-terminal amino acid sequence TEFCA was synthesized by CS Bio custom peptide services group (http;//www. csbio.com/custom-manufactured-peptides/).
  • the sequence used is N-terminal Pra-PEG6-T-E-F-C-A where single letter codes correspond to amino acids, Pra is L-propargylglycine and PEG6 is the amino acid analog formed when using Fmoc- NH-PEG6-propionic acid during peptide synthesis.
  • the HBc VLP-azide was mixed with CpG-alkyne and Pra-PEG6-TEFCA, sodium ascorbate, Tween-20 and potassium phosphate in an opaque reaction chamber.
  • the mixture was overlayed with argon gas.
  • the enhancer, tris(triazoylmethyl)amine [TTMA, Shanghai ChemPartner] was added and an aliquot of the mixture was withdrawn prior to adding the catalyst, tetrakis(acetonitrile)copper(I)hexafluorophosphate [tetrakis Cu(I), Sigma], Both the catalyst- containing and absent mixtures were allowed to react overnight at room temperature with mild agitation.
  • the Hek-BLUETM TLR9 cell-based assay (Invivogen). This assay is based on an engineered reporter cell that responds to TLR9 binding to a number of CpG-based sequences.
  • the HEK-BlueTM TLR cells are engineered HE 293 cells that stably co-express a human or murine TLR gene and an NF-KB/AP-1 -inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Upon activation a reporter substrate in the media reacts with a colorometric substrate, resulting in a color change.
  • DMEM Growth Medium
  • fetal bovine serum 50 U/ml penicillin, 50 ⁇ streptomycin, 100 ⁇ g/ml Normocin
  • HEK ⁇ Blue-hTLR5 cells were cultured according to the procedure specified by the vendor (InvivoGen - hkb-htlr5). Briefly, HE -Blue mTLR9 cells were thawed and transferred to 15 ml of warm growth medium. Cells were pelleted and seeded into a T-25 flask in 5 ml of growth medium without selection. After 2 passages, cells were seeded into growth media with selective antibiotics. Cells were maintained via passaging at 70-80% confluency.
  • HEK-Blue mTLR9 cells were seeded in triplicate wells of a 96 well plate at 2.5x10 4 cells/well in HEK-Blue detection media (InvivoGen - hb-det2).
  • CpG-azide oligonucleotide, and inactive control oligonucleotide were added to PBS at 3, 10, 30, and 100 nanomolar.
  • CpG-VLP preparations were added at 0.05, 0.16, 0.49 and 1.46 nanomolar. The plate was incubated at 37°C for more than 6 hours, and optical density was read at 650 nm (See Figure 5).
  • PEG12-TEFCA and PEG24-TEFCA with C-terminal amino acid sequence TEFCA was synthesized by CS Bio custom peptide services group (http://www.csbio.com/custom- manufactured-peptides/).
  • the sequences used were N-terminal Pra-PEG12-TEFCA and Pra- PEG12- PEG12-TEFCA, respectively, where single letter codes correspond to amino acids, Pra is L-propargylglycine and PEG 12 is the amino acid analog formed when using Fmoc-NH ⁇ PEG12- propionic acid during peptide synthesis.
  • reaction buffer conditions were Na ascorbate 200 ⁇ ; Tween .01%; 10 raM potassium phosphate, at pH 8; TTMA 0.25 mM; Tetrakis Cu(I) 500 ⁇ ; 30°C, overnight. After overnight incubation, 5 ul of reactions with and without copper were analyzed by reducing SDS-PAGE (See Figure 6, lanes 1 - 8). PEG 12-TEFCA-sVLP and PEG24-TEFCA-sVLP products were buffer exchanged into TE with 0.05% BME.
  • 38C 13 scFv fused on the N or C terminus with the InaD domain were constructed by gene synthesis. Sequences are shown in Figure 7 along with other Id scFv constructs. Plasmids were constructed for periplasmic expression of proteins in E.coli and used to transform competent cells. Cells were grown in 1 liter cultures in rich medium and protein expression was induced. Proteins were purified using Nickel affinity chromatography, and buffer was exchanged to TE with 0.05% beta-mercaptoethanol. Docking of InaD-scFv to PEG12-TEFCA-sVLP and PEG24-TEFCA- sVLP.
  • PEG 12-TEFCA-s VLP or PEG24-TEFCA-sVLP from Example 4 and ⁇ 100-fold molar excess, the approximate molarity of the number of docking sites available, of InaD-scFv were mixed and allowed to react at room temperature for 1 hour.
  • An aliquot of docking conjugation sample (C) was saved from each for analysis. Reactions were diafiltered using a 100,000 MWCO spin filter washing 3 times with PBS (-1000 fold buffer exchange). The first permeate (P) and diafiltration retentate (R) were saved for each docking conjugation. Reducing and non-reducing SDS-PAGE analysis demonstrates production of sVLP with attached InaD-scFv proteins for both intermediates (See Figure 6, lanes 9-20).
  • PEG6-TEFCA-sVLP intermediate was performed as described in Example 4 using the N-terminal Pra-PEG6-TEFCA sequence.
  • antiCD3-InaD and antiCD19-InaD (scFv antibody fragments fused to InaD, sequences BB_aHuCD3-InaD-His and BB_ahCD19-InaD-His respectively shown in Figure 8) were produced in E.coli as described for production of the 38C13scFv-InaD fusion in Example 5.
  • antiCD3-InaD and antiCD19-InaD were biotinylated using EZ-linkTM NHS-Biotin (ThermoFisher catalog number 20217) according to the manufacturer's directions.
  • AntiCD19-sVLP was produced using the docking procedure described in Example 5 by docking antiCD19-InaD to PEG6-TEFCA-sVLP intermediate at -100 fold molar excess.
  • AntiCD3-antiCD19-sVLP multispecific was produced using the docking procedure described in Example 5 by docking both antiCD3-InaD and antiCD19-InaD simultaneously, each at ⁇ 50 fold molar excess, to the PEG6-TEFCA-sVLP using the procedure described in Example 5.
  • Frozen PBMCs isolated from Luekopak (20s, female donor) were used to assess the binding capacity of antiCD19-sVLP and antiCD3-antiCD19-sVLP to normal human PBMCs.
  • Cells were thawed, washed twice with 50 mL of DPBS and cultured in RPMI with 5% normal human serum at 37C overnight. Cells were centrifuged and resuspended in FACS buffer. Aliquots of 1.6 million cells in 100 uL were incubated with 1.79 ug of antiCD19-sVLP or antiCD3-antiCD19-sVLP of FACS buffer for 45 minutes on ice.
  • Results from the PBMC staining are shown in Figures 9, 10 and 1 1 for the antiCD3- antiCD19-sVLP or antiCD19-sVLP alone and co-stained with antiCD3, antiCD4, antiCD19 and antiCD20 antibodies.
  • the multispecific antiCD3-antiCD19-sVLP stains cells that are also stained with both T- and B- cell markers while the antiCD19-sVLP stains cells that primarily stain with only B-cell markers.

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Abstract

The invention provides VLP intermediates, VLP intermediates free of viral genome, VLP intermediates attached to agents of interest, and methods for making a population of VLP intermediates to which one or more agents of interest may be or are attached. The method comprises synthesizing virus coat or capsid proteins having at least one non-naturally occurring amino acid to which one of two binding/interacting partners of an InaD domain complex can attach. The virus coat or capsid proteins may then be assembled into a population of VLPs. After assembly, one of two binding/interacting partners of the InaD domain complex is attached to the VLP thereby producing a population of VLP intermediates which may attach to one or more agents of interest at the site of the binding/interacting partner. The invention further provides VLPs produced by the methods of the invention.

Description

VIRUS-LIKE PARTICLE INTERMEDIATES, AGENTS ATTACHED THERETO, METHODS FOR MAKING AND USES THEREOF
Throughout this application various publications are referenced. The disclosures of these publications in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
Virus-like particles (VLPs) attached or joined to therapeutics agents in order to induce an immune response sufficient for use against diseases such as cancer are known. However, there remains a need for more reliable and easier production of such VLPs. In addition, there remains a need for novel VLPs having particular therapeutic agents to provide better agents against disease.
The invention solves the problem of the art by providing novel virus-like particles (VLPs) attached or joined to agents of interest and novel methods for making them.
SUMMARY OF THE INVENTION
The invention provides methods for making a population of VLP intermediates to which one or more agents of interest may be attached when desired. The method comprises synthesizing virus coat or capsid proteins having at least one non-naturally occurring amino acid to which one of two binding/interacting partners of an InaD (Inactivation no afterpotential D) domain complex can attach. The virus coat or capsid proteins may then be assembled into a population of VLPs. After assembly, one of two binding/interacting partners of the InaD domain complex is attached to the VLP thereby producing a population of VLP intermediates which may attach to one or more agents of interest at the site of the binding/interacting partner. The invention further provides VLPs produced by the methods of the invention.
The invention additionally provides methods for making VLPs attached to one or more agents of interest comprising making the population of VLP intermediates by the method above and attaching to the VLP intermediates one or more agents of interest. The agents of interest attaches or is fused to one of two binding/interacting partner of the InaD domain complex that recognizes and binds the other remaining binding/interacting partner on the VLP thereby resulting in VLPs attached to one or more agents of interest. Also provided are VLPs attached to one or more agents of interest produced by the methods of the invention.
Further provided are pharmaceutical compositions for treatment of cancer comprising the VLPs of the invention including pharmaceutical compositions admixed with a therapeutic agent and kits.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the amino acid and nucleotide sequences of a Hepatitis B core antigen (HBc) as well as nucleotide sequence of an expression plasmid, pET21 -Flep B Core 319.44.1.4, used to produce the HBc protein.
Figure 2 shows the amino acid and nucleotide sequences of the stabilized Hepatitis B core antigen (sHBc)
Figure 3 is a table providing exemplary molarities and ratios of components, total HepB monomer (HepB), approximate total molarity of azido groups (Azide), Pra-PEG6-TEFCA (PEG) and CpG- alkyne (CpG), in Click conjugation reaction.
Figure 4 is a reducing SDS-PAGE gel analysis of adapter-CpG-VLP intermediate, obtained from reacting VLP-azide with varying amounts of CpG-alkyne and Pra-PEG6-TEFCA adapter peptide in the presence or absence of Cu(I) as catalyst. Evidence of conjugation can be seen by a shift in migration to higher apparent molecular weight consistent with the combined HBc monomer+CpG oligonucleotide or FIBc monomer+adapter peptide molecular weights.
Figure 5 are two graphs showing dose response curve for adapter-CpG-VLP intermediates in a mTLR HEK-BlueI M assay for CpG activity. Note a general correlation between a higher CpG activity and a higher dose of adapter-CpG-VLP intermediate as well as a higher CpG activity and a greater CpG ratio or concentration used in the preparation of the adapter-CpG-VLP intermediate.
Figure 6 shows and SDS-PAGE gel analysis of Adapter-sVLP intermediates and subsequent docking of an InaD-scFv Id antigen to the intermediates. Evidence of correct intermediate formation is seen for two different adapter-sVLPs based on the shift in migration of conjugated sI IBc monomer in lanes 6 and 7. Evidence of docking of InaD-scFv Id is seen by retention of the InaD-scFv Id after docking with the sVLP.
Figure 7 shows the sequence of eight fusion proteins comprising a portion of an antibody of interest and a binding/interacting partner of the InaD domain complex.
Figure 8 shows the sequence of six fusion proteins comprising a portion of a protein antigen of interest and a binding/interacting partner of the InaD domain complex.
Figure 9 shows flow cytometry results for the staining of PBMCs with biotin labelled antiCD3- antiCD19-sVLP (MS-VLP). Show in clockwise order from the top left are MS-VLP stained (MS- VLP+) vs side scatter, MS-VLP co-stained with antiCD3 antibody (antiCD3+) and MS-VLP co- stained with antiCD19 antibody (antiCD19+) along with tabulation of the percentage of cells that are stained. In total 88.6 percent of all lymphocytes were stained by the MS-VLP. In the subset of either CD3 or CD19 positive cells, 91.8 percent were also stained with the MS-VLP. As expected cells that stain with the labeled MS-VLP also stain with antiCD3 and antiCD19 antibodies.
Figure 10 shows flow cytometry results for the staining of PBMCs with biotin labelled antiCD3- antiCD19-sVLP (MS-VLP). Show in clockwise order from the top left are MS-VLP stained (MS- VLP+) vs side scatter, MS-VLP co-stained with antiCD4 antibody (antiCD4+) and MS-VLP co- stained with antiCD20 antibody (antiCD20+) along with tabulation of the percentage of cells that are stained. In total 91.5 percent of all lymphocytes were stained by the MS-VLP. In the subset of either CD4 or CD20 positive cells, 96.2 percent were also stained with the MS-VLP. As expected cells that stain with the labeled MS-VLP also stain with antiCD4 and antiCD20 antibodies.
Figure 1 1 shows flow cytometry results for the staining of PBMCs with antiCD19-sVLP (aCD19- VLP+) vs side scatter, antiCD19-sVLP co-stained with antiCD4 antibody (antiCD4+) and antiCD19-sVLP co-stained with antiCD20 antibody (antiCD20+). As expected, cells that are stained by the antiCD19-sVLP are co-stained by antiCD20 but not antiCD4.
Figure 12 shows the amino acid and nucleotide sequences of a wild-type Hepatitis B core antigen (HBc).
Figure 13 shows the amino acid and nucleotide sequences of an InaD template.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
"Vaccine" as used herein, is a preparation comprising a virus-like particle (VLP) of the invention that when administered stimulates an immune response in a subject. A therapeutic vaccine may be administered during or after onset of a disease or disorder. A prophylactic treatment vaccine may be administered prior to onset of the disease such as a cancer and is intended to prevent, inhibit or delay onset of the disease.
"VLP" as used herein is a virus-like particle made from non-infectious subunits of a virus that form a structure, commonly in the form of an icosahedral matrix. VLP lacks a viral genome required for producing more infectious particles. The VLP may contain an assemblage of capsid protein monomers/subunits, for example, about a multiple of 60 coat or capsid protein monomers/subunits. VLPs based on an icosahedral structure may be formed by, e.g., 60 (T=l), 120 (T=2), 180 (T=3), 240 (T=4), 360 (T=7d), 420 (T=7), 780 (T=13), 960 (T=16), 1260 (T=21 ), 1500 (T=25), or 1620 (T=27) capsid proteins among other icosahedrons with different number of capsid proteins. In the case of VLP based on Hepatitis B virus (HBV), in one embodiment, 180 or 240 HBV core protein monomers (also referred to herein as viral coat polypeptides) can form two different types of VLPs arranged with, e.g., T=3 or T=4 icosahedron symmetry, respectively. For VLPs formed from F1BV core protein (also referred to as FlepB core protein), the invention provides in one embodiment a HBV coat protein truncated at the C-terminus leaving intact the first 149 amino acid at the N-terminus (aa 1 -149), and the HepB Core VLP is formed by the assembly of, e.g., 180 or 240 C-terminally truncated HepB core proteins.
"Capsid protein," as used herein, participates in the formation of a capsid, which generally form a helical or icosahedral structure. Capsid proteins participate in forming a protective shell around the genome of a virus particle. Throughout the application, a "capsid protein" may be used interchangeably with a "coat protein." In the case of hepatitis B virus (HBV), its "capsid protein" is often referred to as HBV core (HBc) protein. As used herein, HBc, HBC, HepB core, and HBV core are used interchangeably to refer to the Hepatitis B virus core protein monomer or variations thereof.
For example, VLP-azide refers to the presence of at least one azide functional group in VLP, such as through the incorporation of a non-natural amino acid with an azide functional group, e.g., azidohomoalanine. Azidohomoalanine may be used to substitute for methionine in a polypeptide chain in vivo by supplying azidohomoalanine to a methionine auxotroph grown in methionine- deficient medium. Alternatively, azidohomoalanine may be introduced in vitro synthesis using a cell-free protein synthesis (CFPS) system. Presence of an azide functional group permits participation in copper-catalyzed [3+2] cycloaddition or "click chemistry" with an alkyne function group. Other non-natural amino acids with an azide function group, including -azido-L- phenylalanine, are known in the art and are available and may be introduced into a polypeptide including a capsid protein, either intact or truncated, which can form a VLP.
For example, VLP-alkyne refers to the presence of at least one alkyne functional group in VLP, such as through the incorporation of a non-natural amino acid with an alkyne functional group, e.g., by supplying homopropargylglycine as a partial or complete substitute for methionine while expressing VLP in a methionine auxotroph strain, thus replacing methionine with the alkyne- containing non-natural amino acid. Alternatively, a non-natural amino acid may also be incorporated into a polypeptide at a desired site through the introduction of stop codon, e.g., amber stop codon UAG, and use of a suppressor tRNA charged with the desired non-natural amino acid, e.g., ?-propargyloxyphenylalanine, permitting site-specific incorporation of a non-natural amino acid through suppression of an engineered stop codon in a RNA transcript encoding a specific polypeptide (Bundy and Swartz, Bioconjugate Chem. 21 :255-263 (2010)). In either case, presence of an alkyne functional group permits participation in copper-catalyzed [3+2] cycloaddition or "click chemistry" with an azide functional group.
As used herein "Immune checkpoint inhibitors" refers to agents that block immune checkpoint proteins. Immune checkpoint proteins involve or are associated with inhibitory pathways present in immune cells important for maintaining self-tolerance and controlling the degree of an immune response. Blocking these pathways may lead to reduced modulation of immune cells, or increased activation of immune cells.
The term "vector," "construct" or "plasmid" as used herein refers to a recombinant nucleic acid molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A "vector," "construct" or "plasmid" may also be used outside the context of a particular host organism, such as in a cell free protein synthesis system following production RNA transcripts or in an in vitro transcription- translation system.
As used herein, an "active ingredient" includes any compound or composition of matter which, when administered to an organism (human or animal subject) induces a desired pharmacologic and/or physiologic effect by local and/or systemic action.
As used herein, a "subject" means a mammal. The mammal can be a human or an animal such as a non-human primate, mouse, rat, dog, cat, horse, monkey, ape, rabbit or cow, but are not limited to these examples. Mammals, other than humans, can be advantageously used as subjects that represent animal models of disorders associated with, e.g., cancer. In addition, the methods and compositions described herein can be used to treat domesticated animals and/or pets. The terms, "patient" and "subject" are used interchangeably. A subject can be male or female.
The VLP vaccines of the invention may be administered in the form of a pharmaceutical composition comprising the active ingredient in a pharmaceutically acceptable dosage form. Depending upon the type of disease and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses. Administration may be by methods including, but not limited to, intratumoral delivery, peritumoral delivery, intraperitoneal delivery, intrathecal delivery, intramuscular injection, subcutaneous injection, intravenous delivery, nasal spray and other mucosal delivery (e.g. transmucosal delivery), intra-arterial delivery, intraventricular delivery, intrasternal delivery, intracranial delivery, intradermal injection, electroincorporation (e.g., with electroporation), ultrasound, jet injector, and topical patches.
Formulations suitable for administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
When a VLP vaccine of the invention described herein is being given to a subject, a skilled artisan would understand that the dosage depends on several factors, including, but not limited to, the subject's weight, disease and progression thereof or tumor size or tumor progression. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine whether the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume or make other alterations to the treatment regimen. In an embodiment, a non-limiting example of an administration protocol useful for the invention comprises multiple administrations of the multivalent VLP vaccine of the invention during an initial period (such as, for example, a six week period, with, for example, administration every two weeks). Furthermore, an administration protocol may also include multiple administrations of the multivalent VLP vaccine of the invention at first administration (such as at multiple sites within a tumor at first administration of the multivalent VLP vaccine).
By "effective amount" as used herein with respect to a VLP vaccine of the invention, is meant an amount of the multivalent VLP, administered to a subject that results in an immune response by the mammal so as to inhibit the disease such as cancer. Further, an effective amount may include any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.
As used herein, "inhibiting a tumor" may be measured in any way as is known and accepted in the art, including complete regression of the tumor(s) (complete response); reduction in size or volume of the tumor(s) or even a slowing in a previously observed growth of a tumor(s), e.g., at least about a 10-30% decrease in the sum of the longest diameter (LD) of a tumor, taking as reference the baseline sum LD (partial response); mixed response (regression or stabilization of some tumors but not others); or no apparent growth or progression of tumor(s) or neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease, taking as reference the smallest sum LD since the treatment started (stable disease).
Tumor or cancer status may also be assessed by sampling for the number, concentration or density of tumor or cancer cells, alone or with respect to a reference. Tumor or cancer status may also be assessed through the use of surrogate marker(s), such as Her-2 in breast cancer or PSA in prostate cancer. As used herein, "treating" means using a therapy to ameliorate a disease or disorder or one or more of the biological manifestations of the disease or disorder; to directly or indirectly interfere with (a) one or more points in the biological cascade that leads to, or is responsible for, the disease or disorder or (b) one or more of the biological manifestations of the disease or disorder; to alleviate one or more of the symptoms, effects or side effects associated with the disease or disorder or one or more of the symptoms or disorder or treatment thereof; or to slow the progression of the disease or disorder or one or more of the biological manifestations of the disease or disorder. Treatment includes eliciting a clinically significant response. Treatment may also include improving quality of life for a subject afflicted with the disease or disorder (e.g., a subject afflicted with a cancer may receive a lower dose of an anti-cancer drug that cause side-effects when the subject is immunized with a composition of the invention described herein). Throughout the specification, compositions of the invention and methods for the use thereof are provided and are chosen to provide suitable treatment for subjects in need thereof.
In some embodiments, treatment with a composition of the invention described herein induces and/or sustains an immune response in a subject. Immune responses include innate immune response, adaptive immune response, or both. Innate immune response may be mediated by neutrophils, macrophages, natural killer cells (NK cells), and/or dendritic cells. Adaptive immune response includes humoral responses (i.e., the production of antibodies), cellular responses (i.e., proliferation and stimulation of T-lymphocytes), or both. Measurement of activation and duration of cellular response may be by any known methods including, for example, cytotoxic T- lymphocyte (CTL) assays. Humoral responses may be also measured by known methods including isolation and quantitation of antibody titers specific to the compositions of the invention (e.g., vaccines) such as IgG or IgM antibody fractions.
In some embodiments, the methods of treatment (e.g., immunotherapy) described herein is used as a stand-alone therapy without combining with any other therapy.
In other embodiments, the methods of treatment (e.g., immunotherapy) described herein provide adjunct therapy to other therapies, e.g., cancer therapy, prescribed for a subject. For example, the methods of treatment (e.g., immunotherapy) described herein may be administered in combination with radiotherapy, chemotherapy, gene therapy or surgery. The combination is such that the method of treatment (e.g., immunotherapy) described herein may be administered prior to, with or following adjunct therapy.
In accordance with the invention, the effect of anti-disease or disorder treatment (e.g., a cancer treatment) may be assessed by monitoring the patient, e.g., by measuring and comparing survival time or time to disease progression (disease-free survival). Any assessment of response may be compared to individuals who did not receive the treatment or were treated with a placebo, or to individuals who received an alternative treatment.
As used herein, "preventing" is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation. One skilled in the art will appreciate that prevention is not an absolute term. Prophylactic therapy is appropriate, for example, when a subject is considered at high risk for developing a particular disease or disorder (e.g., cancer), such as when a subject has a strong family history of a disease or disorder or when a subject has been exposed to e.g., a disease causing agent, e.g., a carcinogen.
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean a range of +1 - 10%.
The use of the singular includes the plural unless specifically stated otherwise. The word "a" or "an" means "at least one" unless specifically stated otherwise. The use of "or" means "and/or" unless stated otherwise. The meaning of the phrase "at least one" is equivalent to the meaning of the phrase "one or more." Furthermore, the use of the term "including," as well as other forms, such as "includes" and "included," is not limiting. The use of the term "containing," as well as other forms, such as "contains" and "contained," is not limiting. Also, terms such as "element" or "component" encompass both elements or components comprising one unit and elements or components comprising more than one unit unless specifically stated otherwise. COMPOSITIONS AND PRODUCTION METHODS OF THE INVENTION
The invention provides methods for making a population of VLP intermediates to which one or more agents of interest may attach when desired. The method comprises synthesizing virus coat or capsid proteins having at least one non-naturally occurring amino acid to which one of two binding/interacting partners of an InaD domain complex or equivalent can attach. The virus coat or capsid proteins may then be assembled into a population of VLPs. After assembly one of two binding/interacting partners of the InaD domain complex or equivalent (e.g. any PDZ domain complex (any PDZ domain with its binding partner) including those described herein) is attached to the VLP thereby producing a population of VLP intermediates which may attach to one or more agents of interest at the site of the binding/interacting partner. The invention further provides VLPs produced by the methods of the invention.
In an embodiment of the invention, the InaD domain complex or equivalent contains two binding/interacting partners. One binding/interacting partner may be an InaD domain or fragment thereof comprising a domain of InaD protein. The second remaining binding/interacting partner may be a polypeptide or a peptide comprising a sequence TEFCA. In one embodiment of the invention, the domain of InaD protein may be PDZ1 (PSD-95, DiscsLarge, ZO l ) domain or fragment, mutant, variant or derivative thereof.
In one embodiment of the invention, one of the two binding/interacting partners of the the InaD domain complex or equivalent may comprise a non-naturally occurring amino acid. For example, an InaD domain may comprise a non-naturally occurring amino acid which can be used to attach to the assembled VLP. In yet another embodiment, the polypeptide or peptide comprising a TEFCA sequence may comprise a non-naturally occurring amino acid which can be used to attach the InaD domain to the assembled VLP.
In accordance with the practice of the invention, the unnatural amino acid (also referred to herein as non-naturally occurring amino acid) may include an alkyne or azide reactant group for, e.g., Click chemistry reactions. In one embodiment, the unnatural amino acid having an azide reactant group may be a L-azidohomoalanine or £>-azido-L-phenylalanine. In another embodiment, the unnatural amino acid having an alkyne reactant group may be a L-homopropargylglycine or p- propargyloxyphenylalanine. In yet a further embodiment, the unnatural amino acid may be a N6- ((2-propynyloxy)carbonyl)-L-lysine or /?-acetyl-L-phenylalanine.
For attachment of a binding/interacting partner of an InaD domain complex or equivalent (also referred to herein as partner) to the VLP, the virus capsid polypeptides of the VLP may be modified before assembly to comprise at least one first unnatural amino acid (also referred to herein as non- natural amino acid or non-canonical amino acid (nnAA)) at a site of interest, such as the incorporation of azidohomoalanme during virus capsid polypeptide synthesis in the place of methionine, and the partner attached to an alkyne functional group, such as L-propargylglycine, L-propargylphenylalanine or L-propoargylalanine at the N-terminus of the partner, e.g., adapter peptide terminating with TEFCA sequence, or alternatively N- or C-terminus or surface exposed positions of a PDZ domain so as not to interfere with peptide binding, such as N-terminal PDZ domain from Drosophila inactivation no afterpotential D (InaD) protein (Kimple et al., 2001 , EMBO J. 20:20:4414-4422), to produce partner-X. The azide functional group of azidohomoalanine incorporated into a capsid protein of a VLP may participate in a (3+2) cycloaddition click reaction with an alkyne functional group of partner-X, resulting in VLP crosslinked to the binding/interacting partner. Other unnatural amino acid-containing capsid proteins within the same VLP may similarly participate in the (3+2) cycloaddition click reaction to produce a VLP attached or joined to a partner, producing a VLP with two or more partners.
In an embodiment of the invention, the VLP contains at least one or at least two unnatural amino acid per capsid monomer subunit (also referred to herein as a virus coat protein). For example, at least one-fiftieth of the total number of unnatural amino acids in a VLP may be used to attach a partner. In another embodiment, one-twentieth of the total number of unnatural amino acids in a VLP may be used to attach a partner. In another embodiment, one-tenth of the total number of unnatural amino acids in a VLP may be used to attach a partner. In another embodiment, about one fourth of the total number of unnatural amino acids in a VLP may be used to attach a partner. In a further embodiment, about one-third of the total number of unnatural amino acids in a VLP may be used to attach a partner. In yet another embodiment, about one half of the total number of unnatural amino acids in a VLP may be used to attach a partner. For example, about two-thirds of the total number of unnatural amino acids in a VLP may be used to attach a partner. In another example, about four-fifths of the total number of unnatural amino acids in a VLP may be used to attach a partner.
Also, in an embodiment of the invention, in the VLP, at least one-twenty fifth of the viral coat proteins may display a partner attached thereto. In another embodiment, at least one-tenth of the viral coat proteins may display a partner. In another embodiment, at least one-fifth of the viral coat proteins may display a partner. In yet another embodiment, about half of the viral coat proteins may display a partner. In a further embodiment, about two-thirds of the viral coat proteins may display a partner. In yet another embodiment, nearly all of the viral coat proteins may display a partner.
The invention additionally provides methods for making VLPs attached to one or more agents of interest comprising making the population of VLP intermediates by the method above and attaching to the VLP intermediates one or more agents of interest. The agents of interest may attach or may fuse to one of two binding/interacting partner of the InaD domain complex or equivalent. For example, an agent of interest may be attached or may be fused to a binding/interacting partner that recognizes and binds the other remaining binding/interacting partner which is on the VLP thereby resulting in a complex that generates VLPs attached to one or more agents of interest. Also provided by the invention are VLPs which are attached to one or more agents of interest and produced by the methods of the invention.
In accordance with the practice of the invention, one binding/interacting partner of the InaD domain complex or equivalent binds the other remaining binding/interacting partner on the VLP intermediates so that an InaD domain complex or equivalent is formed. This permits one or more agents of interest so bound to the complex to be attached, in turn, to a VLP intermediate. In an embodiment of the invention, an agent of interest may bind an InaD domain of the InaD protein. In another embodiment, an agent of interest may bind a polypeptide or peptide comprising the TEFCA sequence. Either configuration is contemplated. In either configuration, an InaD domain complex or equivalent is formed which permits the agent of interest to attach to the VLP, either directly or indirectly.
Merely by way of example, a InaD domain complex or equivalent is formed when the InaD domain of InaD protein binds the carboxyl terminus of a polypeptide or peptide comprising a TEFCA sequence. In one embodiment, the polypeptide or peptide TEFCA sequence of the InaD domain complex may be directly attached to the VLP. In another embodiment of the invention, the InaD domain of the InaD domain complex or equivalent may be directly attached to the VLP.
In accordance with the practice of the invention, either one of the binding/interacting partners of the InaD domain complex or equivalent may be attached to a spike region of the VLP. Merely by way of example, the spike region of the VLP may be the spike tip. But other portions of the spike region may be the attachment site.
In yet another embodiment, the InaD domain complex or equivalent may additionally comprise a covalent bond formed between the binding/interacting partners of the InaD domain complex or equivalent. For example, the covalent bond may be a disulfide bond formed between two cysteine residues. Merely by way of example, the disulfide bond may be formed under oxidizing condition permissive for the formation of a disulfide bond between two nearby cysteine residues.
In an embodiment of the invention, the agent of interest may be joined to a binding/interacting partner as a fusion protein. Alternatively, the agent of interest may be chemically joined to a binding/interacting partner of the InaD domain complex or equivalent.
Examples of an average amount of the agents of interest attached to the VLP is in an amount equivalent to about 10 to 50 copies per VLP, about 40 to 80 copies per VLP, about 70 to 170 copies per VLP, about 100 copies per VLP or about 160 to 240 copies per VLP.
Additionally, in an embodiment of the invention, the agent of interest may an immunostimulatory oligonucleotide comprising an unmethylated cytosine. Additionally, encompassed within the invention, the immunostimulatory oligonucleotide comprising an unmethylated cytosines (e.g., CpG oligonucleotide comprising a CpG dinucleotide with an unmethylated cytosine) may be attached to the VLP using the InaD domain complex or equivalent, described herein. Alternatively, the immunostimulatory oligonucleotide comprising an unmethylated cytosines (e.g., CpG oligonucleotide) may be attached to the VLP using, other means, e.g., as described herein in Example 2, where, for example, an alkyne reactive group of 5-octadiynyl dU attached to an end of a CpG oligonucleotide and an azide reactive group of a non-naturally occurring amino acid of the VLP participate in a Click chemistry reaction to yield a VLP attached to an immunostimulatory oligonucleotide. For example, in one embodiment, the agents of interest on the VLP may be a tumor associated antigen and/or an immunostimulatory oligonucleotide comprising an unmethylated cytosine. In yet another embodiment, the agents of interest on the VLP are an antibody or fragment or derivative therewith which binds a checkpoint protein and/or an immunostimulatory oligonucleotide comprising an unmethylated cytosine. In a further embodiment, the agents of interest on the VLP are an Id antigen and/or an immunostimulatory oligonucleotide comprising an unmethylated cytosine. Merely by way of example, the unmethylated cytosine may be a CpG oligonucleotide attached to a VLP intermediate in an amount (molar) such that the CpG oligonucleotide to VLP monomer ratio is equivalent to 1 :24 to 1 : 12, 1 : 12 to 1 :6, 1 :6 to 1 :3, 1 :3 to 2:3 or 1 :2 to 1 : 1 . In another example, the CpG oligonucleotide so attached to the VLP may be in an average amount equivalent to 10 to 50 copies per VLP, 40 to 80 copies per VLP, 70 to 170 copies per VLP, or 160 to 240 copies per VLP.
In one embodiment of the invention, the CpG oligonucleotide comprises a sequence, 5' - TGACTGTGAACGTTCGAGATGA-3 ' . Additionally, in another embodiment, the sequence has a mixture of phosphodiester and phosphorothioate bonds as shown in 5>T*G*A*c*T*G*T=t=G*A*A*CG*T*T*c*G*A*G*A*q^G*A 3 where * represents replacement of a phosphodiester bond with a phosphorothioate bond. In a further embodiment, the sequence has phosphorothioate bonds as shown in 5 >T*G*A*C*T*G*T*G*A*A*C*G*T*T*C*G*A*G*A*T*G*A 3 ', where * represents replacement of a phosphodiester bond with a phosphorothioate bond. Also, in one embodiment, the CpG oligonucleotide further comprises a 5-octadiynyl deoxyuridine or a modified deoxyuridine or a linker at the 3 ' end of the sequence.
In another embodiment, the binding/interactive partner of an InaD domain complex or equivalent attached to VLP may be in an amount such that the partner to VLP weight ratio is equivalent to 1 : 1000 to 1 : 100, 1 : 100 to 1 : 10, 1 : 10 to 1 :4, 1 :4 to 1 :2 or 1 :2 to 1 : 1 . In yet another embodiment, the partner attached to a VLP is in an amount (molar) such that the partner to VLP ratio is equivalent to 1 :24 to 1 : 12, 1 : 12 to 1 :6, 1 :6 to 1 :3, 1 :3 to 2:3 or 1 :2 to 1 : 1.
Examples of agents of interest include, but are not limited to, an isolated antibody that specifically recognizes and binds a leucocyte antigen; an isolated antibody that specifically recognizes and binds a cell adhesion molecule; an isolated antibody that specifically recognizes and binds a prostate specific membrane antigen; or an isolated antibody that specifically recognizes and binds an oncogene.
In accordance with the invention, the isolated antibody may be an isolated or purified monoclonal antibody. In further embodiments, the antibody or antigen-binding fragment is a labeled antibody, a bivalent antibody, a polyclonal antibody, a bispecific antibody, a chimeric antibody, a recombinant antibody, an anti-idiotypic antibody, a humanized antibody, or an affinity matured antibody. In other embodiments, the antigen-binding fragment is a camelized single domain antibody, a diabody, an scfv, an scfv dimer, a dsfv, a (dsfv)2, a dsFv-dsfv', a bispecific ds diabody, a Fv, a Fab, a Fab', a F(ab')2, or a domain antibody. In other embodiments, the antigen-binding fragment is operably attached to a constant region, wherein the constant region is a kappa light chain, gamma- 1 heavy chain, gamma-2 heavy chain, gamma- 3 heavy chain or gamma-4 heavy chain.
An example of a leucocyte antigen includes, but is not limited to, an immune checkpoint protein. Examples of immune checkpoint proteins include, but are not limited to, a PD-1 , a PDL1 , a PDL2, a B7-H3, a B7-H4, a CTLA-4, a LAG3, a KIR, a TIM3, a TIGIT, a BTLA, a CD 160, an A2aR, and/or a VISTA protein(s).
Additional examples of leucocyte antigens include, but are not limited to, CD1 , CD2, CD3, CD4, CD5, CD6, CD6L, CD7, CD8, CD9, CD10, CD1 1/CD18, CDl l a, CDl l b, CDl lc, CDl ld, CDwl 2, CD13, CD14, CD15, CD15s, CD16, CDwl 7, CD18, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD26, CD27, CD27L, CD28, CD29, CD30, CD30L, CD31 , CD32, Fc Gamma Receptor, CD33, CD34, CD35, CD36, CD37, CD38, CD40, CD40L, CD41 , CD42, CD42a, CD43b, CD43c, CD43, CD44, CD45, CD45-AP, CD46, CD48, CD49, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51 , CD52, CD53, DC54, CD55, CD56, CD57, CD58, CD59, CD60, C61, CD62, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66, CD66a, CD66b, DC66c, CD66d, CD66e, CD67, CD68, CD69, CD70, CD71 , CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79/BCR, CD80, CD81 , CD82, CD83, CD8w4, CD85, CD86, CD87, CD88, CD89, CD90, CD91 , CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101 , CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDwl 08, CD109, CD1 10, CD1 1 1 , CD1 12, CD1 13, CD1 14, CD1 15, CD1 16, CD1 17, CD1 18, CD 1 19, CD120, CD120a, CD120b, CD121 a, CDwl21 b, CD 122, CD122beta chain, CDwl 23, CD124, CD124alpha chain, CDwl25, CD126alpha chain, CD127alpha chain, CDwl 28, CD128A, CD128B, CD129, CD129alpha chain, CD130, CDwl31 , CD132gamma c chain , CD133, CD142, CD143, CD144, CDwl45, CD146, CD147, CD148, CDwl49, CDwl 50, CD151 , CD155, CD156, CD157, CD158, CD158a, CD158b, CD159, CD160, CD161 , CD162, CD163, CD164, CD165 and/or CD166; or portion(s) thereof.
An example of a prostate specific membrane antigen includes, but is not limited to, a FOHL1.
Examples of oncogenes include, but are not limited to, HER2/ErbB-2/Neu, HER3/ErbB-3, HER4/ErbB-4, and EGFR/ErbB-1 or portion thereof.
An example of a cell adhesion molecule includes, but is not limited to, an EPCAM.
The invention provides specific embodiments such as a VLP which includes antibodies or portion thereof that bind HER2 and antibodies or portion thereof that bind CD3; a VLP which includes antibodies or portion thereof that bind CD 19 and antibodies or portion thereof that bindsCD3; a VLP which includes antibodies or portion thereof that bind EPCAM and antibodies or portion thereof that bind CD3; a VLP which includes antibodies or portion thereof that bind FOLHl and antibodies or portion thereof that bind CD3; a VLP which includes antibodies or portion thereof that bind FIER2 and antibodies or portion thereof that bind FcGR; and a VLP which includes antibodies or portion thereof that binds CD20 and a second antibody or portion thereof that binds CD3. In further embodiments, the VLP may further comprise an additional agent of interest, e.g., CpG, antibodies or portion thereof that binds a checkpoint inhibitor, or both. Suitable average amounts of an antibody attached to a VLP may be an average amount equivalent to about 10 to 50 copies of an antibody per VLP, about 40 to 80 copies per VLP, about 70 to 170 copies per VLP, about 100 copies per VLP or about 160 to 240 copies per VLP.
In yet another embodiment of the invention, the agent of interest so attached to the VLP may be a tumor associated antigen. Examples of tumor-associated antigens include, but are not limited to, 17- 1 A, 707-AP, AFP, Annexin II, ART-4, BAGE, BAGE- 1 , b- catenin, BCG, bcr/abl, Bcr/abl el4a2 fusion junction, bcr-abl (polypeptide from translation of b3a2 transcript), bcr-abl (polypeptide from translation of b2a2 transcript), bcr-abl p210 (polypeptide from translation of b2a2 transcript), bcr-abl p210 (polypeptide from translation of b3a2 transcript), bullous pemphigoid antigen- 1 , CA 19-9, CA 125, CA21 5, CAG-3 cancer peptide, CAMEL tumor antigen, Cancer-testis antigen, Caspase-8, CCL3, CCL4, GD I 6, CD20, CD3, CD30, CD55, CD63, CDC27, CD -4, CDR3, CEA, cluster 5, cluster-5A, cyclin-dependent kinase- 4, Cyp-B, DAM- 1 0, DAM -6, Dek-cain, E7, EGFR, EGFRvlI 1, EGP40, ELF2 M, EpCAM, FucGM 1, G250, GA733, GAGE, GAGE- 1 -8, gastrin cancer associated antigen, GD2, GD3, globoH, glycophorin, GM 1 , GM2, GM3, GnTV, Gn-T-V, gplOO, Her-2/neu, HERV- -ME, high molecular weight-associated antigen, high molecular weight proteoglycan (IMPG), HPV-16 E6, HPV- 16 E7, HPVE6, HSP70-2M, HST- 2, hTERT, human chorionic gonadotropin (HCG), Human milk fat globule (HMFG), iCE, KIAA0205, KK- LC- 1 , KM-HN-1 , L6, LAGE- 1, LcOse4Cer, LDLR/FUT, Lewis A, Lewis v/b, M protein, MAGE- 1 , MVC, MAGE-A 1 - 12, MAGE-C2, MAHGE-3, MART- l /Melan-A, MC 1 R, ME491 , MUC 1 , MUC2, mucin, MUM- 1 , MUM-2, MUM-3, mutated p53, Myosin, MZ2-E, N9 neuraminidase, NA88, NA88-A, nasopharyngeal carcinoma antigen, NGA, NKl/c-3, Novel bcr/ablk fusion BCR exons 1 , 13, 14 with ABL exons 4, NY-ESO-l/LAGE-2, NY-ESO-lb, OC 125, osteosarcoma associated antigen- 1 , P I 5, p i 90 mimor bcr-abl (ela2), p53, Pml/RARa, Polysialic acid, PRAME tumor antigen, PSA, PSM, RU 1 , RU2, SAGE, SART- 1 , SART-2, SART-3, Sialyl LeA, Spl7, SSX-2, SSX-4, surface immunoglobulin, TAG- 1 , TAG-2, TEL/AML 1 , TP1, TRAG-3, TRP-1 (gp75), TRP-2, TRP2-1NT2, hTRT, tumor associated glycoprotein-72 (TAG-72), tyrosinase, u-PA, WT1 , and XAGE-lb, or an immunostimulatory fragment thereof.
In the embodiments where the vaccine contains VLPs also having antibodies against immune checkpoint inhibitors attached thereto, the antibodies against immune checkpoint inhibitors may be modified to comprise at least one second unnatural amino acid, wherein the first unnatural amino acid is different from, and reactive with the second unnatural amino acid. An example of one first unnatural amino acid is azidohomoalanine. An example of a second unnatural amino acid is propargyloxyphenylalanine. The azide functional group of azidohomoalanine incorporated into a capsid protein of a VLP may participate in a (3+2) cycloaddition click reaction with an alkyne functional group of propargyloxyphenylalanine incorporated into a polypeptide agent, such as a polypeptide-based antibody against immune checkpoint inhibitor, resulting in VLP crosslinked to a polypeptide agent. Other unnatural amino acid-containing capsid proteins within the same VLP may similarly participate in the (3+2) cycloaddition click reaction to produce a VLP attached or joined to an antibody against immune check point inhibitors, producing A VLP with two or more antibodies against immune check point inhibitors.
Yet another example of a therapeutic agent includes but is not limited to an agent that suppresses Treg activity. A therapeutic agent is an agent of interest as used herein. An example of a Treg suppressor includes agents that stimulate GITR (e.g., a GITR agonist), or a ligand, or a mimic of a ligand thereof. The agent may be an isolated antibody or fragment or derivative thereof that stimulates the target receptor (e.g., GITR). An example antibody is TRX-518. Another example protein is GITR-L. Further, the agent may be a small molecule that stimulates the target receptor.
Yet another example of a therapeutic agent includes but is not limited to an agent that depletes Treg cells. Examples of Treg depleting agents include agents that induce cell death in Treg cells (e.g., binding to a surface antigen on Treg cells (e.g., FR4, CD4, CD25 (IL-2Ra), CD 127 (IL7Ra), CD45RA, CD45RO, CD39, CD73, GITR, CD101 , GARP)) causing ADCC cytotoxicity (e.g., antibodies that mediate ADCC (antibody-dependent cell-mediated cytotoxicity)), CDC (complement-dependent cytotoxicity), or mediate cell death through other effector functions. Alternatively, examples of Treg depleting agents include agents that induce PCD (programmed cell-death). The agent may be an antibody or fragment or derivative thereof that induces cell death. Further, the agent may be a small molecule that induces cell death.
Yet another example of a therapeutic agent includes but is not limited to an agent (such as an antibody or small molecule) that binds to a tumor necrosis factor superfamily receptor (TNFRSFR) or ligand (TNFRSFRL). Examples include agents (such as an antibody or fragment or derivative thereof or small molecule) that stimulate a TNFRSFR or a ligand (e.g., CD 137 agonist, an NGFR agonist, a BAFFR agonist, an Osteoprotegerin agonist, a BCMA agonist, a OX40 agonist, a CD27 agonist, a RANK agonist, a CD30 agonist, a RELT agonist, a CD40 agonist, a TACI agonist, a DcR3 agonist, a TNF RI agonist, a DcTRAIL Rl agonist, a TNF agonist, a DcTRAIL R2 agonist, a TRAIL Rl agonist, a DR3 agonist, a TRAIL R2 agonist, a DR6 agonist, a TRAIL R3 agonist, a EDAR agonist, a TRAIL R4 agonist, a Fas agonist, a TROY agonist, a GITR agonist, a TWEAK R agonist, a HVEM agonist, a XEDAR agonist, a Lymphotoxin beta receptor agonist, a 4- IBB agonist, a APRIL agonist, a BAFF agonist, a TL1 A agonist, a TWEAK agonist, and a LIGHT agonist).
Examples also include inhibitors of a TNFRSFR or ligand thereof (e.g., CD137 antagonist, an NGFR antagonist, a BAFFR antagonist, an Osteoprotegerin antagonist, a BCMA antagonist, an OX40 antagonist, a CD27 antagonist, a RANK antagonist, a CD30 antagonist, a RELT antagonist, a CD40 antagonist, a TACI antagonist, a DcR3 antagonist, a TNF RI antagonist, a DcTRAIL Rl antagonist, a TNF antagonist, a DcTRAIL R2 antagonist, a TRAIL Rl antagonist, a DR3 antagonist, a TRAIL R2 antagonist, a DR6 antagonist, a TRAIL R3 antagonist, a EDAR antagonist, a TRAIL R4 antagonist, a Fas antagonist, a TROY antagonist, a GITR antagonist, a TWEAK R antagonist, a HVEM antagonist, a XEDAR antagonist, a Lymphotoxin beta receptor antagonist, a 4- IBB antagonist, a APRIL antagonist, a BAFF antagonist, a TL1A antagonist, a TWEAK antagonist, and a LIGHT antagonist). Merely by way of example, these inhibitors may be an antibody or fragment or derivative thereof or a small molecule directed against a TNFRSFR or a ligand thereof.
The therapeutic agent may be an anti-cancer agent that inhibits cell proliferation or induces apoptosis. Examples of therapeutic agents include, but are not limited to, lenalidomide; ipilimumab; rituximab; alemtuzumab; ofatumumab; flavopiridol; Adriamycin; Dactinomycin; Bleomycin; Vinblastine; Cisplatin; ABT-199; acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; amino glutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; lloxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; ibrutinib; idelalisib; idarubicin hydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; obinutuzumab; ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfmer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rituximab; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogerranium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfm; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicin hydrochloride.
In another embodiment, the therapeutic agent may be an alkylating agent which may be nitrogen mustards, ethylenimine and methylmelamines, alkyl sulfonates, nitrosoureas, or triazenes. Using a similar strategy, VLP with reactive azide functional groups could be attached (e.g., coupled) to other non-proteinaceous or non-nucleic acid-based therapeutic agents, such as antagonist ligands or inhibitors, including small molecule inhibitors, of immune checkpoint proteins which are not protein or nucleic acid, through functionalizing these agents with an alkyne functional group. Such non-proteinaceous or non-nucleic acid-based agents may be attached to a VLP through the (3+2) cycloaddition click reaction to produce a VLP attached or joined to non- proteinaceous or non-nucleic acid-based agents.
Further, the VLPs of the invention are VLPs free of a viral genome which may comprise virus capsid polypeptides derived from any of an Adenoviridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papillomaviridae, Rhabdoviridae, Togaviridae or Paroviridae families. Preferably, the VLP is a stable icosahedral VLP free of a viral genome.
Specifically, examples of viruses from which the virus coat or capsid proteins may be derived include but are not limited to any of a bacteriophage, adenovirus, coxsackievirus, Hepatitis A virus, poliovirus, Rhinovirus, Herpes simplex virus, Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, Human herpes virus, Hepatitis B virus, Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, HIV, Influenza virus, Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human metapneumovirus, Human papillomavirus, Rabies virus, Rubella virus, Human bocavirus or Parvovirus, and Norovirus. In one embodiment, the bacteriophage may be a MS2 bacteriophage, PI like viruses, P2 like viruses, T4 like viruses, P22 like viruses, and lambda-like viruses. A VLP derived from Hepatitis B virus is preferred.
The invention additionally provides pharmaceutical composition for treatment of cancer comprising the VLPs of the invention, e.g., the VLP intermediates (not yet attached to an agent of interest) or a VLP attached to one or more agents of interest. In one embodiment of the invention, the pharmaceutical composition further comprises a therapeutic agent admixed therein.
Further, the invention additionally provides for a pharmaceutical composition for treatment of a solid tumor or cancer comprising any of the VLP compositions of the invention and one or more therapeutic agents that may be admixed with the VLP compositions or, alternatively, the therapeutic agent(s) may administered before or after the VLP compositions of the invention. In an embodiment where a second therapeutic agent is added to the treatment, the second therapeutic agent may be the same as the therapeutic agent admixed or sequentially administered or the second therapeutic agent may be a different therapeutic agent. The terms "VLP compositions of the invention" and "VLPs of the invention" include a VLP intermediate (including a VLP intermediate attached to one or more agents of interest but free of an InaD complex or equivalent, such as a VLP intermediate attached to a CpG oligonucleotide free of an InaD complex or a VLP intermediate attached to a TLR agonist free of an InaD complex), a VLP free of a viral genome comprising capsid proteins or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, compositions containing them (e.g., pharmaceutical compositions, dosage forms, immunostimulatory compositions, immunomodulatory compositions,, vaccines); and VLPs having two of more agents of interest attached thereto through means other than by the InaD domain complex or equivalent thereof (free of InaD domain complex or equivalent). The terms "VLP compositions of the invention" and "VLPs of the invention" may be used interchangeably.
Examples of therapeutic agents include, but are not limited to, an agent that inhibits an immune checkpoint protein (also referred to herein as an immune checkpoint inhibitor). Examples of immune checkpoint inhibitors include agents that inhibit PD- 1 (e.g., a PD-1 inhibitor or an anti- PD- 1 agent); CTLA-4 (e.g., a CTLA-4 inhibitor or an anti-CTLA-4 agent); LAG3 (e.g., a LAG 3 inhibitor or an anti-LAG3 agent); KIR (e.g., a KIR inhibitor or an anti-KIR agent); TIM3 (e.g., an ΤΊΜ3 inhibitor or an anti-TIM3 agent); TIGIT (e.g., a TIGIT inhibitor or an anti-TIGIT agent); BTLA (e.g., a BTLA inhibitor or an anti-BTLA agent); CD160 (e.g., a CD160 inhibitor or an anti- CD 160 agent); VISTA (e.g. an VISTA inhibitor or an anti-VISTA agent); and A2aR (e.g., an A2aR inhibitor or an anti-A2aR agent). Alternatively, the immune checkpoint inhibitor may inhibit a ligand of a checkpoint receptor, examples of which would include PDL 1 (e.g., a PDL 1 inhibitor or an anti-PDLl agent), PDL2 (e.g., a PDL2 inhibitor or an anti-PDL2 agent), B7-IT3 (e.g., a B7- H3 inhibitor or an anti-B7I I3 agent); B7-LI4 (e.g., a B7-H4 inhibitor or an anti-B7-H4 agent). The agent may be an isolated antibody or fragment or derivative thereof that blocks the target receptor (e.g., PD- 1 , B7-H3, B7-H4, CTLA-4, LAG3, KIR, TIM3, TIGIT, BTLA, CD160, or A2aR) or a ligand. Further, the agent may be a small molecule that blocks activity of an immune checkpoint protein or a ligand. The ligand may be an antagonist or selective modulator of an immune checkpoint protein, such as a target receptor in an immune checkpoint pathway.
Additional examples of therapeutic agents include, but are not limited to, an agent that is a co- stimulatory molecule. Examples of co-stimulatory agents include HVEM; ICOSL; 4- 1 BBL; OX40L; GITRL; CD40L; and agents that stimulate CD28 (e.g., a CD28 agonist); 1COS (e.g., an ICOS agonist); CD137 (e.g., a CD 137 agonist); OX40 (e.g., an OX40 agonist); CD27 (e.g., an CD27 agonist); CD40 (e.g., a CD40 agonist); CD40L (also known as gp-39) (e.g., an CD40L agonist); LIGHT (e.g., a LIGHT agonist); LT-alpha (e.g., an LT-alpha agonist); GITR (e.g., a G1TR agonist); and a mimic of a ligand of the aforementioned. The agent may be an isolated antibody or fragment or derivative thereof that stimulates the target receptor (e.g., CD28, ICOS, CD 137, OX40, CD27, CD40, CD40L, LIGHT, LT-alpha, and/or GITR also known as TNFRSF 18) such as an anti-CD28 antibody, anti-ICOS antibody, anti-CD 137 antibody, anti-OX40 antibody, anti-CD27 antibody, anti-CD40 antibody, anti-CD40L antibody, anti-LIGHT antibody, anti-LT- alpha antibody, and anti-GITR antibody. Further, the agent may be a small molecule that stimulates the target receptor.
COMPOSITIONS OF THE INVENTION
The invention provides VLP intermediate to which one or more agents of interest may attach when desired comprising a VLP comprising capsid protein or viral coat proteins having at least one non-naturally occurring amino acid to which one of two binding/interacting partners of an InaD domain complex or equivalent is attached and the VLP is free of a viral genome. In accordance with the practice of the invention, the VLP may be a stabilized VLP. In one embodiment of the invention, the capsid protein or viral coat protein is a monomer. In another embodiment, the capsid protein or viral coat protein is a dimer. In accordance with the practice of the invention, the capsid protein or viral coat protein is assembled into a VLP.
In one embodiment, the VLP is formed by the assembly of HBc polypeptides, corresponding to the first 149 amino acids of a Hepatitis B capsid protein or coat protein. In one example, the VLP is formed by the HBc polypeptide with the amino acid sequence provided in Figure 1 with the nucleic acid encoding this polypeptide provided below. This nucleic acid may be used to produce the HBc polypeptide with an amino acid sequence provided in Figure 1. In another example, the VLP is formed by the HBc polypeptide with the amino acid sequence provided in Figure 2. A VLP may contain one type of HBc polypeptide or alternatively a VLP may be formed by a mixture of more than one HBc polypeptides such as for example the two peptides provided in Figure 2.
In an embodiment of the invention, a VLP intermediate may be attached to one or more agents of interest (not through the InaD domain complex), wherein the VLP intermediate attached to such agents can be later used to attach additional agents of interest when so desired (though the InaD domain complex or other means, e.g. cross linking). The VLP intermediate may be stored or stock piled. One preferred embodiment provides a VLP intermediate attached to a CpG oligonucleotide but not through the InaD domain complex.
In an embodiment of the invention, the InaD domain complex or equivalent may contain two binding/interacting partners, wherein one binding/interacting partner is an InaD domain or fragment thereof or equivalent comprising a domain of InaD protein or equivalent and wherein a second binding/interacting partner is a peptide with at least four amino acids to which the InaD domain or fragment or equivalent binds. For example, the peptide with at least four amino acids may be a binding/interacting partner of PDZl domain of the InaD protein. In another embodiment, the peptide is 5 amino acid long. For example, the peptide may comprise a sequence TEFCA. Further, as described above, in one embodiment, the peptide with at least four amino acids additionally comprises a non-naturally occurring amino acid and optionally a linker separating the peptide from the non-naturally occurring amino acid which is or can be used to attach the peptide to a VLP.
In one embodiment of the invention, the domain of an InaD protein may be a PDZl domain or a first PDZ domain of a five PDZ domain-containing InaD protein comprising an amino acid sequence from position 1 1 to 107 of an InaD protein (e.g., having a GenBank Accession Number AAF81203.1 or a closely corresponding sequence in Figure 13 (beginning at amino acid position 2 (alanine) and ending at amino acid position 98 (lysine)). The PDZl domain may have a mutation such as a substitution mutation. An example of substitution mutation include an amino acid change at position 62 of GenBank Accession Number AAF81203.1 from a cysteine residue to an alanine residue, such that the resulting amino acid sequence is AGELIHMVTLDKTGKKSFGICIVRGEVKDSPNTKTTGIFIKGIVPDSPAHLAGRLKVGDRI LSLNGKDVRNSTEQAVIDLIKEADFKIELEIQTFDK. or as provided in Figure 13. Figure 13 also provides a nucleic acid sequence which may be used to produce an InaD domain used in the invention.
In another embodiment of the invention, the peptide binding/interacting partner of PDZl domain comprises a PDZl -binding consensus sequence, X-F/W/Y-C-F/A-COOH, where X denotes any amino acid and COOFI, F/W/Y are amino acid residues at the 3rd amino acid from the C-terminus, C denotes cysteine at the 2"d amino acid from the C-terminus, and -COOH denotes a non-side chain carboxylic acid/carboxylate group of the last amino acid at the C-terminus of the peptide. In a further embodiment, the peptide binding/interacting partner of PDZl domain includes any of XFCF-COOH, XWCF-COOFI, and XYCF-COOH, where X denotes any amino acid, F denotes phenylalanine, W denotes tryptophan, Y denotes tyrosine, and -COOH denotes a non-side chain carboxylic acid/carboxylate group of phenylalanine at the C-terminus of the peptide. In yet a further embodiment, the peptide binding/interacting partner of PDZl domain includes any of VFCF-COOH, KYCF-COOH, LYCF-COOH, QYCF-COOH, EYCF-COOH, QWCF-COOH, RFCF-COOH, SYCF-COOH, GYCF-COOH, KWCF-COOH and PYCF-COOH, where X denotes any amino acid and -COOH denotes a non-side chain carboxylic acid/carboxylate group of phenylalanine at the C-terminus of the peptide.
In an additional embodiment, the peptide binding/interacting partner of PDZl domain comprises a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide. In yet another additional embodiment, the peptide comprises a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide may be one not found in nature, may be from a peptide library or may be derived from C-terminal end of naturally occurring proteins identifiable in publically available databases. Examples of peptides having a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide includes, but are not limited to, peptides having as their last three amino acids, FCA-COOH, TCL-COOH, VCV-COOH, TCA-COOH, SCV-COOH, ACA- COOH, ICA-COOH, LCL-COOH, FCL-COOH, YCA-COOH, SCA-COOH, LCT-COOH, SCV- COOH, ACL-COOH, TCF-COOH, SCL-COOII, TCV-COOH, ACV-COOH, FCL-COOH, XCY- COOH and FCF-COOH, wherein the 4lh amino acid from the C-terminus may be any amino acid and -COOFI denotes a non-side chain carboxylic acid/carboxylate group of the last amino acid at the C-terminus of the peptide. In yet another embodiment, the peptide having a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide may be the sequence TEFCA or TEFCF. In a further embodiment of the invention, the peptide with at least four amino acids may be 4, 5, 6, 7, 8 or 9 amino acid in length. In a further embodiment, the cysteine amino acid as a penultimate amino acid from C-terminus of the peptide may participate in intermolecular disulfide bond formation with the PDZ1 domain of the InaD protein. For example, the intermolecular disulfide bond formation may occur with cysteine-31 of the PDZ1 domain of the InaD protein (GenBank Accession Number AAF81203.1 ), corresponding to cysteine at position 22 in the amino acid sequence of the InaD domain of Figure 13).
In an embodiment of the invention, the InaD complex may additionally comprise an intermolecular disulfide bond between a cysteine of the InaD domain and a cysteine of the peptide binding/interacting partner. For example, the disulfide bond formation between the two binding/interacting partners may further stabilizes the InaD domain complex.
In yet a further embodiment, the peptide binding/interacting partner of PDZ1 domain may be free of a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide. For example, the peptide free of a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide includes any of the peptides having as their last three amino acids, YSF-COOH, WSF- COOH, YEF-COOH, FMF-COOH, YYF-COOH, YMF-COOH and WTF-COOH, wherein the 4lh amino acid from the C-terminus may be any amino acid and -COOH denotes a non-side chain carboxylic acid/carboxylate group of phenylalanine at the C-terminus of the peptide. Additionally, in another embodiment, the peptide free of a cysteine amino acid as a penultimate amino acid from C-terminus of the peptide may be any of the peptides having as their last four amino acids, SYSF- COOH, CYSF-COOH, DWSF-COOH, VYEF-COOH, VFMF-COOH, HYSF-COOH, EYYF- COOH, NYMF-COOI I and DWTF-COOH, wherein -COOH denotes a non-side chain carboxylic acid/carboxylate group of phenylalanine at the C-terminus of the peptide. Also, in one embodiment of the invention, the peptide binding/interacting partner within the InaD domain complex adopts a β-strand structure or an extended conformation, wherein presence of a penultimate cysteine residue from the C-terminus of the peptide may permit formation of an intermolecular disulfide bond with the InaD domain and wherein presence of a phenylalanine 3 amino acids from the C-terminus of peptide may induce an abrupt turn to the β-strand or extended conformation.
In one embodiment of the invention, the equivalent of a domain of InaD protein may be any PDZ domain which forms a β-barrel structure comprising 6 β-strands (B l to B6), a short a-helix (Al ) and a long α-helix (A2) and wherein the β-strands and a-helices are arranged in the order, B 1 -B2- B3-A 1 -B4-B5-A2-B6, or a circular permutation with a carboxylate-binding loop between β- strands, B l and B2, found at one end of a groove formed by a-helix A2 and β -strand B2, wherein a peptide binding/interacting partner of 4 to 9 amino acid in length can fit and form hydrogen bonds, ionic interactions and hydrophobic interactions, and optionally a covalent bond between binding partner cysteine residues, in the InaD domain complex equivalent. Examples of the peptide binding/interacting partner may be any peptide sequence which adopts a β-strand, extended conformation or a β-hairpin conformation and permits binding to a PDZ domain binding pocket comprising a-helix A2 and β-strand B2, wherein the peptide may comprise a non-side chain carboxylic acid/carboxylate group normally present at a C-terminus of a peptide, free of a non-side chain carboxylic acid/carboxylate group normally present at a C-terminus of a peptide, a cysteine residue as a penultimate amino acid from the C-terminus or be free of said cysteine, a peptide with no free ends so as to lack non-side chain amino or carboxyl groups normally associated with ends of a peptide, and 4 to 9 amino acids or a combination thereof.
In another embodiment of the invention, the equivalent of a domain of InaD protein may be a variant of a β-barrel structure of the PDZ domain, wherein the variant can function to bind a peptide binding/interacting partner of 4 to 9 amino acid in length. In yet another embodiment, the equivalent of a domain of InaD protein or PDZ domain or its variant, or the peptide binding/interacting partner of the domain of InaD protein or PDZ domain or its variant comprises a primary sequence or structure, secondary structure or tertiary structure as provided in RCSB Protein Data Bank for structure of a PDZ domain or a PDZ-domain variant in a complex with a peptide binding/interacting partner.
Examples of the structure of PDZ domain or its variant or PDZ domain or its variant in a complex with a peptide binding/interacting partner may be found in RCSB Protein Data Bank with Accession Numbers 1IIIJ, 1N7T, 2101, 2EXG, 2H3L.1W9E, 1W90, 1W9Q, 1Z86, 2FNE, 2FCF, 1 YBO, 2FE5, 2F5Y, 2CSS, 1 Y7N, 2F0A, 2CSJ, 2CS5, 1X5N, 1X5Q, 1X5R, 1XZ9, 1X45, 1VJ6, 1TP3, 1TP5, 1TQ3, lZUB, 2BYG, 1U37, 1U38, 1U39, 1U3B, 1V1T, 1 WF8, 1 WI4, 1ZOK, 1VA8, 1T2M, 1X8S, 1WJL, 1WH1, 1WHA, 1WHD, 1WI2, 1WIF, 1WFV, 1WG6, 1WF7, 1WFG, 1VB7, 1VAE, 1V6B, 1V62, 1V5L, 1V5Q, 1RGR, 1R6J, 1RGW, 1UM7, 1RY4, 1UM1, 1UJU, llJJV, 1UJD, 10ZI, 1Q30, 1Q3P, 1RZX, UJIT, 1UHP, 1UFX, 1Q7X, 1UEW, 1UEZ, 1UF1, 1UEP, llJEQ, 1P1D, 1P1E, 1MC7, 1N7E, 1N7F, 1NTE, 10BY, 10BZ, 10BX, 1L60, 1N99, 1IU0, 1UJ2, 1NF3, 1N7T, 1MFG, 1MFL, 1GQ5, 1M5Z, 1D5G, 1GQ4, 1GM1, 1 KEF, 1IHJ, 1192, 1G90, 3 PDZ, 1QLC, 1QAU, 1QAV, 1B8Q, 2PDZ, 1BE9, 1BFE, 1KWA, 1PDR, 1FC9, 1LCY or 1116.
Examples of sequences of the equivalent of a domain of InaD protein or PDZ domain or its variant, or the peptide binding/interacting partner of the domain of InaD protein or PDZ domain or its variant include sequences for the group of domain of InaD protein or PDZ domain or its variant and respective peptide binding/interacting partners as provided in RCSB Protein Data Bank with Accession Numbers 1IHJ, 1N7T, 2101, 2EXG, 2H3L,1 W9E, 1 W90, 1W9Q, 1Z86, 2FNE, 2FCF, 1YBO, 2FE5, 2F5Y, 2CSS, 1Y7N, 2F0A, 2CSJ, 2CS5, 1X5N, 1X5Q, 1X5R, 1XZ9, 1X45, 1VJ6, 1TP3, 1TP5, 1TQ3, 1ZUB,2BYG, 1U37, 1U38, 1U39, 1U3B, 1V1T, 1WF8, 1WI4, 1ZOK, 1VA8, 1T2M, 1X8S, 1WJL, 1WH1, 1WHA, 1WHD, 1WI2, 1WIF, 1WFV, 1WG6, 1WF7, 1WFG, 1VB7, 1VAE, 1V6B, 1V62, 1V5L, 1V5Q, 1RGR, 1R6J, 1RGW, 1UM7, 1RY4, 1UM1, 1UJU, 1UJV, 1UJD, 10ZI, 1Q30, 1Q3P, 1RZX, 1UIT, UJHP, 1UFX, 1Q7X, 1UEW, 1UEZ, 1UF1, 1UEP, 1UEQ, 1P1D, 1P1E, 1MC7, 1N7E, 1N7F, 1NTE, 10BY, 10BZ, 10BX, 1L60, 1N99, 1IU0, 1IU2, 1NF3, 1N7T, 1MFG, 1 FL, 1GQ5, 1M5Z, 1D5G, 1GQ4, 1GM1, 1 EF, llFIJ, 1192, 1G90, 3PDZ, 1QLC, 1QAU, 1QAV, 1B8Q, 2PDZ, 1BE9, 1BFE, 1KWA, 1PDR, 1FC9, 1LCY or 1116. In an embodiment of the invention, the peptide binding partner with at least four amino acids to which the InaD domain or fragment or equivalent binds may have a free amino terminus, a free carboxyl terminus or lack both free amino and carboxyl ends. In another embodiment, the peptide binding partner with at least four amino acids to which the InaD domain or fragment or equivalent binds comprises a free carboxyl terminus with a non-side chain carboxylate or carboxylic acid group.
In one embodiment of the invention, the non-naturally occurring amino acid and optionally a linker separating the peptide from the non-naturally occurring amino acid is attached N-terminal to the peptide. Examples of linkers include, but are not limited to, a polyethylene glycol (PEG) or one or more amino acids. Examples of PEGs include PEGs comprising more than 3 ethylene glycol repeats and less than 50 ethylene glycol repeats. Suitable examples of PEG include any of PEG&, PEG i2 and PEG24.
As described above, embodiments of the invention include wherein the non-naturally occurring amino acid provides an alkyne or azide reactant group for Click chemistry reactions. Examples of non-naturally occurring amino acids having an azide reactant group include L-azidohomoalanine, /7-azido-L-phenylalanine or N6-((2-azidoethoxy)carbonyl)-L-lysine. Examples of non-naturally occurring amino acids having an alkyne reactant group include L-homopropargylglycine, L- propargylglycine, / propargyloxyphenylalanine or N6-((2-propynyloxy)carbonyl)-L-lysine. In a specific embodiment, the non-naturally occurring amino acid is N6-((2-propynyloxy)carbonyl)-L- lysine, N6-((2-azidoethoxy)carbonyl)-L-lysine or /?-acetyl-L-phenylalanine.
In one specific embodiment of the invention, a VLP of the invention (e.g., a VLP intermediates or a VLP intermediate attached to one or more agents of interest) is formed by a hepatitis B core protein (HBc) or its variant or a portion thereof. However, as described above, other viral species are contemplated and encompassed herein.
For example, in one embodiment, the HBc polypeptide is 149 amino acid in length and is derived from the first 149 amino acids at the N-terminus of a hepatitis B virus (HBV) coat protein of HBV subtype adyw (UniProt accession number P03147) . In another embodiment, the HBc polypeptide variant from HBV subtype adyw has a methionine-to-serine substitution at amino acid 66 (M66S) and a leucine-to-methionine substitution at amino acid 76 (L76M), wherein the variant comprises an amino acid sequence as shown in Figure 1. In a specific embodiment, the VLP comprises an HBc polypeptide comprising the amino acid sequence of Figure 1 or portion thereof.
In yet another embodiment, the VLP is a stabilized VLP comprising hepatitis B core proteins (HBc) or its variant and wherein at least two amino acids of a HBc polypeptide are substituted with cysteine residues that can form intermolecular disulfide bonds when the HBc is assembled into a virus like particle (VLP), thereby stabilizing the VLP structure. In a specific embodiment, the stabilized VLP comprises an HBc polypeptide comprising the amino acid sequence of Figure 2 or portion thereof. In one embodiment, the amino acid substitutions for stabilizing a VLP are selected from any of [D29C, R127C]; [T109C, V120C]; [Y132C, N136C]; [Y132C, A137C]; [R133C, N136C]; [R133C, A137C]; [P134C, P135C]; [P134C, N136CJ; [P134C, A137C]; and [P135C, N136C]. In another embodiment, the amino acid substitutions for stabilizing a VLP include [D29C, R127C]; [P134C, N136C]; or [D29C, R127C, P134C, N 136C]. In yet a further embodiment, the amino acid substitutions for stabilizing a VLP include [D29C, R127C].
In an embodiment of the invention, one of two binding/interacting partners of the InaD domain complex or equivalent is attached to a spike region of the VLP. In one embodiment, the spike region of the VLP is a spike tip. Also, in one embodiment, the amino acids of the spike region at residues 73-81 are substituted to reduce the negative charge, relative to an amino acid sequence as shown in Figure 1. In yet another embodiment, the spike region comprises a hydrophobic pocket and wherein the amino acids of the hydrophobic pocket at residues 57-81 are substituted relative to an amino acid sequence as shown in Figure 1. For example, the amino acid substitutions include any one of [159V, L60S, G63R, D64E, L65V, M66T, T67D, L68F, A69G, T70D, T74N, L76M, E77Q, P79Q, S81 A, S87N, T91A, V93I, F97I] or [Ί74Ν, L76M, E77Q, P79Q, S81A],
In another embodiment, the VLP may comprise an amino acid sequence NxMQxQxA, where x may be any amino acid. Additionally, in yet another embodiment, the VLP may comprise an amino acid sequence VSxxREVTDFGDxxxNxMQxQxAxxxxxNxxxAxIxxxI, where x may be any amino acid. In a further embodiment, the VLP comprises an amino acid sequence NxMQxQxA, where x may be any amino acid so long as the amino acid does not disrupt HBc dimer formation, formation of a HBc hydrophobic pocket and formation of HBc dimer spike. Also, in a further embodiment, the VLP comprises an amino acid sequence VSxxREVTDFGDxxxNxMQxQxAxxxxxNxxxAxIxxxl, where x may be any amino acid so long as the amino acid does not disrupt HBc dimer formation, formation of a HBc hydrophobic pocket and formation of HBc dimer spike.
The invention further provides an embodiment, wherein the VLP further comprises at least one pair of amino acid substitutions selected from [D29C, R127C]; [T109C, V 120C]; [Y132C, N 136C]; [Y132C, A 137C]; [R133C, N136C]; [R133C, A137C]; [P 134C, P 135C]; [P 134C, N 136C]; [P 134C, A 137C] ; and [P 135C, N 136CJ . Additionally, an embodiment is provided wherein the VLP further comprises amino acid substitutions any of [D29C, R 127C]; [P 134C, N 136C]; and [D29C, R127C, P134C, N 136C]. In another embodiment, the VLP further comprising amino acid substitution [D29C, R127C]. In yet a further embodiment, the VLP comprises a hepatitis B core protein (HBc) which has an amino acid other than a methionine at amino acid 66 of wild-type HBc (Figure 12). For example, the amino acid other than a methionine at amino acid 66 is a serine, threonine or an isoleucine. In another example, the amino acid other than a methionine at amino acid 66 is a serine. In a further example, the amino acid other than a methionine at amino acid 66 is a threonine. In specific embodiment, the hepatitis B core protein (HBc) has an amino acid substitution M66S.
The invention further provides a VLP free of a viral genome comprising capsid proteins or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, wherein the capsid proteins or viral coat proteins have at least one non-naturally occurring amino acid to which one of two binding/interacting partners of the InaD domain complex or equivalent is attached, wherein an agent of interest is attached to a second binding/interacting partner of an InaD domain complex or equivalent, and wherein the two binding/interacting partners form the InaD domain complex or equivalent, such that the VLP displays one or more agents of interest. In one embodiment, the VLP free of a viral genome additionally comprises a CpG oligonucleotide. In one embodiment, the VLP free of a viral genome is produced from a VLP intermediate previously attached to one or more agents of interest free of an InaD domain complex or equivalent. In one embodiment, the VLP free of a viral genome is produced from a VLP intermediate previously attached to a CpG oligonucleotide or a TLR agonist free of an InaD domain complex or equivalent.
Examples of agents of interest include, but are not limited to, a peptide, polypeptide, nucleic acid molecule, polymer of a nucleic acid molecule, lipopolysaccharide, lipopeptide, peptidoglycan, small molecule, antibody, Id antigen, tumor-associated antigen, TLR agonist and immunostimulatory oligonucleotide. Specific example of these agents are described herein and are encompassed here. For example, a description of tumor-associated antigens are described hereinabove and contemplated herein but not repeated to avoid duplication. Further, examples of the leucocyte antigens are described hereinabove and contemplated herein but not repeated to avoid duplication.
In an embodiment of the invention, the VLP (also referred to herein as a VLP intermediate or a VLP free of a viral genome) may contain at least one non-naturally occurring amino acid or unnatural amino acid per capsid protein or viral coat protein. In one embodiment, at least one-fourth of the total number of non-naturally occurring amino acids in a VLP is used to attach a an agent of interest (examples include, but are not limited to, a peptide, polypeptide, nucleic acid molecule, polymer of nucleic acid molecules, lipopolysaccharide, lipopeptide, peptidoglycan, small molecule, antibody, Id antigen, tumor-associated antigen, TLR agonist and/or immunostimulatory oligonucleotides). In another embodiment, at least one-fourth of the total number of non-naturally occurring amino acids in a VLP is used to attach one of two binding/interacting partners of an InaD domain complex or equivalent.
In another embodiment, at least one-third of the total number of non-naturally occurring amino acids in a VLP is used to attach an agent of interest. In yet another embodiment, at least one-third of the total number of non-naturally occurring amino acids in a VLP may be used to attach one of two binding/interacting partners of an InaD domain complex or equivalent.
In an additional embodiment, at most 120 of the 240 capsid protein or viral coat proteins display an agent of interest. One or more types of agents of interest may be attached. In yet a further embodiment, at most 120 of the 240 capsid proteins or viral coat proteins display one of two binding/interacting partners of an InaD domain complex or equivalent.
Accordingly, in one embodiment, the VLP may display only one type or kind of agent of interest. In another embodiment, the VLP may display a combination of two or more different types or kinds of agents of interest. Additionally, encompassed within the invention are embodiments, wherein the VLP displays agents of interest using the INAD domain complex described herein. In other embodiments, the VLP may display agents of interest using the INAD domain complex and other agents through other attachment means, for example, via crosslinking as described herein, such as crosslinking of CpG to the VLP as described in Example 2.
In one embodiment of the invention, the VLP comprises one or more additional moieties conjugated to the non-naturally occurring amino acid. For example, the one or more additional moieties may be a peptide binding partner of the InaD domain complex or equivalent. In a specific example, the one or more additional moieties is an InaD domain or equivalent.
In accordance with the practice of the invention, the VLP may be an isolated VLP or purified VLP. In one embodiment of the invention, the VLP 10 further comprising two or more types of agents of interest. In an embodiment of the invention, the second agent of interest is attached to a second binding/interacting partner of an InaD domain complex.
In an embodiment of the invention, one agent of interest comprises a reactive functional group, which can participate in a crosslinking reaction with the non-naturally occurring amino acid. Merely by way of example, the crosslinking reaction may be an alkyne-azide cycloaddition or click chemistry between an alkyne or azide functional group of the agent of interest and respective alkyne or azide functional group of the non-naturally occurring amino acid of the capsid protein or viral coat protein.
In another embodiment, the agent of interest is an antibody that recognizes and binds a leucocyte antigen, cell adhesion molecule, prostate specific membrane antigen or oncoprotein. In one example, the leucocyte antigen is an immune checkpoint protein. Suitable examples of immune checkpoint protein include, but are not limited to, a PD-1 , a PDL l , a PDL2, a B7-H3, a B7-H4, a CTLA-4, a LAG3, a KIR, a TIM3, a TIGIT, a BTLA, a CDl 60, an A2aR, and a VISTA protein. In specific embodiments, the antibody that recognizes and binds an immune checkpoint is an antibody that recognizes and binds any of a PD-1, a PDL1, a PDL2, a B7-I I3, a B7-H4, a CTLA- 4, a LAG3, a KIR, a TIM3, a TIGIT, a BTLA, a CD 160, an A2aR, and a VISTA protein.
An examples of an agent of interest include, but are not limited to, an antibody specifically directed against any of CDl, CD2, CD3, CD4, CD5, CD6, CD6L, CD7, CD8, CD9, CDIO, CD11/CD18, CDlla, CDl lb, CDllc, CDl Id, CDwl2, CD13, CD14, CD15, CD15s, CD16, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD27L, CD28, CD29, CD30, CD30L, CD31, CD32, Fc Gamma Receptor, CD33, CD34, CD35, CD36, CD37, CD38, CD40, CD40L, CD41, CD42, CD42a, CD43b, CD43c, CD43, CD44, CD45, CD45-AP, CD46, CD48, CD49, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, DC54, CD55, CD56, CD57, CD58, CD59, CD60, C61, CD62, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66, CD66a, CD66b, DC66c, CD66d, CD66e, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79/BCR, CD80, CD81, CD82, CD83, CD8w4, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDwl08, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CDl 19, CD120, CD120a, CD120b, CD121a, CDwl21b, CD122, CD122beta chain, CDwl23, CD 124, CD124alpha chain, CDwl25, CD126alpha chain, CD127alpha chain, CDwl28, CD 128 A, CD 128B, CD 129, CD 129alpha chain, CD 130, CDw 131 , CD 132gamma c chain , CD 133 , CD 142, CD143, CD144, CDwl45, CD146, CD147, CD148, CDwl49, CDwl50, CD151, CD155, CD156, CD157, CD158, CD158a, CD158b, CD159, CD160, CD161, CD162, CD163, CD164, CD165, CD 166, EPCAM, FOLHl, CTLA4, B7 (B7-1, B7-2, B7-3, B7-4 or B7-5), PD-1 and HER2 or portion thereof.
The invention further provides a VLP free of a viral genome comprising two or more antibodies selected from the group of antibodies for CDl, CD2, CD3, CD4, CD5, CD6, CD6L, CD7, CD8, CD9, CDIO, CDl 1/CD18, CDl la, CDl lb, CDl lc, CDl Id, CDwl2, CD13, CD14, CD15, CD15s, CD16, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD27L, CD28, CD29, CD30, CD30L, CD31, CD32, Fc Gamma Receptor, CD33, CD34, CD35, CD36, CD37, CD38, CD40, CD40L, CD41 , CD42, CD42a, CD43b, CD43c, CD43, CD44, CD45, CD45- AP, CD46, CD48, CD49, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51 , CD52, CD53, DC54, CD55, CD56, CD57, CD58, CD59, CD60, C61 , CD62, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66, CD66a, CD66b, DC66c, CD66d, CD66e, CD67, CD68, CD69, CD70, CD71 , CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79/BCR, CD80, CD81 , CD82, CD83, CD8w4, CD85, CD86, CD87, CD88, CD89, CD90, CD91 , CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101 , CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDwl08, CD109, CD1 10, CD1 1 1 , CD1 12, CD1 13, CD1 14, CD1 15, CD1 16, CD1 17, CD1 18, CD1 19, CD120, CD120a, CD120b, CD121a, CDwl21 b, CD122, CD 122beta chain, CDwl23, CD 124, CD124alpha chain, CDwl25, CD126alpha chain, CD127alpha chain, CDwl28, CD128A, CD128B, CD129, CD129alpha chain, CD130, CD l 31 , CD132gamma c chain , CD133, CD142, CD143, CD144, CDwl45, CD146, CD147, CD148, CDwl49, CDwl 50, CD151 , CD155, CD156, CD157, CD158, CD158a, CD158b, CD1 59, CD160, CD161 , CD162, CD163, CD 164, CD 165, CD 166, EPCAM, FOLHl and HER2 proteins or portion thereof; wherein the VLP has capsid proteins or virus coat proteins with at least one non-naturally occurring amino acid; and wherein one of two binding/interacting partners of an InaD domain complex or equivalent is attached to a non-naturally occurring amino acid of a virus coat protein.
Specific examples of two antibodies displayed (attached) on a VLP of the invention include any of (a) a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds CD3; (b) a first antibody or portion thereof that binds CD 19 and a second antibody or portion thereof that binds CD3; (c) a first antibody or portion thereof that binds EPCAM and a second antibody or portion thereof that binds CD3; (d) a first antibody or portion thereof that binds FOLHl and a second antibody or portion thereof that binds CD3; (e) a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds FcGR; and (f) a first antibody or portion thereof that binds CD20 and a second antibody or portion thereof that binds CD3.
In yet a further embodiment of the invention, the Id antigen is derived from a T cell receptor (TCR). An embodiment of the invention includes an Id antigen which comprises an immunoglobulin variable heavy (VTI) chain domain or sequence having an amino acid motif Q-(A or P)-(P or L)- G-(Q or K)-G-L-E-W-(M or V or I) immediately preceding a tri-peptide motif, (G or A or S)-(X)- I, wherein X is any amino acid. Examples of Id antigens include, but are not limited to, an Id antigen designated N2517-H comprises an amino acid sequence, EVQLVESGGALVQPGGSLRLSCAASGFTFRDFWMSWVRQAPGKGLEWVANIREDGND YYVGAVRGRFTVSRDNARNSLYLQMNSLRAEDTAVYYCARDLGGYCSSTNCEGYFD YWGQGTLVTVSS or a portion thereof; an Id antigen designated N2517-K comprises an amino acid sequence, DIQMTQSPSTLSASVGDRVTITCRASQSISTWLAWYQQKPGRAPKLLIYKASTLESGVPS RFSGSGSGTEFTLTISGLQPDDFATYYCQQYADYRTFGQGTKVEIK or a portion thereof ; an Id antigen designated N3803-H comprises an amino acid sequence, EVQLVESGGGVVRPGGSRRLSCAASGFSFDDYGMSWVRQAPG GLEWVSGIKWNGGS RGYLDSVKGRFTISRDNAK FLYLQMNSLRVEDTAVYHCARDPKNYHYDSSGYYDYY YFPMDVWGQGTTVTVSS or a portion thereof; and anld antigen designated N3803-K comprises an amino acid sequence,
DIQMTQSPSSLSASVGDRVTITCRASQDIRNDLGWYQQKPGKAP RLIYAAFSLQSGVPS RFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIK or a portion thereof.
In addition to VLP compositions of the invention where an agent of interest is attached through a InaD domain complex or equivalent thereof, the invention provides specific VLPs having two of more agents of interest attached thereto through means other than by the InaD domain complex or equivalent thereof (free of InaD domain complex or equivalent). For example, these alternative attachment means are described in PCT/US2014/030788 or PCT/US 14/069406 which are incorporated by reference and in Example 2 herein. In an embodiment of this invention provides VLPs free of a viral genome comprising two or more display polypeptides comprising two or more antibodies selected from the group of antibodies for CD1 , CD2, CD4, CD5, CD6, CD6L, CD7, CD8, CD9, CD10, CD1 1/CD18, GDI l a, CD1 l b, CD1 lc, CD1 I d, CDwl 2, CD13, CD14, CD15, CD15s, CDwl 7, CD18, CD19, CD21 , CD22, CD23, CD24, CD25, CD26, CD27, CD27L, CD28, CD29, CD30L, CD31 , CD32, Fc Gamma Receptor, CD33, CD34, CD35, CD36, CD37, CD38, CD40, CD40L, CD41 , CD42, CD42a, CD43b, CD43c, CD43, CD44, CD45, CD45-AP, CD46, CD48, CD49, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51 , CD52, CD53, CD54, CD56, CD57, CD58, CD59, CD60, C61 , CD62, CD62E, CD62L, CD62P, CD64, CD65, CD66, CD66a, CD66b, DC66c, CD66d, CD66e, CD67, CD68, CD69, CD70, CD71 , CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79/BCR, CD80, CD81 , CD82, CD83, CD8w4, CD85, CD86, CD87, CD88, CD89, CD90, CD91 , CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101 , CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDwl 08, CD109, CD1 10, CD1 1 1 , CD1 12, CD1 13, CD1 14, CD1 15, CD1 16, CD1 17, CD1 18, CD1 19, CD 120, CD 120a, CD 120b, CD121a, CDwl 21b, CD 122, CD122beta chain, CDwl23, CD124, CD124alpha chain, CDwl25, CD126alpha chain, CD127alpha chain,
CDwl28, CD128A, CD128B, CD 129, CD129alpha chain, CD 130, CDwl 31 , CD132gamma c chain , CD133, CD142, CD143, CD144, CDwl45, CD146, CD147, CD148, CDwl49, CDwl 50, CD151 , CD155, CD156, CD157, CD158, CD158a, CD158b, CD159, CD160, CD161 , CD162, CD163, CD164, CD165, CD166, EPCAM, FOLH1 , CTLA4, B7 (B7-1 , B7-2, B7-3, B7-4 or B7- 5), PD-1 and HER2 or portion thereof. In a specific embodiment, the invention provides a VLP which comprises any of two antibodies displayed (attached, for example, not through an InaD domain complex but by, e.g., crosslinking) thereon including any of (a) a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds CD3; (b) a first antibody or portion thereof that binds CD 19 and a second antibody or portion thereof that binds CD3; (c) a first antibody or portion thereof that binds EPCAM and a second antibody or portion thereof that binds CD3; (d) a first antibody or portion thereof that binds FOLH1 and a second antibody or portion thereof that binds CD3; (e) a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds FcGR; and (f) a first antibody or portion thereof that binds CD20 and a second antibody or portion thereof that binds CD3.
The invention further provides a nucleic acid molecules encoding the VLP and sVLP of the invention, e.g., as shown in Figure 1 and Figure 2.
The nucleic acids of the invention may comprise nucleotide sequences and encode polypeptides (amino acid sequences) which are at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the reference nucleotide and amino acid sequences of the present invention (i.e., see examples herein, e.g., the sequences in Figures 1 and 2) when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. Polypeptides comprising amino acid sequences which are at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%o) to the reference amino acid sequences of the present invention when the comparison is performed with a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences, are also included in the present invention.
The nucleic acid molecule may be a DNA molecule (e.g., an isolated cDNA) encoding the VLP of the invention. Additionally, the nucleic acid molecule may be a RNA (e.g., an isolated RNA such as isolated mRNA). Alternatively, the nucleic acid molecule may be a hybrid of cDNA and mRNA. For example, the invention provides for a DNA construct comprising a vector that expresses the VLP free of a viral genome of the invention.
The nucleic acid molecules of the invention also include derivative nucleic acid molecules which differ from DNA or RNA molecules. Derivative molecules include peptide nucleic acids (PNAs), and non-nucleic acid molecules including phosphorothioate, phosphotriester, phosphoramidate, and methylphosphonate molecules, that bind to single-stranded DNA or RNA in a base pair- dependent manner (Zamecnik, P. C, et al., 1978 Proc. Natl. Acad. Sci. 75 :280284; Goodchild, P. C, et al., 1986 Proc. Natl. Acad. Sci. 83 :4143-4146). Reviews of methods for synthesis of DNA, RNA, and their analogues can be found, e.g., in: Oligonucleotides and Analogues, eds. F. Eckstein, 1991 , IRL Press, New York; Oligonucleotide Synthesis, ed. M. J. Gait, 1984, IRL Press, Oxford, England.
Additionally, the invention provides a vector which comprises the nucleic acid molecule of the invention. The term vector includes, but is not limited to, plasmids, cosmids, and phagemids. The host vector system comprises the vector of the invention in a suitable host cell. Examples of suitable host cells include but are not limited to bacterial cell and eukaryotic cells. In another embodiment, the invention provides a process comprising recovering a VLP of the invention and/or VLP monomers from a culture medium and from cultured cells. In the case of VLP monomers from a culture medium or cultured cells, such monomers may be first isolated and then allowed to form VLPs.
The invention also provides for a method for producing a VLP free of a viral genome protein comprising culturing the host vector system of the invention under suitable culture conditions so as to produce the VLP free of a viral genome in the host and recovering the VLP free of a viral genome so produced. Alternatively, the method comprises culturing the host vector system of the invention under suitable culture condition so as to produce VLP coat protein in the host, assembling VLP from VLP coat protein isolated from the host in the absence of a viral genome, and recovering the VLP free of a viral genome so produced. VLP may also be produced from assembly of VLP capsid monomers following isolation from a host cell. For example, the VLP may be assembled from capsid proteins outside of the host cell. Alternatively, the VLP of the invention may be produced following synthesis and assembly of its capsid protein or coat proteins in a cell free in vitro transcription and/or translation system.
In another example, the invention provides methods for producing, in a cell-free in vitro reaction, a VLP free of a viral genome. Preferably, the VLP is a population of icosahedral virus like particles free of a viral genome. This method may comprise synthesizing virus coat proteins in a prokaryotic cell-free in vitro translation reaction (e.g. substantially free of polyethylene glycol). The prokaryotic cell-free in vitro translation reaction may contain a bacterial cell extract, components of polypeptide and/or mRNA synthesis machinery; a template for transcription for the translation of the polypeptide; monomers for synthesis of the polypeptide; and co-factors, enzymes and other reagents necessary for translation to produce the virus coat proteins (e.g., at least about 250 ug/ml of the virus coat proteins) under conditions permissive for the virus coat proteins to self-assemble into a stable icosahedral virus like particle free of a viral genome which comprises at least 60 separate proteins.
According to embodiments of the invention, the degeneracy of the genetic code provides a predictable number of nucleic acid sequences encoding the VLP of the invention, the codons of which may be selected to optimally express the isolated nucleic acid in a host organism (including without limitation, bacteria, yeast, mammalian cells cultured in vitro, and cells of a mammal (including a human). Such expression is useful for production of the nucleic acid or the polypeptide in a host organism for subsequent isolation and use according to the invention or in cell free in vitro transcription and/or translation system.
The terms "pharmaceutical formulations," "pharmaceutical compositions" and "dosage forms" are used interchangeably herein and refer to a composition containing the active ingredient(s) of the invention in a form suitable for administration to a subject.
The pharmaceutical compositions of the present invention may be mixed with one or more pharmaceutically acceptable carriers, binders, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, polymers, disintegrating agents, glidants, wetting agents, emulsifying agents, suspending agents, lubricating agents, acidifying agents, dyes, preservatives and dispensing agents, or compounds of a similar nature depending on the nature of the mode of administration and dosage forms. Such ingredients, including pharmaceutically acceptable carriers and excipients that may be used to formulate dosage forms, are described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association ( 1986), incorporated herein by reference in its entirety.
Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. Examples of pharmaceutically acceptable carriers include water, saline, Ringer's solution, dextrose solution, ethanol, polyols, vegetable oils, fats, ethyl oleate, liposomes, waxes polymers, including gel forming and non-gel forming polymers, and suitable mixtures thereof. The carrier may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG. The carrier may be a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient.
Examples of binders include, but are not limited to, microcrystalline cellulose and cellulose derivatives, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polvinylpyrrolidine, povidone, crospovidones, sucrose and starch paste.
Examples of diluents include salt.
Examples of excipients include, but are not limited to, surfactants, lipophilic vehicles, hydrophobic vehicles, sodium citrate, calcium carbonate, and dicalcium phosphate.
Examples of wetting agents include, but are not limited to, propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene laural ether.
The artisan of ordinary skill in the art will recognize that many different ingredients can be used in formulations according to the present invention, in addition to the active agents, while maintaining effectiveness of the formulations in treating cancer. The list provided herein is not exhaustive.
For parenteral administration, in one embodiment, the agents of the invention can be formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier(s) described above.
Any dosage form used for therapeutic administration should be sterile. Sterility can readily be accomplished by filtration through sterile filtration membranes. Therapeutics generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. KITS OF THE INVENTION
According to another aspect of the invention, kits are provided. Kits according to the invention include package(s) comprising a composition of the invention or VLP compositions of the invention.
The phrase "package" means any vessel containing compositions presented herein. In preferred embodiments, the package can be a box or wrapping. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes (including pre-filled syringes), bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
The kit can also contain items that are not contained within the package but are attached to the outside of the package, for example, pipettes.
Kits may optionally contain instructions for administering compositions of the present invention to a subject having a condition in need of treatment. Kits may also comprise instructions for approved uses of components of the composition herein by regulatory agencies, such as the United States Food and Drug Administration. Kits may optionally contain labeling or product inserts for the present compositions. The package(s) and/or any product insert(s) may themselves be approved by regulatory agencies. The kits can include compositions in the solid phase or in a liquid phase (such as buffers provided) in a package. The kits also can include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.
The kit may optionally also contain one or more other compositions for use in combination therapies as described herein. In certain embodiments, the package(s) is a container for intravenous administration. In other embodiments, compositions are provided in an inhaler. In still other embodiments compositions are provided in a polymeric matrix or in the form of a liposome. THERAPEUTIC METHODS OF THE INVENTION
The invention also provides methods of stimulating an immune response, e.g., in a subject. In one embodiment, the method comprises contacting immune cells with an effective amount of a VLP intermediate free of a viral genome, comprising a VLP having capsid proteins or viral coat proteins comprising at least one non-naturally occurring amino acid to which one of two binding/interacting partners of an InaD domain complex or equivalent is attached thereby stimulating an immune response. In another embodiment of the invention, the method comprises contacting immune cells with an effective amount of a VLP free of a viral genome comprising capsids or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, wherein the capsid proteins or viral coat proteins have at least one non-naturally occurring amino acid to which one of two binding/interacting partners of the InaD domain complex or equivalent is attached, and wherein an agent of interest is attached to a second binding/interacting partner of an InaD domain complex or equivalent, thereby stimulating an immune response.
In an embodiment of the invention, the VLP intermediate further comprises an immunostimulatory oligonucleotide. Further, in an embodiment of the invention, the VLP intermediate comprising an immunostimulatory oligonucleotide remains as a VLP intermediate so long as additional agents of interest may be attached to the VLP through the non-naturally occurring amino acid on the VLP or through one of the binding/interacting partner of the InaD complex or equivalent attached to the VLP. Additionally, in some embodiments, the VLP intermediate further comprises additional agents of interest and wherein the VLP intermediate so further attached to additional agents of interest comprises unreacted non-naturally occurring amino acid and/or unoccupied binding/interacting partner of the InaD complex or equivalent.
Further, the invention provides methods of inhibiting an immune response, e.g., in a subject. In one embodiment, the method comprises contacting immune cells with an effective amount of a VLP free of a viral genome comprising capsid proteins or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, wherein the capsid proteins or viral coat proteins have at least one non-naturally occurring amino acid to which one of two binding/interacting partners of the InaD domain complex or equivalent is attached, and wherein an agent of interest is attached to a second binding/interacting partner of an InaD domain complex or equivalent, thereby inhibiting an immune response.
Additionally, the invention provides method of inhibiting tumor cells. In one embodiment, the method comprises contacting the tumor cells with an effective amount of a VLP intermediate free of a viral genome, comprising a VLP having capsid proteins or viral coat proteins comprising at least one non-naturally occurring amino acid to which one of two binding/interacting partners of an InaD domain complex or equivalent is attached thereby inhibiting the tumor cells. In another embodiment, the method comprises contacting the tumor cells with an effective amount of a VLP free of a viral genome comprising capsid proteins or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, wherein the capsid proteins or viral coat proteins have at least one non-naturally occurring amino acid to which one of two binding/interacting partners of the InaD domain complex or equivalent is attached, wherein an agent of interest is attached to a second binding/interacting partner of an InaD domain complex or equivalent, and wherein the two binding/interacting partners form the InaD domain complex or equivalent, such that the VLP displays one or more agents of interest thereby inhibiting the tumor cells. In yet a further embodiment, the method comprises contacting the tumor cells with an effective amount of the VLP of the invention thereby inhibiting the tumor cells.
The invention also provides methods of treating, inhibiting or preventing the progression of a tumor in a subject, which comprises administering to said subject an effective amount of a VLP compositions of the invention thereby treating, inhibiting or preventing the progression of a tumor in the subject.
The invention further provides for a method of treating, inhibiting or preventing the progression of a disease or disorder comprising administering to said subject an effective amount of a VLP composition of the invention.
In one embodiment, the disorder is an autoimmune disorder and may be a myasthenia gravis, chronic active hepatitis, primary biliary cirrhosis, dilated cardiomyopathy, myocarditis, dilated cardiomyopathy, autoimmune polyendocrine syndrome type I (APS- 1 ), autoimmune hepatitis, cystic fibrosis vasculitidis, acquired hypoparathyroidism, Goodpasture syndrome, Crohn's disease, coronary artery disease, pemphigus foliaceus, pemphigus vulgaris, Guillain-Barr syndrome, type 1 diabetes, stiff man syndrome, Rasmussen encephalitis, autoimmune gastritis, Addison disease, insulin hypoglycemic syndrome (Hi rata disease), type B insulin resistance, acanthosis, systemic lupus erythematosus (SLE), pernicious anemia, treatment- resistant Lyme arthritis, polyneuropathy, multiple sclerosis, demyelinating disease, rheumatic fever, atopic dermatitis, primary biliary cirrhosis, Graves' disease, neuromyelitis optica, autoimmune hypothyroidism, vitilago, autoimmune thyroiditis, autoimmune Hashimoto thyroiditis, cel iac disease, and metastatic melanoma. In a preferred embodiment, the autoimmune disorder is Grave's disease. In another preferred embodiment, the autoimmune disorder is myasthenia gravis. In yet a further preferred embodiment, the autoimmune disorder is neuromyelitis optica.
In another embodiment, the disorder may be a systemic autoimmune disorder and may include ACTH deficiency, myositis, dermatomyositis, polymyositis, dermatomyositis, SLE, Sjogren syndrome, systemic sclerosis, rheumatoid arthritis (RA), progressive systemic sclerosis, systemic sclerosis, deimatomyositis, scleroderma, morphea, primary antiphospholipid syndrome, bullous pemphigoid, herpes gestationis, cicatricial pemphigoid, chronic idiopathic urticaria, necrotizing and cescentic glomerulonephritis (NCGN), system vasculitis, Wegener granulomatosis, Churg-Strauss syndrome, polymyositis, scleroderma, Raynaud syndrome, chronic liver disease, visceral leishmaniasis, and systemic autoimmune disease.
In yet another embodiment, the disorder may be a cancer or a paraneoplastic autoimmune disorder which may include neuropathy, small lung cell cancer, hepatocellular carcinoma, liver cancer, paraneoplastic pemphigus, paraneoplastic stiff man syndrome, paraneoplastic encephalomyelitis, sub-acute autonomic neuropathy, cancer, SLE, hepatocellular carcinoma, cancer-associated retinopathy, paraneoplastic opsoclonus myoclonus ataxia, lower motor neuron syndrome, Lambert-Eaton myasthenic syndrome, and paraneoplastic cerebellar degeneration.
In yet another embodiment, the disorder may be a solid tumor cancer which may be a adrenal cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, cancer of unknown primary origin, Castieman Disease, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Leukemia, Liver Cancer, Lung Cancer, Lymphoma, Malignant Mesothelioma, Multiple Myeloma, Myelodysplasia Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non- Hodgkin Lymphoma, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, pancreatic cancer, Penile Cancer, Pituitary Tumors, prostate cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Skin Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, Wilms Tumor, non-Hodgkin lymphoma, Hodgkin lymphoma, Burkitt's lymphoma, lymphoblastic lymphomas, mantle cell lymphoma (MCL), multiple myeloma (MM), small lymphocytic lymphoma (SLL), splenic marginal zone lymphoma, marginal zone lymphoma (extra-nodal or nodal), mixed cell type diffuse aggressive lymphomas of adults, large cell type diffuse aggressive lymphomas of adults, large cell immunoblastic diffuse aggressive lymphomas of adults, small non-cleaved cell diffuse aggressive lymphomas of adults, or follicular lymphoma.
In a further embodiment, the cancer may be any of head and neck cancer, breast, salivary gland, thyroid, pancreas, stomach, bladder, endometrial or uterine carcinoma, cervical cancer, ovarian, vulvar cancer, prostate, colon, rectal, colorectal, lung, non-small cell lung cancer, osteosarcoma, glioblastoma, kidney, liver, metastatic cancer. In a preferred embodiment, the cancer is a B-cel l lymphoma (such as CLL). In another preferred embodiment, the cancer is a T-cell lymphoma. In yet a further preferred embodiment, the cancer is prostate cancer. In a further embodiment, the subject is a human, a farm animal, a horse, a dog, or a cat.
In another embodiment, the disorder may be a plasma protein autoimmune disorder or cytokine autoimmune disorder. Examples of plasma protein autoimmune disorder or cytokine autoimmune disorder include but not limited to autoimmune CI deficiency, SLE membrane proliferative glomerulonephritis, RA, systemic sclerosis, autoimmune thrombocytopenia purpura, immunodeficiency disorder, and atherosclerosis.
In another embodiment, the disorder may be a B-cel l malignancy. Examples of B-cell mal ignancy include but not limited to non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), mantle cell lymphoma and multiple myeloma, B-cell prolymphocyte leukemia, lymphoplasmocytic leukemia, splenic marginal zone lymphoma, marginal zone lymphoma (extra-nodal or nodal), plasma cell neoplasms (e.g., plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases), and follicular lymphoma (e.g., Grades 1 , II, III or IV).
In yet another embodiment, the disorder may be a T-cell malignancy. Examples of T-cell malignancy include but not limited to chronic lymphocytic leukemia (CLL), large granular lymphocyte leukemia (T gamma lymphoproliferative disease, mycosis fungoides/Sezary syndrome, diffuse aggressive lymphomas of adults, peripheral T-cell lymphomas (mixed cell type and large cell, immunoblastic), adult T-cell leukemia/lymphoma, angiocentric lymphomas (lymphomatoid granulomatosis polymorphic reticulosis, acute lymphocytic leukemia, or lymphoblastic lymphoma.
In one embodiment, the VLP is produced by a method for producing a population of icosahedral virus like particles free of a viral genome in a cell-free in vitro reaction. The method for producing a population of icosahedral virus like particles free of a viral genome in a cell-free in vitro reaction comprise synthesizing virus coat proteins in a prokaryotic cell-free in vitro translation reaction substantially free of polyethylene glycol and comprising a bacterial cell extract, components of polypeptide and/or mRNA synthesis machinery; a template for transcription for the translation of the polypeptide; monomers for synthesis of the polypeptide; and co-factors, enzymes and other reagents necessary for translation to produce at least about 250 ug/ml of the virus coat proteins-under conditions permissive for the virus coat proteins to self- assemble into a stable icosahedral virus like particle free of a viral genome, and comprising at least 60 separate proteins.
In an embodiment, the invention provides a method of treating a cancer in a subject further comprising administering to the subject a therapeutically effective amount of one or more chemotherapeutic agents (which are included herein as an agent of interest), wherein the chemotherapeutic agents are one or more of the following: alkylating agents; thiotepa; cyclosphosphamide; alkyl sulfonates; busulfan; improsulfan; piposulfan; aziridines; benzodopa; carboquone; meturedopa; uredopa; ethylenimines; methylamelamines; altretamine; triethylenemelamine; trietylenephosphoramide; triethylenethiophosphaoramide; trimethylolomelamine; nitrogen mustards; chlorambucil; chlornaphazine; cholophosphamide; estramustine; ifosfamide; mechlorethamine; mechlorethamine oxide hydrochloride; melphalan; novembichin; phenesterine; prednimustine; trofosfamide; uracil mustard; nitrosureas; carmustine; chlorozotocin; fotemustine; lomustine; nimustine; ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcel lomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate; 5-fluorouraci l; denopterin, methotrexate, pteropterin, trimetrexate; fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; aminoglutethimide, mitotane, trilostane; frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etogl cid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; polysaccharide K (PSK); razoxane; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; paclitaxel; docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; cisplatin; carboplatin; vinblastine; platinum; etoposide (VP- 1 6); ifosfarnide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT- 1 1 ; topoisomerase inhibitor 9-nitrocamptothecin; difluoromethylornithine; retinoic acid; esperamicins; capecitabine; tamoxifen; raloxifene; aromatase inhibiting 4(5)-imidazoles; 4-hydroxytamoxifen; trioxifene, keoxifene; LY 1 1 701 8; onapristone; toremifene; flutamide; nilutamide; bicalutamide; leuprolide; and goserelin.
In yet another embodiment, the disorder is an infectious disease and may be polio, respiratory syncytial virus (RSV) infection AIDS, hepatitis B, hepatitis C, hepatitis E, rabies, herpes, HSV, EBV, influenza, smallpox, myxoma infection, rhinovirus infection, coronavirus infection, whooping cough (rubella virus infection), adenovirus infection, papilloma virus infection or human T-cell leukemia virus (HTLV) infection. In a preferred embodiment, the infectious disease is HIV. In another preferred embodiment, the infectious disease is influenza. In yet a further preferred embodiment, the infectious disease is RSV infection.
The invention provides for a method for treating, inhibiting, or preventing the progression of a solid tumor cancer, in a subject. The method comprises administering to the subject, in need thereof, an effective amount of VLP vaccine or pharmaceutical composition of the invention so as to inhibit tumor growth or metastasis, kill tumor cells or reduce tumor burden. In another embodiment, the invention provides for a method of inhibiting tumor cells for a solid tumor which comprises contacting the tumor cells with an effective amount of a vaccine or composition of the invention.
The VLP compositions of the invention may be administered, e.g., in the case for cancer, by directly injection into or near a solid tumor. In a preferred embodiment, the administration may be intratumoral. Alternatively, in the case of cancer where a tumor site is not readily apparent, the administration may be made directly into or around the lymph node, spleen, thyroid, bone marrow, or other organ of the body with a high concentration of tumor cells. Further, depending on the site of the cancer, the administration may be intramuscular, intraperitoneal, intranasal, intradermal, or transmucosal.
In accordance with the practice of the invention, the VLP compositions of the invention may be admixed with the therapeutic agent (included herein as an agent of interest) just prior to administration of the composition to the subject. Alternatively, the composition may be available premixed so as to contain both the VLP vaccine and the therapeutic agent.
The solid tumor cancer may be an adrenal cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, cancer of unknown primary origin, Castleman Disease, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Liver Cancer, Lung Cancer, Lymphoma, Malignant Mesothelioma, Multiple Myeloma, Myelodysplastic Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, pancreatic cancer, Penile Cancer, Pituitary Tumors, prostate cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Skin Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, Wilms Tumor, non-Hodgkin lymphoma, Hodgkin lymphoma, Burkitt's lymphoma, lymphoblastic lymphomas, mantle cell lymphoma (MCL), multiple myeloma (MM), small lymphocytic lymphoma (SLL), splenic marginal zone lymphoma, marginal zone lymphoma (extra-nodal or nodal), mixed cell type diffuse aggressive lymphomas of adults, large cell type diffuse aggressive lymphomas of adults, large cell immunoblastic diffuse aggressive lymphomas of adults, small noncleaved cell diffuse aggressive lymphomas of adults, or follicular lymphoma.
In a further embodiment, the cancer may be any of head and neck cancer, breast, salivary gland, thyroid, pancreas, stomach, bladder, endometrial or uterine carcinoma, cervical cancer, ovarian, vulvar cancer, prostate, colon, rectal, colorectal, lung, non-small cell lung cancer, osteosarcoma, glioblastoma, kidney, liver, melanoma or metastatic cancer. The invention also provides a method for regulating functional CD 19 positive B cell and CD3 positive T cells interactions comprising contacting CD 19 positive B cells and CD3 positive T cells with a VLP composition of the invention that comprises a first antibody or portion thereof that binds CD 19 and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous CD19 positive B cells with CD3 positive T cells. In one embodiment, the interaction of said CD 19 positive B cell and CD3 positive T cells stimulates an immune response (e.g., activating cytotoxic T cells).
The invention additionally provides a method for regulating functional HER2 positive B cell and CD3 positive T cells interactions comprising contacting HER2 positive B cells and CD3 positive T cells with a VLP composition of the invention that comprises a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous HER2 positive B cells with CD3 positive T cells. In an embodiment of the invention, the interaction of said HER2 positive B cell and CD3 positive T cells stimulates an immune response (e.g., activating cytotoxic T cells).
Further, the invention provides a method for regulating functional EPCAM positive B cell and CD3 positive T cells comprising contacting EPCAM positive B cells and CD3 positive T cells with a VLP composition of the invention that comprises a first antibody or portion thereof that binds EPCAM and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous EPCAM positive B cells with CD3 positive T cells. In one embodiment, the interaction of said EPCAM positive B cell and CD3 positive T cells stimulates an immune response (e.g., activating cytotoxic T cells).
Additionally, the invention further provides a method for regulating functional FOLHl positive B cell and CD3 positive T cells comprising contacting FOLHl positive B cells and CD3 positive T cells with a VLP composition of the invention that comprises a first antibody or portion thereof that binds FOLHl and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous FOLHl positive B cells with CD3 positive T cells. An embodiment of this invention provides that the interaction of said FOLH 1 positive B cell and CD3 positive T cells stimulates an immune response (e.g., activating cytotoxic T cells).
Also, the invention provides a method for regulating functional CD20 positive B cell and CD3 positive T cells comprising contacting CD20 positive B cells and CD3 positive T cells with a VLP composition of the invention that comprises a first antibody or portion thereof that binds CD20 and a second antibody or portion thereof that binds CD3 to interfere with reaction of endogenous CD20 positive B cells with CD3 positive T cells. In an embodiment of the invention, the interaction of said CD20 positive B cell and CD3 positive T cells stimulates an immune response (e.g., activating cytotoxic T cells).
It is contemplated that in any of the methods of this section (Therapeutic methods of the invention), any of the methods that involve increased activation of immune cells may further comprise contact or administration of one or more of an immune checkpoint inhibitor, examples which are provided herein, i.e., agents that block immune checkpoint proteins. Contact or administration of one or more of immune checkpoint inhibitors may be effected before, during or after contact or administration of one or more VLP compositions of the invention.
The following examples are provided to further illustrate aspects of the invention. These examples are non-limiting and should not be construed as limiting any aspect of the invention.
EXAMPLES EXAMPLE 1
Synthesis of HBc azido-VLP and azido-sVLP intermediates
HBc Azido-VLP intermediate production
Plasmid construction and host cell bank preparation. The sequence encoding the human Hepatitis B core (HBc) monomer of subtype adyw (Pasek et al., 1979) with the C-terminus truncated at amino acid 149 (Figure 1 ) was codon optimized for E. coli expression, produced by overlapping oligonucleotide gene synthesis and cloned into a pET21a plasmid. The plasmid was used to transform T7 Express Crystal Competent E. coli (High Efficiency) bacterial cells along with a second plasmid containing the pLysS gene. Cells were selected for growth using both ampicillin and chloramphenicol. Several colonies were selected for production of the HBc protein and a high-producing line was identified and stored in a Research Cell Bank.
HBc-producing E. coli from the Research Cell Bank were expanded in shake flasks in minimal media, M9 (Sigma) supplemented with glucose, magnesium sulfate, calcium chloride ferric ammonium citrate, riboflavin, niacinamide, pyridoxic hydrochloride, thiamine, biotin, 13 amino acids mixture (arginine, glutamine, lysine, histidine, glycine, isoleucine, phenylalanine, leucine, cysteine, aspartic acid, valine, serine and threonine), ampicillin, chloramphenicol and methionine at 37 degrees Centigrade for approximately 8 hours at approximately 175 rpm. These expanded cultures were used to inoculate a 1.5 L of minimal media (with lower methionine concentration) in a Biostat B fermenter. Cells were grown at 37 degrees Centigrade to an OD of ~2.5. A mixture of methionine and azidohomoalanine were then added at a ratio of 1 :20 Met: AHA and the cells were induced with IPTG. At approximately 6 hours post-induction, cells were collected by centrifugation at 9,000g. Cells were lysed in Lysis Buffer (50 mM Tris 15 mM EDTA / 10 mM Magnesium Chloride / 1 mM phenylmethylsulfonyl fluoride / pH 7.5) using a French Press method. The supernatant of the cell lysis was collected by centrifugation at 15,000g and HBc protein was purified by multiple rounds of precipitation in 30 percent ammonium sulfate/re- suspension in 50 mM Tris / 500 mM sodium chloride / pH7.5 to form HBc azido-VLP intermediates. sHBc Azido-sVLP intermediate production
Stabilization and modification of Hepatitis B core (HBc) virus-like particles (VLPs) were described by Lu, W. Chan, B. Y. Ko, C. C. VanLang and J. R. Swartz (2015) Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery. Proc Natl Acad Sci U S A Vol 1 12, Num 40, pp 12360. Plasmid construction and host cell bank preparation. The sequence encoding the stabilized human Hepatitis B core (sHBc) monomer of subtype adyw (Pasek et al., 1979) with the C-terminus truncated at amino acid 149 (Figure 2) was codon optimized for E. coli expression, produced by overlapping oligonucleotide gene synthesis and cloned into a pET24a plasmid. The plasmid was used to transform T7 Express Crystal Competent E. coli (High Efficiency) bacterial cells. Cells were selected for growth using kanamycin. Several colonies were selected for production of the sHBc protein and a high-producing line was identified and stored in a Research Cell Bank. sFIBc-producing E. coli from the Research Cell Bank were expanded in shake flasks in minimal media, M9 (Sigma) supplemented with glucose, magnesium sulfate, calcium chloride, ferric ammonium citrate, riboflavin, niacinamide, pyridoxic hydrochloride, thiamine, biotin, 13 amino acids mixture (arginine, glutamine, lysine, histidine, glycine, isoleucine, phenylalanine, leucine, cysteine, aspartic acid, valine, serine and threonine), kanamycin and methionine at 37 degrees Centigrade for approximately 8 hours at approximately 175 rpm. These expanded cultures were used to inoculate two 1 .5 L of minimal media (with lower methionine concentration) in a Biostat B fermenter. Cells were grown at 37 degrees Centigrade with media feeding to an OD of ~6. The temperature was dropped to 30 degrees Centigrade and a mixture of methionine and azidohomoalanine was then added at a ratio of 1 :20 and 1 :40 Met: AHA to Fermenters 1 and 2 respectively and the cells were induced with IPTG. At approximately 6 hours post-induction, cells were collected by centrifugation at 9,000g for 15 minutes. Cells were lysed in Lysis Buffer (50 mM Tris / 25 mM Imidazole / 5 mM DTT / pl l 7.5) using a French Press method. The supernatant of the cell lysis was collected by centrifugation at 15,000g and azido-sHBc protein was purified by Nickel Column affinity chromatography. Formation of azido-sVLP intermediates was accomplished by dialysis into 1.5 M sodium chloride followed by dialysis into PBS and treatment with diamide to induce disulfide bond formation. Azido-sVLP was then dialysed into conjugation buffer for click reactions. EXAMPLE 2
Adapter-CpG-VLP intermediate
Conjugation of CpG Oligonucleotide and Adapter Peptide directly to azido-VLP
intermediate to form active docking CpG-VLP intermediate
A CpG-containing oligonucleotide with a cross-linkable functional group (CpG-X) was synthesized and purified by Nitto Avecia Oligrow© custom services group (http://www.avecia.com/avecia/nitto-avecia-oligrow.html). The sequence used is 5' TsGsAsCsTsGsTsGsAsAsCGsTsTsCsGsAsGsAsTsGsA-{5-0ct-dU} 3', where 's' denotes a phosphorothioate linkage in the sequence and 5-Oct-dU is 5-octadiynyl dU at the 3 ' end of the oligonucleotide. Presence of a 5-Oct-dU moiety introduces an alkyne functional group to the CpG oligonucleotide, and the resulting CpG-X oligonucleotide is also referred to as CpG-alkyne. 5 octadiynyl dU attached at the 3' end of the oligo formed the basis of alkyne-azide conjugation to the VLP.
A peptide, PEG6-TEFCA with C-terminal amino acid sequence TEFCA was synthesized by CS Bio custom peptide services group (http;//www. csbio.com/custom-manufactured-peptides/). The sequence used is N-terminal Pra-PEG6-T-E-F-C-A where single letter codes correspond to amino acids, Pra is L-propargylglycine and PEG6 is the amino acid analog formed when using Fmoc- NH-PEG6-propionic acid during peptide synthesis.
We conducted five 120 ul click reactions using 0.3 mM CpG3" in CB, 0.5 mM PEG (1/10 dilution of 5 mg/ml stock), and approximately 50 uM final HepB monomer concentration. The 5 reactions (A-E) involved VLP having PEG6-TEFCA and CpG attached thereto in 5 ratios. The PEG6- TEFCA : CpG ratio for Reaction [A] is 1 :4; Reaction [B is 1 : 1 ; Reaction [C] is 2: 1 ; Reaction [D] is 5 : 1 ; and Reaction [E] is 3 : 1. For each reaction, the HBc VLP-azide was mixed with CpG-alkyne and Pra-PEG6-TEFCA, sodium ascorbate, Tween-20 and potassium phosphate in an opaque reaction chamber. The mixture was overlayed with argon gas. The enhancer, tris(triazoylmethyl)amine [TTMA, Shanghai ChemPartner] was added and an aliquot of the mixture was withdrawn prior to adding the catalyst, tetrakis(acetonitrile)copper(I)hexafluorophosphate [tetrakis Cu(I), Sigma], Both the catalyst- containing and absent mixtures were allowed to react overnight at room temperature with mild agitation. Final reaction buffer conditions were Na ascorbate 200 μΜ; Tween .01 %; 10 mM potassium phosphate, at pH 8; TTMA 0.25 mM; Tetrakis Cu(I) 500 μΜ; 30°C, and molar amounts of HBC VLP-azide, CpG-alkyne and Pra-PEG6-TEFCA as shown in Figure 3. Figure 4 shows a reduced SDS-PAGE gel of 5ul reactions, with and without Cu addition. The molecular weight standards reference is included on the left. Evidence for the CpG or adapter linked to HBC monomer is observed based on the presence of increased density migrating at approximately 22- 24 kD for CpG-FIBc monomer and 17-18 kD for the Adapter-HBc monomer in the reactions with copper compared to the corresponding reactions without copper.
EXAMPLE 3
Analysis of Adapter-CpG-VLP CpG Oligonucleotide Activity Assay
CpG Activity Assay:
Activity of the CpG-azide and CpG-VLP were assessed using the Hek-BLUE™ TLR9 cell-based assay (Invivogen). This assay is based on an engineered reporter cell that responds to TLR9 binding to a number of CpG-based sequences. The HEK-Blue™ TLR cells are engineered HE 293 cells that stably co-express a human or murine TLR gene and an NF-KB/AP-1 -inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Upon activation a reporter substrate in the media reacts with a colorometric substrate, resulting in a color change.
Materials:
1. HEK-Blue-mTLR9 cells (InvivoGen);
2. HEK-Blue Detection Media (InvivoGen);
3. Growth Medium (DMEM, 4.5 g/1 glucose, 2-4 mM L-glutamine, 10% (v/v) fetal bovine serum, 50 U/ml penicillin, 50 μ^πιΐ streptomycin, 100 μg/ml Normocin); and
4. Selection Antibiotics (Blasticidin-30 μ^ηιΐ, Zeocin 100μg/ml). Cell Maintenance:
HEK~Blue-hTLR5 cells were cultured according to the procedure specified by the vendor (InvivoGen - hkb-htlr5). Briefly, HE -Blue mTLR9 cells were thawed and transferred to 15 ml of warm growth medium. Cells were pelleted and seeded into a T-25 flask in 5 ml of growth medium without selection. After 2 passages, cells were seeded into growth media with selective antibiotics. Cells were maintained via passaging at 70-80% confluency.
Assay:
HEK-Blue mTLR9 cells were seeded in triplicate wells of a 96 well plate at 2.5x104 cells/well in HEK-Blue detection media (InvivoGen - hb-det2). CpG-azide oligonucleotide, and inactive control oligonucleotide were added to PBS at 3, 10, 30, and 100 nanomolar. CpG-VLP preparations were added at 0.05, 0.16, 0.49 and 1.46 nanomolar. The plate was incubated at 37°C for more than 6 hours, and optical density was read at 650 nm (See Figure 5).
EXAMPLE 4
Adapter-sVLP intermediate
Conjugation of varying lengths of Adapter Peptide to azido-sVLP intermediate to form active docking intermediates.
Two peptides, PEG12-TEFCA and PEG24-TEFCA with C-terminal amino acid sequence TEFCA was synthesized by CS Bio custom peptide services group (http://www.csbio.com/custom- manufactured-peptides/). The sequences used were N-terminal Pra-PEG12-TEFCA and Pra- PEG12- PEG12-TEFCA, respectively, where single letter codes correspond to amino acids, Pra is L-propargylglycine and PEG 12 is the amino acid analog formed when using Fmoc-NH~PEG12- propionic acid during peptide synthesis.
We conducted 100 ul click reactions using 6 ul of 5 mg/ml PEG12-TEFCA or PEG24-TEFCA, approximately 50 uM final HepB monomer concentration, sodium ascorbate, Tween-20 and potassium phosphate in an opaque reaction chamber. The mixture was overlayed with argon gas. The enhancer, tris(triazoylmethyl)amine [TTMA, Shanghai ChemPartner] was added and an aliquot of the mixture was withdrawn prior to adding the catalyst, tetrakis(acetonitrile)copper(I)hexafluorophosphate [tetrakis Cu(I), Sigma], Both the catalyst- containing and absent mixtures were allowed to react overnight with mild agitation. Final reaction buffer conditions were Na ascorbate 200 μΜ; Tween .01%; 10 raM potassium phosphate, at pH 8; TTMA 0.25 mM; Tetrakis Cu(I) 500 μΜ; 30°C, overnight. After overnight incubation, 5 ul of reactions with and without copper were analyzed by reducing SDS-PAGE (See Figure 6, lanes 1 - 8). PEG 12-TEFCA-sVLP and PEG24-TEFCA-sVLP products were buffer exchanged into TE with 0.05% BME.
EXAMPLE 5
Docking of InaD-scFv fusion protein to Adapter-sVLP intermediates
Production of InaD-scFv and scFv-InaD fusion proteins.
38C 13 scFv fused on the N or C terminus with the InaD domain were constructed by gene synthesis. Sequences are shown in Figure 7 along with other Id scFv constructs. Plasmids were constructed for periplasmic expression of proteins in E.coli and used to transform competent cells. Cells were grown in 1 liter cultures in rich medium and protein expression was induced. Proteins were purified using Nickel affinity chromatography, and buffer was exchanged to TE with 0.05% beta-mercaptoethanol. Docking of InaD-scFv to PEG12-TEFCA-sVLP and PEG24-TEFCA- sVLP. PEG 12-TEFCA-s VLP or PEG24-TEFCA-sVLP from Example 4 and ~ 100-fold molar excess, the approximate molarity of the number of docking sites available, of InaD-scFv were mixed and allowed to react at room temperature for 1 hour. An aliquot of docking conjugation sample (C) was saved from each for analysis. Reactions were diafiltered using a 100,000 MWCO spin filter washing 3 times with PBS (-1000 fold buffer exchange). The first permeate (P) and diafiltration retentate (R) were saved for each docking conjugation. Reducing and non-reducing SDS-PAGE analysis demonstrates production of sVLP with attached InaD-scFv proteins for both intermediates (See Figure 6, lanes 9-20). EXAMPLE 6
Production of single and multispecific VLPs
Production of azido-sVLP intermediate was performed as described in Example 1 with 1 :40 Met:AHA ratio during fermentation in 2 L bioreactors.
Production of PEG6-TEFCA-sVLP intermediate was performed as described in Example 4 using the N-terminal Pra-PEG6-TEFCA sequence. antiCD3-InaD and antiCD19-InaD (scFv antibody fragments fused to InaD, sequences BB_aHuCD3-InaD-His and BB_ahCD19-InaD-His respectively shown in Figure 8) were produced in E.coli as described for production of the 38C13scFv-InaD fusion in Example 5. These antiCD3-InaD and antiCD19-InaD were biotinylated using EZ-link™ NHS-Biotin (ThermoFisher catalog number 20217) according to the manufacturer's directions.
AntiCD19-sVLP was produced using the docking procedure described in Example 5 by docking antiCD19-InaD to PEG6-TEFCA-sVLP intermediate at -100 fold molar excess.
AntiCD3-antiCD19-sVLP multispecific was produced using the docking procedure described in Example 5 by docking both antiCD3-InaD and antiCD19-InaD simultaneously, each at ~50 fold molar excess, to the PEG6-TEFCA-sVLP using the procedure described in Example 5.
EXAMPLE 7
Binding of antiCD19-sVLP and antiCD3-antiCD19-sVLP to normal human PBMCs
Frozen PBMCs isolated from Luekopak (20s, female donor) were used to assess the binding capacity of antiCD19-sVLP and antiCD3-antiCD19-sVLP to normal human PBMCs. Cells were thawed, washed twice with 50 mL of DPBS and cultured in RPMI with 5% normal human serum at 37C overnight. Cells were centrifuged and resuspended in FACS buffer. Aliquots of 1.6 million cells in 100 uL were incubated with 1.79 ug of antiCD19-sVLP or antiCD3-antiCD19-sVLP of FACS buffer for 45 minutes on ice. Cells were washed with 4 mL FACS buffer and subsequently incubated with streptavidin-allophycocyanin (BioLegend catalog number 405243) on ice for 30 minutes then washed with 4 mL of FACS buffer. Cells incubated with the antiCD19-sVLP and cells incubated with antiCD3-antiCD19-sVLP were then stained with either antiCD3 and antiCD19 (BioLegend catalog numbers 300440 and 302208 respectively) or antiCD4 and antiCD20 (BioLegend catalog numbers 344632 and 302312 respectively) on ice for 30 minutes. Cells were then washed with 4 mL of FACS buffer and resuspended at 400 uL prior to analysis by flow cytometry on a Canto II from BD Bioscience instrument using the DIVA software from BD Bioscience. Results from the PBMC staining are shown in Figures 9, 10 and 1 1 for the antiCD3- antiCD19-sVLP or antiCD19-sVLP alone and co-stained with antiCD3, antiCD4, antiCD19 and antiCD20 antibodies. As expected, the multispecific antiCD3-antiCD19-sVLP stains cells that are also stained with both T- and B- cell markers while the antiCD19-sVLP stains cells that primarily stain with only B-cell markers.

Claims

What is claimed is:
1 . A VLP intermediate to which one or more agents of interest may attach when desired comprising a VLP comprising capsid proteins or viral coat proteins having at least one non- naturally occurring amino acid to which one of two binding/interacting partners of an InaD domain complex or equivalent is attached and the VLP is free of a viral genome.
2. The VLP intermediate of claim 1 , wherein the InaD domain complex or equivalent contains two binding/interacting partners, wherein one binding/interacting partner is an InaD domain or fragment thereof or equivalent comprising a domain of InaD protein or equivalent and wherein a second binding/interacting partner is a peptide with at least four amino acids to which the InaD domain or fragment or equivalent binds.
3. The VLP intermediate of claim 2, wherein the InaD domain or fragment thereof or equivalent additionally comprises a non-naturally occurring amino acid which is or can be used to attach the InaD domain or fragment or equivalent to a VLP.
4. The VLP intermediate of claim 2, wherein the peptide with at least four amino acids additionally comprises a non-naturally occurring amino acid and optionally a linker separating the peptide from the non-naturally occurring amino acid which is or can be used to attach the peptide to a VLP.
5. The VLP intermediate of claim 1 , 3 or 4, wherein the non-naturally occurring amino acid provides an alkyne or azide reactant group for Click chemistry reactions.
6. The VLP intermediate of claim 5, wherein the non-naturally occurring amino acid having an azide reactant group is L-azidohomoalanine, -azido-L-phenylalanine or N6-((2- azidoethoxy)carbonyl)-L-lysine.
7. The VLP intermediate of claim 5, wherein the non-naturally occurring amino acid having an alkyne reactant group is L-homopropargylglycine, L-propargylglycine, p- propargyloxyphenylalanine or N6-((2-propynyloxy)carbonyl)-L-lysine.
8. The VLP intermediate of claim 1 , wherein the non-naturally occurring amino acid is N6- ((2-propynyloxy)carbonyl)-L-lysine, N6-((2-azidoethoxy)carbonyl)-L-lysine or -acetyl- L-phenylalanine.
9. The VLP intermediate of claim 1 , wherein the VLP is formed by a hepatitis B core protein (I IBc) or a portion thereof.
10. A VLP free of a viral genome comprising capsid proteins or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, wherein the capsid proteins or viral coat proteins have at least one non-naturally occurring amino acid to which one of two binding/interacting partners of the InaD domain complex or equivalent is attached, wherein an agent of interest is attached to a second binding/interacting partner of an InaD domain complex or equivalent, and wherein the two binding/interacting partners form the InaD domain complex or equivalent, such that the VLP displays one or more agents of interest.
1 1 . The VLP of claim 10, wherein the agent of interest is any of a peptide, polypeptide, nucleic acid molecule, polymer of a nucleic acid molecule, lipopolysaccharide, lipopeptide, peptidoglycan, small molecule, antibody, Id antigen, tumor-associated antigen, TLR agonist and immunostimulatory oligonucleotide or a combination thereof.
12. The VLP of claim 10, wherein the InaD domain complex contains two binding/interacting partners, wherein one binding/interacting partner is an InaD domain or fragment thereof or equivalent and wherein a second binding/interacting partner is a peptide with at least four amino acids to which the InaD domain or fragment or equivalent binds.
13. The VLP of claim 12, wherein the peptide with at least four amino acids additionally comprises a non-naturally occurring amino acid and optionally a linker separating the peptide from the non-naturally occurring amino acid which is or can be used to attach the peptide to a VLP.
14. The VLP of claim 12, wherein the InaD domain or fragment thereof or equivalent additionally comprises a non-naturally occurring amino acid which is or can be used to attach the InaD domain or fragment or equivalent to a VLP.
15. The VLP of claim 10, 13 or 14, wherein the non-naturally occurring amino acid provides an alkyne or azide reactant group for Click chemistry reactions.
16. The VLP of claim 15, wherein the non-naturally occurring amino acid having an azide reactant group is L-azidohomoalanine, / azido-L-phenylalanine or N6-((2- azidoethoxy)carbonyl)-L-lysine.
17. The VLP of claim 15, wherein the non-naturally occurring amino acid having an alkyne reactant group is L-homopropargylglycine, L-propargylglycine, p- propargyloxyphenylalanine, or N6-((2-azidoethoxy)carbonyl)-L-lysine.
1 8. The VLP of claim 10, 13 or 14, wherein the non-naturally occurring amino acid is N6-((2- propynyloxy)carbonyl)-L-lysine, N6-((2-azidoethoxy)carbonyl)-L-lysine or ;?-acetyl-L- phenylalanine.
19. The VLP of claim 1 or 10, wherein the VLP contains at least one non-naturally occurring amino acid per capsid protein or viral coat protein.
20. The VLP of claim 1 or 10, wherein at least one-fourth of the total number of non-naturally occurring amino acids in a VLP is used to attach a peptide, polypeptide, nucleic acid molecule, polymer of nucleic acid molecules, lipopolysaccharide, lipopeptide, peptidoglycan, small molecule, antibody, Id antigen, tumor-associated antigen, TLR agonist and/or immunostimulatory oligonucleotide.
21. The VLP of claim 1 or 10, wherein at least one-third of the total number of non-naturally occurring amino acids in a VLP is used to attach a peptide, polypeptide, nucleic acid molecule, polymer of nucleic acid molecules, lipopolysaccharide, lipopeptide, peptidoglycan, small molecule, antibody, Id antigen, tumor-associated antigen, TLR agonist and/or immunostimulatory oligonucleotide.
22. The VLP of claim 10, wherein at most 120 of the 240 capsid proteins or viral coat proteins display a peptide, polypeptide, nucleic acid molecule, polymer of nucleic acid molecules, lipopolysaccharide, lipopeptide, peptidoglycan, small molecule, antibody, Id antigen, tumor-associated antigen, TLR agonist and/or immunostimulatory oligonucleotide.
23. The VLP of claim 1 or 10, wherein the VLP is formed by a hepatitis B core protein (HBc) or its variant or a portion thereof.
24. The VLP of claim 23, wherein the HBc polypeptide is 149 amino acid in length and is derived from the first 149 amino acids at the N-terminus of a hepatitis B virus (HBV) coat protein of HBV subtype adyw (UniProt accession number P03147) .
25. The VLP of claim 23, wherein the HBc polypeptide variant from HBV subtype adyw has a methionine-to-serine substitution at amino acid 66 (M66S) and a leucine-to-methionine substitution at amino acid 76 (L76M), wherein the variant comprises an amino acid sequence as shown in Figure 1 .
26. The VLP of claim 1 or 10, wherein the VLP comprises an HBc polypeptide comprising the amino acid sequence of Figure 1 or portion thereof.
27. The VLP of claim 1 or 10, wherein the VLP is a stabilized VLP comprising hepatitis B core proteins (HBc) or its variant and wherein at least two amino acids of a HBc polypeptide are substituted with cysteine residues that can form intermolecular disulfide bonds when the HBc is assembled into a virus like particle (VLP), thereby stabilizing the VLP structure.
28. The VLP of claim 27, wherein the stabilized VLP comprises an HBc polypeptide comprising the amino acid sequence of Figure 2 or portion thereof.
29. The VLP of claim 27, wherein the amino acid substitutions for stabilizing a VLP are selected from any of [D29C, R127C]; [T109C, V120C]; [Y132C, N 136C]; [Y132C, A137C]; [R133C, N136C]; [R133C, A137C]; [P134C, P135C]; [P134C, N136C]; [P134C, A137C]; and [P135C, N136C].
30. The VLP of claim 27, wherein the amino acid substitutions for stabilizing a VLP are [D29C, R127C]; [P134C, N136C]; or [D29C, R127C, P134C, N136C], wherein a single VLP may comprise an HBc with one pair of said amino acid substitutions or two pairs of said amino acid substitutions, or alternatively, a single VLP may comprise two types of HBc polypeptides having one or the other pair of said amino acid substitutions.
31. The VLP of claim 27, wherein the amino acid substitutions for stabilizing a VLP are [D29C, R127C].
32. The VLP of claim 1 or 10, wherein one of two binding/interacting partners of the InaD domain complex or equivalent is attached to a spike region of the VLP.
33. The VLP of claim 32, wherein the spike region of the VLP is a spike tip.
34. The VLP of claim 32, wherein the amino acids of the spike region at residues 73-81 are substituted to reduce the negative charge, relative to an amino acid sequence as shown in Figure 1 .
35. The VLP of claim 32, wherein the spike region comprises a hydrophobic pocket and wherein the amino acids of the hydrophobic pocket at residues 57-81 are substituted relative to an amino acid sequence as shown in Figure 1.
36. The VLP of claim 35, wherein the amino acid substitutions are one of [I59V, L60S, G63R, D64E, L65V, M66T, T67D, L68F, A69G, T70D, T74N, L76M, E77Q, P79Q, S81 A, S87N, T91 A, V93I, F97I] or [T74N, L76M, E77Q, P79Q, S81 A].
37. The VLP of claim 1 , wherein at least one-fourth of the total number of non-naturally occurring amino acids in a VLP is used to attach one of two binding/interacting partners of an InaD domain complex or equivalent.
38. The VLP of claim 1 or 10, wherein the capsid protein or viral coat protein is a monomer.
39. The VLP of claim 1 or 10, wherein the capsid protein or viral coat protein is a dimer.
40. The VLP of claim 1 or 10, wherein the capsid protein or viral coat protein is assembled into a VLP.
41 . The VLP of Claim 1 or 10 further comprising one or more additional moieties conjugated to the non-naturally occurring amino acid.
42. VLP of claim 1 or 10, wherein one or more additional moieties is a peptide binding partner of the InaD domain complex or equivalent.
43. The VLP of claim 1 1 , wherein the Id antigen is derived from a T cell receptor (TCR).
44. VLP of claim 1 or 10, wherein one or more additional moieties is an InaD domain or equivalent.
45. The VLP of claim 1 or 10, wherein the VLP is an isolated VLP or purified VLP.
46. The VLP of claim 1 or 10, wherein the VLP is a stable icosahedral VLP.
47. The VLP of claim 10 further comprising two or more agents of interest.
48. VLP of claim 47, wherein the second agent of interest is attached to a second binding/interacting partner of an InaD domain complex.
49. The VLP of claim 1 1 or 48, wherein one agent of interest comprises a reactive functional group, which can participate in a crosslinking reaction with the non-naturally occurring amino acid.
50. The VLP of claim 49, wherein the crosslinking reaction is an alkyne-azide cycloaddition or click chemistry between an alkyne or azide functional group of the agent of interest and respective alkyne or azide functional group of the non-naturally occurring amino acid of the capsid protein or viral coat protein.
51 . The VLP of claim 1 1 or 48, wherein the agent of interest is an antibody that recognizes and binds a leucocyte antigen, cell adhesion molecule, prostate specific membrane antigen or oncoprotein.
52. The VLP of claim 51 , wherein the leucocyte antigen is an immune checkpoint protein.
53. The VLP of claim 52, wherein the immune checkpoint protein is selected from the group consisting of a PD- 1 , a PDL1 , a PDL2, a B7-H3, a B7-H4, a CTLA-4, a LAG3, a KIR, a TIM3, a TIGIT, a BTLA, a CD1 60, an A2aR, and a VISTA.
54. The VLP of claim 51 , wherein the antibody that recognizes and binds an immune checkpoint point is selected from the group consisting of an antibody that recognizes and binds a PD- 1 , a PDL1 , a PDL2, a B7-H3, a B7-H4, a CTLA-4, a LAG3, a KIR, a TIM3, a TIGIT, a BTLA, a CD 160, an A2aR, or a VISTA protein.
55. The VLP of claim 49, wherein the one agent of interest is an immunostimulatory oligonucleotide comprising an unmethylated cytosine as part of a CpG dinucleotide- containing oligonucleotide and wherein 5-octadiynyl dU attached to an end of the CpG oligonucleotide provides an alkyne functional group which can participate in a crosslinking reaction with a non-naturally occurring amino acid comprising an azide functional group..
56. The VLP of claim 1 1 , wherein the agent of interest is an antibody selected from the group of antibodies against CDl , CD2, CD3, CD4, CD5, CD6, CD6L, CD7, CD8, CD9, CDI O, CD1 1/CD18, CDl la, CDl lb, CDl lc, CDl Id, CDwl2, CD 13, CD14, CD15, CD15s, CD16, CDwl 7, CD18, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD26, CD27, CD27L, CD28, CD29, CD30, CD30L, CD31 , CD32, Fc Gamma Receptor, CD33, CD34, CD35, CD36, CD37, CD38, CD40, CD40L, CD41 , CD42, CD42a, CD43b, CD43c, CD43, CD44, CD45, CD45-AP, CD46, CD48, CD49, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51 , CD52, CD53, DC54, CD55, CD56, CD57, CD58, CD59, CD60, C61 , CD62, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66, CD66a, CD66b, DC66c, CD66d, CD66e, CD67, CD68, CD69, CD70, CD71 , CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79/BCR, CD80, CD81 , CD82, CD83, CD8w4, CD85, CD86, CD87, CD88, CD89, CD90, CD91 , CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101 , CD102, CD103, CD104, CD105, CD 106, CD107a, CD107b, CDwl 08, CD109, CDl 10, CDl 1 1 , CDl 12, CDl 13, CDl 14, CDl 15, CDl 16, CDl 17, CDl 18, CDl 19, CD120, CD120a, CD120b, CD121a, CDwl21b, CD122, CD122beta chain, CDwl23, CD124, CD124alpha chain, CDwl 25, CD126alpha chain, CD127alpha chain, CDwl28, CD128A, CD128B, CD 129, CD129alpha chain, CDl 30, CDwl 31 , CD132gamma c chain , CD133, CD142, CD143, CD144, CDwl 45, CD146, CD147, CD148, CDwl49, CDwl 50, CD151 , CD155, CD156, CD157, CD158, CD158a, CD158b, CD159, CD160, CD161 , CD162, CD163, CD164, CD165, CD166, EPCAM, FOLH1 , CTLA4, B7 (B7-1 , B7-2, B7-3, B7-4 or B7-5), PD-1 and HER2 or portion thereof.
57. A VLP free of a viral genome comprising two or more display polypeptides comprising two or more antibodies selected from the group of antibodies for CDl , CD2, CD3, CD4, CD5, CD6, CD6L, CD7, CD8, CD9, CDI O, CDl 1/CD18, CDl l a, CDl l b, CDl l c, CDl I d, CDwl2, CD13, CD14, CD15, CD15s, CD16, CDwl 7, CD18, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD26, CD27, CD27L, CD28, CD29, CD30, CD30L, CD31 , CD32, Fc Gamma Receptor, CD33, CD34, CD35, CD36, CD37, CD38, CD40, CD40L, CD41 , CD42, CD42a, CD43b, CD43c, CD43, CD44, CD45, CD45-AP, CD46, CD48, CD49, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51 , CD52, CD53, DC54, CD55, CD56, CD57, CD58, CD59, CD60, C61 , CD62, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66, CD66a, CD66b, DC66c, CD66d, CD66e, CD67, CD68, CD69, CD70, CD71 , CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79/BCR, CD80, CD81 , CD82, CD83, CD8w4, CD85, CD86, CD87, CD88, CD89, CD90, CD91 , CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD 100, CD101 , CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDwl 08, CD109, CD1 10, CD1 1 1 , CD1 12, CD1 13, CD1 14, CD1 15, CD1 16, CD1 17, CD1 18, CD1 19, CD120, CD120a, CD120b, CD 121 a, CDwl 21b, CD 122, CD122beta chain, CDwl23, CD124, CD124alpha chain, CDwl 25, CD126alpha chain, CD127alpha chain, CDwl 28, CD128A, CD128B, CD129, CD129alpha chain, CD130, CDwl 31 , CD132gamma c chain , CD133, CD142, CD143, CD144, CDwl45, CD146, CD147, CD148, CDwl49, CDwl 50, CD151 , CD155, CD156, CD157, CD158, CD158a, CD158b, CD159, CD160, CD161 , CD162, CD163, CD164, CD165, CD166, EPCAM, FOLHl and HER2 or portion thereof; wherein the VLP has viral coat proteins with at least one non-naturally occurring amino acid; wherein one of two binding/interacting partners of an InaD domain complex or equivalent is attached to a non- naturally occurring amino acid of a viral coat protein; wherein the antibody so selected is attached to a second binding/interacting partner of an InaD domain complex or equivalent; and wherein the two binding/interacting partners form the InaD domain complex or equivalent, such that the VLP displays two or more display polypeptides.
58. The VLP of claim 10 or 57, wherein the VLP comprises any of:
a. a first antibody or portion thereof that binds FffiR2 and a second antibody or portion thereof that binds CD3;
b. a first antibody or portion thereof that binds CD 19 and a second antibody or portion thereof that binds CD3;
c. a first antibody or portion thereof that binds EPCAM and a second antibody or portion thereof that binds CD3;
d. a first antibody or portion thereof that binds FOLHl and a second antibody or portion thereof that binds CD3; e. a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds FcGR; and
f. a first antibody or portion thereof that binds CD20 and a second antibody or portion thereof that binds CD3.
59. The VLP of claim 1 , 1 1 or 58, wherein the VLP further comprises an unmethylated cytosine.
60. The VLP of claim 1 1 , wherein the Id antigen comprises an immunoglobulin variable heavy (VI I) chain domain or sequence having an amino acid motif Q-(A or P)-(P or L)-G-(Q or K)-G-L-E-W-(M or V or I) immediately preceding a tri-peptide motif, (G or A or S)-(X)- I, wherein X is any amino acid.
61 . The VLP of claim 1 1 , wherein the tumor-associated antigen is selected from the group consisting of an Id antigen, 17- 1 A, 707-AP, AFP, Annexin II, ART-4, BAGE, BAGE- 1 , b- catenin, BCG, bcr/abl, Bcr/abl el4a2 fusion junction, bcr-abl (polypeptide from translation of b3a2 transcript), bcr-abl (polypeptide from translation of b2a2 transcript), bcr-abl p210 (polypeptide from translation of b2a2 transcript), bcr-abl p210 (polypeptide from translation of b3a2 transcript), bullous pemphigoid antigen-1 , CA 19-9, CA125, CA215, CAG-3 cancer peptide, CAMEL tumor antigen, Cancer-testis antigen, Caspase-8, CCL3, CCL4, CD16, CD20, CD3, CD30, CD55, CD63, CDC27, CDK-4, CDR3, CEA, cluster 5, cluster-5A, cyclin-dependent kinase-4, Cyp-B, DAM- 1 0, DAM -6, Dek-cain, E7, EGFR, EGFRvlI 1, EGP40, ELF2 M, EpCAM, FucGM 1, G250, GA733, GAGE, GAGE- 1 -8, gastrin cancer associated antigen, GD2, GD3, globoH, glycophorin, GM1 , GM2, GM3, GnTV, Gn-T-V, gplOO, Her-2/neu, HERV-K-ME, high molecular weight- associated antigen, high molecular weight proteoglycan (IMPG), HPV- 16 E6, I IPV- 16 E7, ITPVE6, HSP70-2M, I IST-2, hTERT, human chorionic gonadotropin (HCG), Human milk fat globule (ITMFG), iCE, IAA0205, KK-LC- 1 , KM-HN- 1 , L6, LAGE- I, LcOse4Cer, LDLR/FUT, Lewis A, Lewis v/b, M protein, MAGE- 1 , MVC, MAGE-A 1 - 12, MAGE-C2, MAHGE-3, MART- 1 /Melan- A, MC 1 R, ME491 , MUC 1 , MUC2, mucin, MUM- 1 , MUM-2, MUM-3, mutated p53, Myosin, MZ2-E, N9 neuraminidase, NA88, NA88-A, nasopharyngeal carcinoma antigen, NGA, N l/c-3, Novel bcr/ablk fusion BCR exons 1 , 13, 14 with ABL exons 4, NY-ESO-l/LAGE-2, NY-ESO-lb, OC 125, osteosarcoma associated antigen-1 , P 1 5, pl 90 mimor bcr-abl (ela2), p53, Pml/RARa, Polysialic acid, PRAME tumor antigen, PSA, PSM, RU 1 , RU2, SAGE, SART- 1 , SART- 2, SART-3, Sialyl LeA, Spl7, SSX-2, SSX-4, surface immunoglobulin, TAG- 1 , TAG-2, TEL/AML1 , TPI, TRAG-3, TRP-1 (gp75), TRP-2, TRP2-INT2, hTRT, tumor associated glycoprotein-72 (TAG-72), tyrosinase, u-PA, WT1 , and XAGE-lb, and an immunostimulatory fragment thereof.
62. A nucleic acid molecule encoding the capsid protein or viral coat protein of the VLP free of a viral genome of claim 1 or 10.
63. The nucleic acid molecule of claim 62, wherein sequence of the nucleic acid molecule comprises nucleic acid sequence as provided in Figure 1 or Figure 2.
64. The nucleic acid molecule of claim 62, wherein sequence of the nucleic acid molecule is nucleic acid sequence as provided in Figure 1 or Figure 2 or a portion thereof.
65. The cDNA of claim 64.
66. A vector which comprises the nucleic acid molecule of claim 64.
67. A host vector system comprising a vector of claim 66 in a suitable host cell.
68. The host vector system of claim 67, wherein the suitable host cell is a bacterial cell.
69. The host vector system of claim 68, wherein the suitable host cell is an eukaryotic cell.
70. A method for producing a VLP free of a viral genome protein comprising culturing the host vector system of claim 67 under suitable culture conditions so as to produce the VLP free of a viral genome in the host and recovering the VLP free of a viral genome so produced.
71. A method for making a population of VLP intermediates of claim 1 to which one or more agents of interest may attach when desired comprising:
a. synthesizing capsid proteins or viral coat proteins having at least one non-naturally occurring amino acid to which one of two binding/interacting partners of an InaD domain complex or equivalent can attach;
b. assembling the capsid protein or viral coat proteins of step (a) into a population of VLP; and
c. attaching the binding/interacting partner of the InaD domain complex or equivalent of step (a) to the VLP, thereby producing a population of VLP intermediates which may attach to one or more agents of interest.
72. A method for making VLPs attached to one or more agents of interest comprising making the population of VLP intermediates by the method of claim 71 and attaching to the VLP intermediates to one or more agents of interest attached to the other remaining
binding/interacting partner of the InaD domain complex or equivalent so that it specifically recognizes and binds the binding/interacting partner in the VLP intermediates thereby making VLPs attached to one or more agents of interest.
73. The method claim 71 or 72, wherein the InaD domain complex contains two binding/interacting partners, wherein one binding/interacting partner is an InaD domain or fragment thereof or equivalent comprising a domain of InaD protein and wherein a second binding/interacting partner is a peptide (a) with at least four amino acids to which the InaD domain or fragment or equivalent binds or (b) comprising a peptide sequence TEFCA.
74. The method of claim 71 or 72, wherein the InaD domain or fragment thereof or equivalent additionally comprises a non-naturally occurring amino acid which can be used to attach the InaD domain or fragment or equivalent to a VLP.
75. The method of claim 73, wherein the peptide additionally comprises a non-naturally occurring amino acid, and optionally a linker separating the peptide from the non-naturally occurring amino acid, which can be used to attach the peptide to a VLP.
76. The method of claim 71 ,72, 74 or 75, wherein the non-naturally occurring amino acid provides an alkyne or azide reactant group for Click chemistry reactions.
77. The method of claim 76, wherein the non-naturally occurring amino acid having an azide reactant group is L-azidohomoalanine, p-azido-L-phenylalanine or N6-((2- azidoethoxy)carbonyl)-L-lysine.
78. The method of claim 76, wherein the non-naturally occurring amino acid having an alkyne reactant group is L-homopropargylglycine, L-propargylglycine, p- propargyloxyphenylalanine or N6-((2-propynyloxy)carbonyl)-L-lysine.
79. The method of claim 71 or 72, wherein the non-naturally occurring amino acid is N6-((2- propynyloxy)carbonyl)-L-lysine, N6-((2-azidoethoxy)carbonyl)-T-lysine or /?-acetyl-L- phenylalanine.
80. The method of claim 71 or 72, wherein the non-naturally occurring amino acid provides a reactive chemical functional group that can participate in chemical crosslinking and free of alkyne or azide reactant group.
81 . The method claim 71 or 72, wherein one of two binding/interacting partners of the InaD domain complex or equivalent is attached to a spike region of the VLP.
82. The method claim 80, wherein the spike region of the VLP is a spike tip.
83. The method of claim 71 or 72, wherein the agent of interest is a tumor-associated antigen or an antibody that recognizes and binds a leucocyte antigen, cell adhesion molecule, prostate specific membrane antigen or oncoprotein.
84. The method of claim 83, wherein the leucocyte antigen is an immune checkpoint protein.
85. The method of claim 84, wherein the immune checkpoint protein is selected from the group consisting of PD-1, PDL1, PDL2, B7-I13, B7-H4, CTLA-4, LAG3, KIR, TIM3, TIGIT, BTLA, CD 160, A2aR, and VISTA.
86. The method of claim 83, wherein the antibody recognizes and binds an immune checkpoint protein selected from the group consisting of PD-1, PDL1, PDL2, B7-H3, B7-H4, CTLA- 4, LAG3, KIR, TIM3, TIGIT, BTLA, CD160, A2aR, and VISTA.
87. The method of claim 83, wherein the leucocyte antigen is selected from the group consisting of CD1, CD2, CD3, CD4, CD5, CD6, CD6L, CD7, CDS, CD9, CD10, CD11/CD18, CDlla, CDllb, CDllc, CDlld, CDwl2, CD13, CD14, CD15, CD15s, CD16, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD27L, CD28, CD29, CD30, CD30L, CD31, CD32, Fc Gamma Receptor, CD33, CD34, CD35, CD36, CD37, CD38, CD40, CD40L, CD41, CD42, CD42a, CD43b, CD43c, CD43, CD44, CD45, CD45-AP, CD46, CD48, CD49, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, DC54, CD55, CD56, CD57, CD58, CD59, CD60, C61, CD62, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66, CD66a, CD66b, DC66c, CD66d, CD66e, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79/BCR, CD80, CD81, CD82, CD83, CD8w4, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CDIOO, CDlOl, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDwl08, CD109, CDllO, CDlll, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120, CD120a, CD120b, CD121a, CDwl21b, CD122, CD122beta chain, CDwl23, CD124, CD124alpha chain, CDwl25, CD126alpha chain, CD127alpha chain, CDwl28, CD128A, CD128B, CD129, CD129alpha chain, CD130, CDwl31, CD132gamma c chain , CD133, CD142, CD143, CD144, CDwl45, CD146, CD147, CD148, CDwl49, CDwlSO, CD151, CD155, CD156, CD157, CD158, CD158a, CD158b, CD159, CD160, CD161 , CD162, CD163, CD164, CD165 and CD166 and/or a portion thereof.
88. The method of claim 83, wherein the oncoprotein is an antigen selected from the group consisting of HER2/ErbB-2/Neu, HER3/ErbB-3, HER4/ErbB-4, and EGFR/ErbB-1 and/or a portion thereof.
89. The method of claim 83, wherein the cell adhesion molecule is an EPCAM.
90. The method of claim 71 , wherein the agents of interest so attached is any of:
a. a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds CD3;
b. a first antibody or portion thereof that binds CD19 and a second antibody or portion thereof that binds CD3;
c. a first antibody or portion thereof that binds EPCAM and a second antibody or portion thereof that binds CD3;
d. a first antibody or portion thereof that binds FOLH1 and a second antibody or portion thereof that binds CD3;
e. a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds FcGR; and
f. a first antibody or portion thereof that binds CD20 and a second antibody or portion thereof that binds CD3.
91. The method of claim 71 or 72, wherein the antibody when attached to the VLP is in an average amount equivalent to about 10 to 50 copies per VLP, about 40 to 80 copies per VLP, about 70 to 170 copies per VLP, about 100 copies per VLP or about 160 to 240 copies per VLP.
92. The method of claim 90, wherein the VLP further comprises an additional agent of interest which is an immunostimulatory oligonucleotide comprising an unmethylated cytosine.
93. The method of claim 71 or 72, wherein the agent of interest is an immunostimulatory oligonucleotide comprising an unmethylated cytosine.
94. The method of claim 71 or 72, wherein the agents of interest attached to the VLP are an antibody or fragment or derivative thereof which binds a leucocyte antigen/or and an immunostimulatory oligonucleotide comprising an unmethylated cytosine.
95. The method of claim 71 or 72, wherein the agents of interest attached to the VLP are an antibody or fragment or derivative thereof which binds an immune checkpoint protein and/or an immunostimulatory oligonucleotide comprising an unmethylated cytosine.
96. The method of claim 93, 94 or 95, wherein the immunostimulatory oligonucleotide comprises a CpG dinucleotide which comprises the unmethylated cytosine.
97. The method of claim 92, 93, 94, 95 or 96, wherein the unmethylated cytosine is a CpG oligonucleotide which is attached to a VLP intermediate in an amount (molar) such that the CpG oligonucleotide to VLP monomer ratio is equivalent to 1 :24 to 1 : 12, 1 : 12 to 1 :6, 1 :6 to 1 :3, 1 :3 to 2:3 or 1 :2 to 1 : 1.
98. The method of claim 92, 93, 94, 95 or 96, wherein the unmethylated cytosine is a CpG oligonucleotide which is attached to the VLP in an average amount equivalent to 10 to 50 copies per VLP, 40 to 80 copies per VLP, 70 to 170 copies per VLP, or 160 to 240 copies per VLP.
99. The method of claim 92, 93, 94, 95 or 96, wherein the unmethylated cytosine is a CpG oligonucleotide which comprises a sequence, 5' -TGACTGTGAACGTTCGAGATGA- 3 '.
100. The method of claim 99, wherein the sequence has a mixture of phosphodiester and phosphorothioate bonds as shown in 5'T*G*A*C*T*G*T*G*A*A*CG*T*T*C*G*A*G*A*T*G*A 3', where * represents replacement of a phosphodiester bond with a phosphorothioate bond.
101. The method of claim 99, wherein the sequence has phosphorothioate bonds as shown in 5'T*G*A*C*T*G*T*G*A*A*C*G*T*T*C*G*A*G*A*T*G*A 3 ', where * represents replacement of a phosphodiester bond with a phosphorothioate bond.
102. The method of claim 99, wherein the CpG oligonucleotide further comprises a 5-octadiynyl deoxyuridine or a modified deoxyuridine or a linker at the 3' end of the sequence.
103. The method of claim 83, wherein the tumor-associated antigen is selected from the group consisting of 17- 1 A, 707-AP, AFP, Annexin II, ART-4, BAGE, BAGE- 1 , b- catenin, BCG, bcr/abl, Bcr/abl el4a2 fusion junction, bcr-abl (polypeptide from translation of b3a2 transcript), bcr-abl (polypeptide from translation of b2a2 transcript), bcr-abl p210 (polypeptide from translation of b2a2 transcript), bcr-abl p210 (polypeptide from translation of b3a2 transcript), bullous pemphigoid antigen-l , CA 19-9, CA125, CA215, CAG-3 cancer peptide, CAMEL tumor antigen, Cancer-testis antigen, Caspase-8, CCL3, CCL4, CD 16, CD20, CD3, CD30, CD55, CD63, CDC27, CD -4, CDR3, CEA, cluster 5, cluster-5A, cyclin-dependent kinase-4, Cyp-B, DAM- 1 0, DAM -6, Dek-cain, E7, EGFR, EGFRvlI 1, EGP40, ELF2 M, EpCAM, FucGM 1, G250, GA733, GAGE, GAGE- 1 -8, gastrin cancer associated antigen, GD2, GD3, globoH, glycophorin, GM1 , GM2, GM3, GnTV, Gn-T-V, gplOO, Her-2/neu, IIERV-K-ME, high molecular weight-associated antigen, high molecular weight proteoglycan (IMPG), HPV-16 E6, HPV- 16 E7, HPVE6, HSP70-2M, HST-2, hTERT, human chorionic gonadotropin (HCG), Human milk fat globule (HMFG), iCE, IAA0205, KK-LC-1 , KM-HN-1 , L6, LAGE- I, LcOse4Cer, LDLR/FUT, Lewis A, Lewis v/b, M protein, MAGE-1 , MVC, MAGE-A1 -12, MAGE-C2, MAHGE-3, MART- 1 /Melan-A, MC1 R, ME491 , MUC1 , MUC2, mucin, MUM-1 , MUM- 2, MUM-3, mutated p53, Myosin, MZ2-E, N9 neuraminidase, NA88, NA88-A, nasopharyngeal carcinoma antigen, NGA, NKl/c-3, Novel bcr/ablk fusion BCR exons 1 , 13, 14 with ABL exons 4, NY-ESO-l/LAGE-2, NY-ESO-lb, OC125, osteosarcoma associated antigen-l , P15, pi 90 mimor bcr-abl (ela2), p53, Pml/RARa, Polysialic acid, PRAME tumor antigen, PSA, PSM, RU1 , RU2, SAGE, SART-1 , SART-2, SART-3, Sialyl LeA, Spl7, SSX-2, SSX-4, surface immunoglobulin, TAG-1 , TAG-2, TEL/AML 1 , TPI, TRAG-3, TRP-1 (gp75), TRP-2, TRP2-INT2, hTRT, tumor associated glycoprotein- 72 (TAG-72), tyrosinase, u-PA, WT1 , and XAGE-lb, or an immunostimulatory fragment thereof.
104. The method of claim 71 , wherein the VLP is formed by a hepatitis B core protein which comprises a sequence as shown in Figure 1 or 2 or a portion thereof.
105. The method of claim 71 , wherein the VLP comprises capsid protein or viral coat proteins or portions thereof from a virus selected from the group consisting of a bacteriophage, adenovirus, coxsackievirus, Hepatitis A virus, poliovirus, Rhinovirus, Herpes simplex virus, Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, Human herpes virus, Hepatitis B virus, Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, HIV, Influenza virus, Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human metapneumovirus, Human papillomavirus, Rabies virus, Rubella virus, Human bocarivus or Parvovirus, and Norovirus.
106. VLPs attached to one or more agents of interest produced by the method of claim 71 or 72.
107. A pharmaceutical composition comprising an effective amount of the VLP of claim 1 or 10 and pharmaceutically acceptable carriers, binders, diluents, adjuvants, excipients, and/or vehicles.
108. The pharmaceutical composition of claim 107 and a therapeutic agent admixed therewith.
109. A composition comprising the VLP of claim 1 or 10 and a suitable carrier.
1 10. A vaccine comprising the composition of claim 109 for inducing an immune response to the polypeptide(s) attached to the VLP or one or more agents of interest in a subject.
1 1 1. An immunostimulatory composition for inducing an immune response in a subject comprising the VLP free of a viral genome of claim 1 or 10 and a suitable carrier.
1 12. An immunomodulatory composition for modulating an immune response in a subject comprising the VLP free of a viral genome of claim 1 or 10 and a suitable carrier.
1 13. The immunomodulatory composition of claim 1 12, wherein the VLP engages one or more immune cells.
1 14. The immunomodulatory composition of claim 1 13, wherein the VLP engages a B-cell to modulate adaptive humoral response.
1 15. The immunomodulatory composition of claim 1 14, wherein the VLP engages a CD19+ B cell.
1 16. The immunomodulatory composition of claim 1 14, wherein the VLP engages a CD20+ B cell.
1 17. The immunomodulatory composition of claim 1 13, wherein the VLP engages a T-cell to modulate adaptive cellular immune response.
1 18. The immunomodulatory composition of claim 1 17, wherein the VLP engages CD3+ T cells.
1 19. The immunomodulatory composition of claim 1 17, wherein the VLP engages CD4+ T cells.
120. The immunomodulatory composition of claim 1 12, wherein the VLP engages both a B-cell and a T-cell.
121. The immunomodulatory composition of claim 1 12, wherein the VLP elicits an immunostimulatory response.
122. The immunomodulatory composition of claim 1 12, wherein the VLP elicits a cytotoxic response.
123. The immunostimulatory composition of claim 1 1 1 further comprising an adjuvant.
124. An immunostimulatory composition for inducing an immune response in a subject, the vaccine comprising a vector that expresses the VLP free of a viral genome of claim 1 or 10 and a suitable carrier.
125. An immunostimulatory composition for inducing an immune response in a subject, the vaccine comprising a viral gene delivery system to deliver a nucleic acid sequence that encodes the VLP free of a viral genome of claim 1 or 10 and a suitable carrier.
126. A kit comprising the composition of claim 1 or 10, optionally with reagents and/or instructions for use.
127. A method of stimulating an immune response which comprises contacting immune cells with an effective amount of a VLP intermediate free of a viral genome, comprising a VLP having capsid proteins or viral coat proteins comprising at least one non-naturally occurring amino acid to which one of two binding/interacting partners of an InaD domain complex or equivalent is attached, thereby stimulating an immune response.
128. A method of stimulating an immune response which comprises contacting immune cells with an effective amount of a VLP free of a viral genome comprising capsid proteins or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, wherein the capsid proteins or viral coat proteins have at least one non-naturally occurring amino acid to which one of two binding/interacting partners of the InaD domain complex or equivalent is attached, wherein an agent of interest is attached to a second binding/interacting partner of an InaD domain complex or equivalent, and wherein the two binding/interacting partners form the InaD domain complex or equivalent, such that the VLP displays one or more agents of interest, thereby stimulating an immune response.
129. A method of inhibiting an immune response which comprises contacting immune cells with an effective amount of a VLP free of a viral genome comprising capsid proteins or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, wherein the capsid proteins or viral coat proteins have at least one non-naturally occurring amino acid to which one of two binding/interacting partners of the InaD domain complex or equivalent is attached, wherein an agent of interest is attached to a second binding/interacting partner of an InaD domain complex or equivalent, and wherein the two binding/interacting partners form the InaD domain complex or equivalent, such that the VLP displays one or more agents of interest, thereby inhibiting an immune response.
130. The method of claim 127 or 128, wherein the immune response is stimulated in a subject.
13 1 . The method of claim 129, wherein the immune response is inhibited in a subject.
132. The method of claim 127, wherein the VLP intermediate further comprises an immunostimulatory oligonucleotide.
133. The method of claim 132, wherein the VLP intermediate comprising an immunostimulatory oligonucleotide remains as a VLP intermediate so long as additional agents of interest may be attached to the VLP through the non-naturally occurring amino acid on the VLP or through one of the binding/interacting partner of the InaD complex or equivalent attached to the VLP.
134. The method of claim 133, wherein the VLP intermediate further comprises additional agents of interest and wherein the VLP intermediate so further attached to additional agents of interest comprises unreacted non-naturally occurring amino acid and/or unoccupied binding/interacting partner of the InaD complex or equivalent.
135. A method of inhibiting tumor cells which comprises contacting the tumor cells with an effective amount of a VLP intermediate, thereby inhibiting the tumor cells.
136. A method of inhibiting tumor cells which comprises contacting the tumor cells with an effective amount of a VLP free of a viral genome comprising capsid proteins or viral coat proteins, an InaD domain complex or equivalent, and one or more agents of interest, wherein the capsid proteins or viral coat proteins have at least one non-naturally occurring amino acid to which one of two binding/interacting partners of the InaD domain complex or equivalent is attached, wherein an agent of interest is attached to a second binding/interacting partner of an InaD domain complex or equivalent, and wherein the two binding/interacting partners form the InaD domain complex or equivalent, such that the VLP displays one or more agents of interest, thereby inhibiting the tumor cells.
137. A method of inhibiting tumor cells which comprises contacting the tumor cells with an effective amount of the VLP of claim 1 or 10 thereby inhibiting the tumor cells.
138. The method of claim 135, 136 or 137, wherein the VLP intermediate further comprises an immunostimulatory oligonucleotide.
139. The method of claim 138, wherein the VLP intermediate comprising an immunostimulatory oligonucleotide remains as a VLP intermediate so long as additional agents of interest may be attached to the VLP through the non-naturally occurring amino acid on the VLP or through one of the binding/interacting partner of the InaD complex or equivalent attached to the VLP.
140. The method of claim 139, wherein the VLP intermediate further comprises additional agents of interest and wherein the VLP intermediate so further attached to additional agents of interest comprises unreacted non-naturally occurring amino acid and/or unoccupied binding/interacting partner of the InaD complex or equivalent.
141 . A method of treating, inhibiting or preventing the progression of a tumor in a subject, which comprises administering to said subject an effective amount of the VLP of claim 1 or 10 thereby treating, inhibiting or preventing the progression of a tumor in the subject.
142. A method of treating, inhibiting or preventing the progression of a disease or disorder comprising administering to said subject an effective amount of the composition of claim 109, 1 1 1 or 1 12, thereby treating, inhibiting or preventing the progression of the disease or disorder in the subject.
143. A method of treating a subject suffering from a disease or disorder, which comprises administering to said subject in need thereof an effective amount of the vaccine of claim 1 10 thereby treating the subject.
144. A method of treating a subject suffering from a disease or disorder comprising administering to said subject in need thereof an effective amount of the composition of claim 107, 108, 109, 1 1 1 , 1 12 or 1 13, thereby treating the subject.
145. The method of claim 141 , wherein said VLP is administered intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally, intradermally, intraocularly, transmucosally or as an aerosol.
146. The method of claim 142 or 144, wherein said composition is administered intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally, intradermally, intraocularly, transmucosally or as an aerosol.
147. The method of claim 143, wherein said vaccine is administered intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally, intradermally, intraocularly, transmucosally or as an aerosol.
148. The method of claim 142 or 144, wherein the disorder is an autoimmune disorder selected from the group consisting of myasthenia gravis, primary biliary cirrhosis, dilated cardiomyoapthy, myocarditis, dilated cardiomyopathy, autoimmune polyendocrine syndrome type I (APS-1)), autoimmune hepatitis, cystic fibrosis vasculitidis, acquired hypoparathyroidism, Goodpasture syndrome, Crohn's disease, coronary artery disease, pemphigus foliaceus, neuromyelitis optica, pemphigus vulgaris, Guillain-Barr syndrome, type 1 diabetes, stiff man syndrome, Rasmussen encephalitis, autoimmune gastritis, Addison disease, insulin hypoglycemic syndrome (Hirata disease), type B insulin resistance, acanthosis, systemic lupus erythematosus (SLE)), pernicious anemia, treatment- resistant lyme arthritis, polyneuropathy, multiple sclerosis, demyelinating disease, rheumatic fever, atopic dermatitis, primary biliary cirrhosis, Graves' disease, autoimmune hypothyroidism, vitilago, autoimmune thyroiditis, autoimmune Hashimoto thyroiditis, celiac disease, and metastatic melanoma.
149. The method of claim 142 or 144, , wherein the disorder is a systemic autoimmune disorder selected from the group consisting of ACTH deficiency, myositis, dermatomyositis, polymyositis, dermatomyositis, SLE, Sjogren syndrome, systemic sclerosis, rheumatoid arthritis (RA), progressive systemic sclerosis, systemic sclerosis, deimatomyositis, scleroderma, morphea, primary antiphospholipid syndrome, bullous pemphigoid, herpes gestationis, cicatricial pemphigoid, chronic idiopathic urticaria, necrotizing and cescentic glomerulonephritis (NCGN), system vasculitis, Wegener granulomatosis, Churg-Strauss syndrome, polymyositis, scleroderma, Raynaud syndrome, chronic liver disease, visceral leishmaniasis, and systemic autoimmune disease.
150. The method of claim 142, 143 or 144, wherein the disorder is a cancer or a paraneoplastic autoimmune disorder selected from the group consisting of neuropathy, small lung cell cancer, hepatocellular carcinoma, liver cancer, paraneoplastic pemphigus, paraneoplastic stiff man syndrome, paraneoplastic encephalomyelitis, subacute autonomic neuropathy, cancer, SLE, hepatocellular carcinoma, cancer-associated retinopathy, paraneoplastic opsoclonus myoclonus ataxia, lower motor neuron syndrome, Lambert-Eaton myasthenic syndrome, and paraneoplastic cerebellar degeneration.
151. The method of claim 142 or 144, wherein the disorder is a plasma protein autoimmune disorder or cytokine autoimmune disorder.
152. The method of claim 151 , wherein the plasma protein autoimmune disorder or cytokine autoimmune disorder is selected from the group consisting of autoimmune CI deficiency, SLE membrane proliferative glomerulonephritis, RA, systemic sclerosis, autoimmune thrombocytopenia purpura, immunodeficiency disorder, and atherosclerosis.
153. The method of claim 142, 143 or 144, wherein the disorder is a B-cell malignancy.
154. The method of claim 153, wherein the B-cell malignancy is non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma (MM), small lymphocytic lymphoma (SLL), B-cell prolymphocyte leukemia, lymphoplasmocytic leukemia, splenic marginal zone lymphoma, marginal zone lymphoma (extra-nodal or nodal), plasma cell neoplasms (e.g., plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases), or follicular lymphoma (e.g., Grades 1 , II, III or IV).
155. The method of claim 142, 143 or 144, wherein the disorder is a T-cell malignancy.
156. The method of claim 155, wherein the T-cell malignancy is chronic lymphocytic leukemia (CLL), large granular lymphocyte leukemia (T gamma lymphoproliferative disease, mycosis fungoides/Sezary syndrome, diffuse aggressive lymphomas of adults, peripheral T-cell lymphomas (mixed cell type and large cell, immunoblastic), adult T-cell leukemia/lymphoma, angiocentric lymphomas (lymphomatoid granulomatosis polymorphic reticulosis), acute lymphocytic leukemia, or lymphoblastic lymphoma.
157. The method of claim 142, 143 or 144, wherein the disorder is an infectious disease.
158. The method of claim 157, wherein the infectious disease is polio, RSV infection, AIDS, hepatitis B, hepatitis C, hepatitis E infection, rabies, herpes, HSV, EBV, influenza, smallpox, myxoma infection, rhinovirus infection, coronavirus infection, whooping cough, adenovirus infection, papilloma virus infection or HTLV infection.
159. A method for inhibiting tumor cells associated with a disease or disorder in a subject which comprises: a. Obtaining a sample from the subject;
b. Identifying an Id antigen associated with a disease or disorder from the sample; c. Producing a recombinant Id antigen or fragment thereof;
d. Generating the VLP of claim 1 or 10 which comprises the recombinant Id antigen or fragment thereof; and
e. Administering an effective amount of the VLP free of a viral genome of claim 1 or 10 from step (d) to the subject so as to permit an immune response against the tumor cells thereby inhibiting the tumor cells.
160. A method for inhibiting a disease or disorder in a subject which comprises:
a. Obtaining a sample from the subject;
b. Identifying an Id antigen associated with the disease or disorder from the sample; c. Producing a recombinant Id antigen or fragment thereof;
d. Generating the VLP free of a viral genome of claim 1 or 10 which comprises the recombinant Id antigen or fragment thereof; and
e. Administering an effective amount of the VLP free of a viral genome of 1 or 10 from step (d) to the subject so as to permit or stimulate an immune response thereby inhibiting the disease or disorder.
161 . A method of treating a subject with a cancer, comprising administering an effective amount of the VLP of claim 1 or 10 to the subject so as to inhibit the cancer thereby treating the subject.
162. The method of claim 161 , wherein the cancer is a solid tumor and administration is intratumoral.
163. The method of claim 142, 143 or 144, wherein the subject is a mammal.
164. The method of claim 163, wherein the mammal is any of a human, monkey, ape, dog, cat, cow, horse, rabbit, mouse, or rat.
165. The method of claim 161 , wherein the cancer comprises breast cancer, colon cancer, pancreatic cancer, prostate cancer, lung cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, Burkitt's lymphoma, lymphoblastic lymphomas, mantle cell lymphoma (MCL), multiple myeloma (MM), small lymphocytic lymphoma (SLL), head and neck cancer, melanoma or follicular lymphoma.
166. A method of inhibiting the growth of a solid tumor in a subject comprising intratumorally administering to a subject the VLP of claim 1 or 10 in an amount effective to inhibit growth of the solid tumor.
167. The method of claim 166, wherein the tumor is a breast cancer tumor.
168. The method of claim 161 , wherein the cancer is a B-cell malignancy selected from the group consisting of a non-Hodgkin lymphoma, Hodgkin lymphoma, Burkitt's lymphoma, acute lymphocytic leukemias, lymphoblastic lymphomas, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma (MM), small lymphocytic lymphoma (SLL), B-cell prolymphocyte leukemia, lymphoplasmocytic leukemia, splenic marginal zone lymphoma, marginal zone lymphoma (extra-nodal or nodal), plasma cell neoplasms (e.g., plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases), mixed cell type diffuse aggressive lymphomas of adults, large cell type diffuse aggressive lymphomas of adults, large cell immunoblastic diffuse aggressive lymphomas of adults, small non-cleaved cell diffuse aggressive lymphomas of adults, and follicular lymphoma.
169. The method of claim 161 , wherein the cancer is a T-cell malignancy selected from the group consisting of a chronic lymphocytic leukemia (CLL), large granular lymphocyte leukemia (T gamma lymphoproliferative disease, mycosis fungoides/Sezary syndrome, diffuse aggressive lymphomas of adults, peripheral T-cell lymphomas (mixed cell type and large cell, immunoblastic), adult T-cell leukemia/lymphoma, angiocentric lymphomas (lymphomatoid granulomatosis polymorphic reticulosis), acute lymphocytic leukemia, or lymphoblastic lymphoma.
170. A method for regulating functional CD 19 positive B cell and CD3 positive T cells comprising contacting CD 19 positive B cells and CD3 positive T cells with a VLP that comprises a first antibody or portion thereof that binds CD 19 and a second antibody or portion thereof that binds CD3 of claim 164 to interfere with reaction of endogenous CD 19 positive B cells with CD3 positive T cells.
171. The method of claim 170, wherein the interaction of said CD 19 positive B cell and CD3 positive T cells stimulates an immune response.
172. A method for regulating functional HER2 positive B cell and CD3 positive T cells comprising contacting HER2 positive B cells and CD3 positive T cells with a VLP that comprises a first antibody or portion thereof that binds HER2 and a second antibody or portion thereof that binds CD3 of claim 158 to interfere with reaction of endogenous HER2 positive B cells with CD3 positive T cells.
173. The method of claim 172, wherein the interaction of said HER2 positive B cell and CD3 positive T cells stimulates an immune response.
174. A method for regulating functional EPCAM positive B cell and CD3 positive T cells comprising contacting EPCAM positive B cells and CD3 positive T cells with a VLP that comprises a first antibody or portion thereof that binds EPCAM and a second antibody or portion thereof that binds CD3 of claim 158 to interfere with reaction of endogenous EPCAM positive B cells with CD3 positive T cells.
175. The method of claim 174, wherein the interaction of said EPCAM positive B cell and CD3 positive T cells stimulates an immune response.
176. A method for regulating functional FOLH1 positive B cell and CD3 positive T cells comprising contacting FOLH1 positive B cells and CD3 positive T cells with a VLP that comprises a first antibody or portion thereof that binds FOLH1 and a second antibody or portion thereof that binds CD3 of claim 158 to interfere with reaction of endogenous FOLH1 positive B cells with CD3 positive T cells.
177. The method of claim 176, wherein the interaction of said FOLH 1 positive B cell and CD3 positive T cells stimulates an immune response.
178. A method for regulating functional CD20 positive B cell and CD3 positive T cells comprising contacting CD20 positive B cells and CD3 positive T cells with a VLP that comprises a first antibody or portion thereof that binds CD20 and a second antibody or portion thereof that binds CD3 of claim 158 to interfere with reaction of endogenous CD20 positive B cells with CD3 positive T cells.
179. The method of claim 178, wherein the interaction of said CD20 positive B cell and CD3 positive T cells stimulates an immune response.
180. The method claim 72, wherein one binding/interacting partner of the InaD domain complex binds the other remaining binding/interacting partner in the VLP intermediates so that an InaD domain complex is formed and the one or more agents of interest bound to the complex is thereby attached to a VLP intermediate.
181. The method claim 72, wherein in the InaD domain complex, the InaD domain binds the carboxyl terminus of the peptide comprising a TEFCA sequence.
182. The method of claim 72, wherein the InaD domain complex additionally comprises a covalent bond formed between the binding/interacting partners of the InaD domain complex.
183. The method of claim 176, wherein the covalent bond is a disulfide bond formed between two cysteine residues.
184. The method of claim 177, wherein the disulfide bond is formed under oxidizing condition permissive for the formation of a disulfide bond between two nearby cysteine residues.
185. The method claim 72, wherein the InaD domain of the InaD domain complex is directly attached to the VLP.
186. The method claim 72, wherein the peptide sequence TEFCA of the InaD domain complex is directly attached to the VLP.
187. The method claim 72, wherein the InaD domain complex is attached to a spike region of the VLP.
188. The method claim 187, wherein the spike region of the VLP is a spike tip.
189. VLPs attached to one or more agents of interest produced by the method of claim 72.
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