Patent Grant
7786267
The present invention relates in general to the field of novel vaccines, and more particularly, to the design, manufacture and use of multivariable antigens complexed with targeting humanized monoclonal antibodies.
Without limiting the scope of the invention, its background is described in connection with vaccine development.
Protein engineering technology relating to monoclonal antibodies is highly advanced regarding humanization (i.e., rendering e.g., a rodent mAb sequence into a human mAb sequence while preserving specific antigen combining sites of the original mAb) and production (typically secreted from mammalian cell lines). In research and development are new applications of rAbs related to vaccination and are presently based on engineered rAb-antigen fusion proteins (typically with the antigen coding region placed in-frame with the C-terminal codon of the rAb heavy or H chain). A roadblock to this technology is the successful expression and production of fully functional rAb-antigen. In many, perhaps most, cases the desired antigen confounds secretion of the engineered rAb-antigen. Also, the likelihood of poor or null expression is increased if the desired entity includes multiple antigen coding regions.
The invention provides methods for the assembly of rAb antigen complexes in a controlled manner by simple mixing components and accommodates the ability to express and produce the rAb and antigen(s) in different expression—production systems that are best suited to the individual rAb and particular antigen. In addition, the invention demonstrates the novel application of the high affinity and high specificity cohesin-dockerin interaction to secreted mammalian expression systems, thus permitting the development of unique protein engineering formats and production of new protein tools for research and clinical application.
More particularly, the present invention uses the cohesin-dockerin protein domains and their surrounding linker. For example, the invention permits the controlled assembly of recombinant monoclonal antibodies (rAbs) complexed to antigens, toxins, or cellular activating agents. The invention has wide potential application in vaccination and cancer therapy. Also claimed are derivatives of this technology that permit the production of novel proteins with specific affinities for other proteins.
The invention is based on particular components of the well studied bacterial cellulose degrading protein complex called the cellulosome. Specifically, two protein domains (cohesin and dockerin) and natural protein linker sequences are utilized via the invention in novel contexts and applications.
The present invention is based on the discovery that particular cohesin and dockerin domains can be successfully and efficiently secreted from mammalian cells as fusion proteins while maintaining the specific and high affinity cohesin-dockerin protein-protein interaction. While the extensive cohesin-dockerin literature teaches the expectation that such fusion proteins should have this functionality, it does not describe production of such fusion proteins in mammalian secretion systems. The state of scientific knowledge does not allow the prediction of the discovery since the rules (other than features such as signal peptide) for successful secretion are not fully established. Furthermore, the cohesin linker regions are known to be glycosylated in their native bacteria, and the cohesin and dockerin domains contain predicted glycosylation sites. While this may actually favor secretion from mammalian cells, it is unclear if ‘unnatural’ glyosylation will perturb the cohesis-dockerin interaction.
While cohesin-dockerin interaction for various commercial applications has been published, the present invention is based on a previously unrealized potential for this interaction built around assembling specific protein complexes unrelated to the controlled assembly enzyme applications.
The invention includes the use of all cohesin-dockerin sequences from diverse cellulose degrading microbes, but describes the application of specific cohesin and dockerin and linker sequences from the microbe Clostridium thermocellum. For example, the sequence described herein encodes the H chain of a human IgG4 linked at the C-terminal codon to a Clostridium thermocellum dockerin sequence (called rAb.doc). Other embodiments of rAb.doc proteins are described similarly with examples that are engineered by simply transferring the dockerin coding region as a DNA fragment to vectors encoding the different H chain entities.
More particularly, the present invention includes a modular rAb carrier that includes an antigen-specific binding domain linked to one or more antigen carrier domains and one half of a cohesin-dockerin binding pair. The antigen-specific binding domain may be at least a portion of an antibody and the antibody is a fusion protein with and the binding pair in a fusion protein with one half of a cohesin-dockerin binding pair. The rAb may also include a complementary half of the cohesin-dockerin binding pair bound to an antigen that forms a complex with the modular rAb carrier. The complementary half of the cohesin-dockerin binding pair may itself be a fusion protein with the antigen carried as part of the complex (modular rAb carrier (cohesin/dockerin) antigen complex). Examples of antigen specific domain include a full length antibody, an antibody variable region domain, an Fab fragment, a Fab′ fragment, an F(ab)2 fragment, and Fv fragment, and Fabc fragment and/or a Fab fragment with portions of the Fc domain. Non limiting examples of sources for the cohesin-dockerin binding pair include Clostridium thermocellum, Clostridium josui, Clostridium cellulolyticum and Bacteroides cellulosolvens and combinations thereof.
Non-limiting examples for targeting by the antigen-specific binding domain include: cell surface marker selected from MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD 19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fcγ receptor or other receptor relatively specifically expressed by antigen presenting cells.
The rAb of the present invention may also includes combinations of the domains that are defined as: an rAb.Doc; an rAb.Coh; an rAb.(Coh)x; an rAb.(Doc)x; an rAb.(Coh.Doc)x; or an rAb.(Coh)x(Doc)x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Examples of the modular rAb carrier in a complex include:
The present invention also include a vaccine of a modular rAb carrier that includes an antigen specific domain linked to one or more domains comprising one half of the cohesin-dockerin binding pair bound to a complementary half of the cohesin-dockerin binding pair bound to an antigen. Non-limiting examples for targeting the rAb include immune cell surface protein selected from MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD 19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fcγ receptor or other receptor relatively specifically expressed by antigen presenting cells. Targets for vaccination with the rAb antigen carrier include, e.g., a bacterial, viral, fungal, protozoan or cancer protein and fragments thereof. The vaccine wherein the modular rAb carrier is further defined: an rAb.Doc:Coh.antigen; an rAb.Coh:Doc.antigen; an rAb.(Coh)x:(Doc.antigen)x; an rAb.(Doc)x:(Coh.antigen)x; an rAb.(Coh.Doc)x:(Doc.antigen1)(Coh.antigen2); or an rAb.(Coh)x(Doc)x:(Doc.antigen1)x(Coh.antigen2)x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
The present invention also includes an isolated nucleic acid comprising a coding segment for a target-specific domain and one or more domains and one half of a cohesin-dockerin binding pair. For example, the target may be an antigen and the target specific domain may encode at least a portion of an antibody. The one or more domains can encode one or more cohesin domains, one or more dockerin domains or a combination of one or more cohesin and dockerin domains. The rAb is further defined as: an rAb.Doc; an rAb.Coh; an rAb.(Coh)x; an rAb.(Doc)x; an rAb.(Coh.Doc)x; or an rAb.(Coh)x(Doc)x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
The present invention also includes a vector that includes a nucleic acid encoding an antigen specific domain and one or more domains that comprise one half of a cohesin-dockerin binding pair, a one half of a cohesin-dockerin binding pair with a protein molecule to be carried and combinations thereof. The one half of a cohesin-dockerin binding pair, a one half of a cohesin-dockerin binding pair with a protein molecule to be carried and combinations thereof are under the control of the same promoter, different promoters, transcribed in-line, transcribed in opposite directions.
The present invention also includes a host cell comprising a vector comprising a nucleic acid encoding an antigen specific domain and one or more domains and one half of a cohesin-dockerin binding pair.
A method of making a modular rAb carrier by combining an antigen specific domain linked to one or more domains of one half of a cohesin-dockerin binding pair. The rAb is further defined as: an rAb.Doc; an rAb.Coh; an rAb.(Coh)x; an rAb.(Doc)x; an rAb.(Coh.Doc)x; or an rAb.(Coh)x(Doc)x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Examples of the rAb is complexed with a complementary half of a cohesion:dokerin pair bound to an antigen and is selected from: an rAb.Doc:Coh.antigen; an rAb.Coh:Doc.antigen; an rAb.(Coh)x:(Doc.antigen)x; an rAb.(Doc)x:(Coh.antigen)x; an rAb.(Coh.Doc)x:(Doc.antigen1)(Coh.antigen2); or an rAb.(Coh)x(Doc)x:(Doc.antigen1)x(Coh.antigen2)x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
The present invention may also be an immunotoxin that includes an rAb.Doc:Coh.toxin self-assembled conjugate, wherein the rAb is specific for a cell target. Examples of toxins include a radioactive isotope, metal, enzyme, botulin, tetanus, ricin, cholera, diphtheria, aflatoxins, perfringens toxin, mycotoxins, shigatoxin, staphylococcal enterotoxin B, T2, seguitoxin, saxitoxin, abrin, cyanoginosin, alphatoxin, tetrodotoxin, aconotoxin, snake venom and spider venom. Cell targets for the immunotoxin include diseased or infected cells. Examples of diseased cells for targeting include cancer cell for, e.g., hematological cancers such as leukemias and lymphomas, neurological tumors such as astrocytomas or glioblastomas, melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumors such as gastric or colon cancer, liver cancer, pancreatic cancer, genitourinary tumors such cervix, uterus, ovarian cancer, vaginal cancer, testicular cancer, prostate cancer or penile cancer, bone tumors, vascular tumors, or cancers of the lip, nasopharynx, pharynx and oral cavity, esophagus, rectum, gall bladder, biliary tree, larynx, lung and bronchus, bladder, kidney, brain and other parts of the nervous system, thyroid, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma and leukemia. The immunotoxin may target pathogens directly, e.g., bacteria, a protozoan, a helminth, a virally-infected cell or a fungus.
The present invention also includes a method for protein purification by separating a cohesin or dockerin fusion protein by interacting the fusion protein with a rAb that is conjugated to the complementary cohesin or dockerin bound to a substrate. The present invention may also use the cohesin as a fusion partner for toxins for conferring beneficial biochemical properties favoring ready purification of active cohesin.toxin fusion protein. The present invention may also use the anti-DC rAb.Doc to target DC for therapeutic applications where ablating DC. Therapeutic applications include, e.g., transplantation, autoimmune disease, infectious disease or cancer. The invention also includes an anti-DC-SIGN/L antibody provided in an amount that is sufficient to enhance the survival of dendritic cells, wherein the antibody matures and activates the dendritic cells for immunization. The antibody may target cells in vivo, e.g., dendritic cells as an adjuvant in vaccines.
Also invented is a bivalent and multivalent (rAb1.Doc:Coh.rAb2) self-assembled conjugates as therapeutic, diagnostic, and industrial agents. Alternatively, the invention is a bivalent and multivalent (rAb.Doc:Coh.cytokine), (rAb.Coh:Doc.cytokine) or (cytokine1.Coh:cytokine2 Doc) self-assembled conjugates as therapeutic, cell proliferation or maturing agents. The modular rAbs carrier may be made by method that includes screening one or more multivalent rAb and/or rAb.cytokine and/or cytokine.cytokine combinations that are capable of specifically binding to a target cell and delivering the cytokine such that it exerts its effect on the target cell. Cytokines for use with the present invention include: interleukins, transforming growth factors (TGFs), fibroblast growth factors (FGFs), platelet derived growth factors (PDGFs), epidermal growth factors (EGFs), connective tissue activated peptides (CTAPs), osteogenic factors, and biologically active analogs, fragments, and derivatives of such growth factors, B/T-cell differentiation factors, B/T-cell growth factors, mitogenic cytokines, chemotactic cytokines, colony stimulating factors, angiogenesis factors, IFN-α, IFN-β, IFN-γ, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, etc., leptin, myostatin, macrophage stimulating protein, platelet-derived growth factor, TNF-α, TNF-β, NGF, CD40L, CD137L/4-1BBL, human lymphotoxin-β, G-CSF, M-CSF, GM-CSF, PDGF, IL-1α, IL1-β, IP-10, PF4, GRO, 9E3, erythropoietin, endostatin, angiostatin, VEGF, transforming growth factor (TGF) supergene family include the beta transforming growth factors (for example TGF-β1, TGF-β2, TGF-β3); bone morphogenetic proteins (for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors (fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF)); Inhibins (for example, Inhibin A, Inhibin B); growth differentiating factors (for example, GDF-1); and Activins (for example, Activin A, Activin B, Activin AB).
For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:
While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
At present, protein engineering technology enables the ready and controlled addition of an antigen (or different antigens to one of the chains) of a recombinant mAb (H or L, usually the C-terminus of H is often used). If different antigens or different antigen sets need to be linked to the mAb, then the mAb needs to be re-engineered, expressed, and purified as a different entity.
The present invention provides for the complexing of multiple antigens or proteins (engineered, expressed, and purified independently from the primary mAb) in a controlled, multivariable fashion, to one single primary recombinant mAb. Presently, there are methods for engineering site-specific biotinylation sites that provide for the addition of different proteins (each engineered separately linked to streptavidin) to the one primary mAb. However, the present invention provides for addition to the primary mAb of multiple combinations, in fixed equimolar ratios and locations, of separately engineered proteins.
As used herein, the term “modular rAb carrier” is used to describe a recombinant antibody system that has been engineered to provide the controlled modular addition of diverse antigens, activating proteins, or other antibodies to a single recombinant monoclonal antibody (mAb). The rAb may be a monoclonal antibody made using standard hybridoma techniques, recombinant antibody display, humanized monoclonal antibodies and the like. The modular rAb carrier can be used to, e.g., target (via one primary recombinant antibody against an internalizing receptor, e.g., a human dendritic cell receptor) multiple antigens and/or antigens and an activating cytokine to dendritic cells (DC). The modular rAb carrier may also be used to join two different recombinant mAbs end-to-end in a controlled and defined manner.
The antigen binding portion of the “modular rAb carrier” may be one or more variable domains, one or more variable and the first constant domain, an Fab fragment, a Fab′ fragment, an F(ab)2 fragment, and Fv fragment, and Fabc fragment and/or a Fab fragment with portions of the Fc domain to which the cognate modular binding portions are added to the amino acid sequence and/or bound. The antibody for use in the modular rAb carrier can be of any isotype or class, subclass or from any source (animal and/or recombinant).
In one non-limiting example, the modular rAb carrier is engineered to have one or more modular cohesin-dockerin protein domains for making specific and defined protein complexes in the context of engineered recombinant mAbs. The mAb is a portion of a fusion protein that includes one or more modular cohesin-dockerin protein domains carboxy from the antigen binding domains of the mAb. The cohesin-dockerin protein domains may even be attached post-translationally, e.g., by using chemical cross-linkers and/or disulfide bonding.
The modular rAb carrier will be used to carry a separate molecule, e.g., a peptide, protein, lipid, carbohydrate, nucleic acid (oligonucleotide, aptamer, vector with or without base or backbone modifications) or combinations thereof by binding that separate molecule to the complementary half of the cohesion:dockerin pair. For example, either the dockerin or cohesin made be made into a fusion protein or chemically bound to an antigen, a peptide, a protein, a toxin, a cytokine, an enzyme, a structural protein, an extracellular matrix protein, another antibody, a cell or fragments thereof. The modular rAb carrier may have one or more cohesin, dockerin or both cohesin and dockerin domains that allow the formation of a complex with one or more complementary cohesin/dockerin-molecules for delivery via the antigen recognition domain of the modular rAb carrier.
The term “antigen” as used herein refers to a molecule that can initiate a humoral and/or cellular immune response in a recipient of the antigen. Antigen may be used in two different contexts with the present invention: as a target for the antibody or other antigen recognition domain of the rAb or as the molecule that is carried to and/or into a cell or target by the rAb as part of a dockerin/cohesin-molecule complement to the modular rAb carrier. The antigen is usually an agent that causes a disease for which a vaccination would be advantageous treatment. When the antigen is presented on MHC, the peptide is often about 8 to about 25 amino acids. Antigens include any type of biologic molecule, including, for example, simple intermediary metabolites, sugars, lipids and hormones as well as macromolecules such as complex carbohydrates, phospholipids, nucleic acids and proteins. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoal and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, and other miscellaneous antigens.
The modular rAb carrier is able to carry any number of active agents, e.g., antibiotics, anti-infective agents, antiviral agents, anti-tumoral agents, antipyretics, analgesics, anti-inflammatory agents, therapeutic agents for osteoporosis, enzymes, cytokines, anticoagulants, polysaccharides, collagen, cells, and combinations of two or more of the foregoing active agents. Examples of antibiotics for delivery using the present invention include, without limitation, tetracycline, aminoglycosides, penicillins, cephalosporins, sulfonamide drugs, chloramphenicol sodium succinate, erythromycin, vancomycin, lincomycin, clindamycin, nystatin, amphotericin B, amantidine, idoxuridine, p-amino salicyclic acid, isoniazid, rifampin, antinomycin D, mithramycin, daunomycin, adriamycin, bleomycin, vinblastine, vincristine, procarbazine, imidazole carboxamide, and the like.
Examples of anti-tumor agents for delivery using the present invention include, without limitation, doxorubicin, Daunorubicin, taxol, methotrexate, and the like. Examples of antipyretics and analgesics include aspirin, Motrin®, Ibuprofen®, naprosyn, acetaminophen, and the like.
Examples of anti-inflammatory agents for delivery using the present invention include, without limitation, include NSAIDS, aspirin, steroids, dexamethasone, hydrocortisone, prednisolone, Diclofenac Na, and the like.
Examples of therapeutic agents for treating osteoporosis and other factors acting on bone and skeleton include for delivery using the present invention include, without limitation, calcium, alendronate, bone GLa peptide, parathyroid hormone and its active fragments, histone H4-related bone formation and proliferation peptide and mutations, derivatives and analogs thereof.
Examples of enzymes and enzyme cofactors for delivery using the present invention include, without limitation, pancrease, L-asparaginase, hyaluronidase, chymotrypsin, trypsin, tPA, streptokinase, urokinase, pancreatin, collagenase, trypsinogen, chymotrypsinogen, plasminogen, streptokinase, adenyl cyclase, superoxide dismutase (SOD), and the like.
Examples of cytokines for delivery using the present invention include, without limitation, interleukins, transforming growth factors (TGFs), fibroblast growth factors (FGFs), platelet derived growth factors (PDGFs), epidermal growth factors (EGFs), connective tissue activated peptides (CTAPs), osteogenic factors, and biologically active analogs, fragments, and derivatives of such growth factors. Cytokines may be B/T-cell differentiation factors, B/T-cell growth factors, mitogenic cytokines, chemotactic cytokines, colony stimulating factors, angiogenesis factors, IFN-α, IFN-β, IFN-γ, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, etc., leptin, myostatin, macrophage stimulating protein, platelet-derived growth factor, TNF-α, TNF-β, NGF, CD40L, CD137L/4-1BBL, human lymphotoxin-β, G-CSF, M-CSF, GM-CSF, PDGF, IL-1α, IL1-β, IP-10, PF4, GRO, 9E3, erythropoietin, endostatin, angiostatin, VEGF or any fragments or combinations thereof. Other cytokines include members of the transforming growth factor (TGF) supergene family include the beta transforming growth factors (for example TGF-β1, TGF-β2, TGF-β3); bone morphogenetic proteins (for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors (for example, fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF)); Inhibins (for example, Inhibin A, Inhibin B); growth differentiating factors (for example, GDF-1); and Activins (for example, Activin A, Activin B, Activin AB).
Examples of growth factors for delivery using the present invention include, without limitation, growth factors that can be isolated from native or natural sources, such as from mammalian cells, or can be prepared synthetically, such as by recombinant DNA techniques or by various chemical processes. In addition, analogs, fragments, or derivatives of these factors can be used, provided that they exhibit at least some of the biological activity of the native molecule. For example, analogs can be prepared by expression of genes altered by site-specific mutagenesis or other genetic engineering techniques.
Examples of anticoagulants for delivery using the present invention include, without limitation, include warfarin, heparin, Hirudin, and the like. Examples of factors acting on the immune system include for delivery using the present invention include, without limitation, factors which control inflammation and malignant neoplasms and factors which attack infective microorganisms, such as chemotactic peptides and bradykinins.
Examples of viral antigens and/or viral antigenic targets include, but are not limited to, e.g., retroviral antigens such as retroviral antigens from the human immunodeficiency virus (HIV) antigens such as gene products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV components; hepatitis viral antigens such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis A, B, and C, viral components such as hepatitis C viral RNA; influenza viral antigens such as hemagglutinin and neuraminidase and other influenza viral components; measles viral antigens such as the measles virus fusion protein and other measles virus components; rubella viral antigens such as proteins E1 and E2 and other rubella virus components; rotaviral antigens such as VP7sc and other rotaviral components; cytomegaloviral antigens such as envelope glycoprotein B and other cytomegaloviral antigen components; respiratory syncytial viral antigens such as the RSV fusion protein, the M2 protein and other respiratory syncytial viral antigen components; herpes simplex viral antigens such as immediate early proteins, glycoprotein D, and other herpes simplex viral antigen components; varicella zoster viral antigens such as gpI, gpII, and other varicella zoster viral antigen components; Japanese encephalitis viral antigens such as proteins E, M-E, M-E-NS1, NS1, NS1-NS2A, 80% E, and other Japanese encephalitis viral antigen components; rabies viral antigens such as rabies glycoprotein, rabies nucleoprotein and other rabies viral antigen components. See Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe, D. M. (Raven Press, New York, 1991) for additional examples of viral antigens.
Antigens and/or antigenic targets that may be delivered using the rAb-DC/DC-antigen vaccines of the present invention include genes encoding antigens such as viral antigens, bacterial antigens, fungal antigens or parasitic antigens. Viruses include picornavirus, coronavirus, togavirus, flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus, arenavirus, reovirus, retrovirus, papilomavirus, parvovirus, herpesvirus, poxvirus, hepadnavirus, and spongiform virus. Other viral targets include influenza, herpes simplex virus 1 and 2, measles, dengue, smallpox, polio or HIV. Pathogens include trypanosomes, tapeworms, roundworms, helminthes, malaria. Tumor markers, such as fetal antigen or prostate specific antigen, may be targeted in this manner. Other examples include: HIV env proteins and hepatitis B surface antigen. Administration of a vector according to the present invention for vaccination purposes would require that the vector-associated antigens be sufficiently non-immunogenic to enable long term expression of the transgene, for which a strong immune response would be desired. In some cases, vaccination of an individual may only be required infrequently, such as yearly or biennially, and provide long term immunologic protection against the infectious agent. Specific examples of organisms, allergens and nucleic and amino sequences for use in vectors and ultimately as antigens with the present invention may be found in U.S. Pat. No. 6,541,011, relevant portions incorporated herein by reference, in particular, the tables that match organisms and specific sequences that may be used with the present invention.
Bacterial antigens for use with the rAb vaccine disclosed herein include, but are not limited to, e.g., bacterial antigens such as pertussis toxin, filamentous hemagglutinin, pertactin, FIM2, FIM3, adenylate cyclase and other pertussis bacterial antigen components; diptheria bacterial antigens such as diptheria toxin or toxoid and other diptheria bacterial antigen components; tetanus bacterial antigens such as tetanus toxin or toxoid and other tetanus bacterial antigen components; streptococcal bacterial antigens such as M proteins and other streptococcal bacterial antigen components; gram-negative bacilli bacterial antigens such as lipopolysaccharides and other gram-negative bacterial antigen components, Mycobacterium tuberculosis bacterial antigens such as mycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secreted protein, antigen 85A and other mycobacterial antigen components; Helicobacter pylori bacterial antigen components; pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharides and other pneumococcal bacterial antigen components; haemophilus influenza bacterial antigens such as capsular polysaccharides and other haemophilus influenza bacterial antigen components; anthrax bacterial antigens such as anthrax protective antigen and other anthrax bacterial antigen components; rickettsiae bacterial antigens such as rompA and other rickettsiae bacterial antigen component. Also included with the bacterial antigens described herein are any other bacterial, mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens. Partial or whole pathogens may also be: haemophilus influenza; Plasmodium falciparum; neisseria meningitidis; streptococcus pneumoniae; neisseria gonorrhoeae; salmonella serotype typhi; shigella; vibrio cholerae; Dengue Fever; Encephalitides; Japanese Encephalitis; lyme disease; Yersinia pestis; west nile virus; yellow fever; tularemia; hepatitis (viral; bacterial); RSV (respiratory syncytial virus); HPIV 1 and HPIV 3; adenovirus; small pox; allergies and cancers.
Fungal antigens for use with compositions and methods of the invention include, but are not limited to, e.g., candida fungal antigen components; histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other histoplasma fungal antigen components; cryptococcal fungal antigens such as capsular polysaccharides and other cryptococcal fungal antigen components; coccidiodes fungal antigens such as spherule antigens and other coccidiodes fungal antigen components; and tinea fungal antigens such as trichophytin and other coccidiodes fungal antigen components.
Examples of protozoal and other parasitic antigens include, but are not limited to, e.g., plasmodium falciparum antigens such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte/gamete surface antigens, blood-stage antigen pf 155/RESA and other plasmodial antigen components; toxoplasma antigens such as SAG-1, p30 and other toxoplasmal antigen components; schistosomae antigens such as glutathione-S-transferase, paramyosin, and other schistosomal antigen components; leishmania major and other leishmaniae antigens such as gp63, lipophosphoglycan and its associated protein and other leishmanial antigen components; and trypanosoma cruzi antigens such as the 75-77 kDa antigen, the 56 kDa antigen and other trypanosomal antigen components.
Target antigens on immune cell surfaces that can be targeted using the antigen recognition site of the antibody portion of the rAb of the present invention will generally be selected based on a number of factors, including: likelihood of internalization, level of immune cell specificity, type of immune cell targeted, level of immune cell maturity and/or activation and the like. Examples of cell surface markers for dendritic cells include, but are not limited to, MHC class I, MHC Class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD 19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fcγ receptor or other receptor relatively specifically expressed by antigen presenting cells. Examples of cell surface markers for antigen presenting cells include, but are not limited to, MHC class I, MHC Class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD 19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fcγ receptor or other receptor relatively specifically expressed by antigen presenting cells. Examples of cell surface markers for T cells include, but are not limited to, CD3, CD4, CD8, CD 14, CD20, CD11b, CD16, CD45 and HLA-DR.
Target antigens on cell surfaces for delivery includes those characteristic of tumor antigens typically will be derived from the cell surface, cytoplasm, nucleus, organelles and the like of cells of tumor tissue. Examples of tumor targets for the antibody portion of the present invention include, without limitation, hematological cancers such as leukemias and lymphomas, neurological tumors such as astrocytomas or glioblastomas, melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumors such as gastric or colon cancer, liver cancer, pancreatic cancer, genitourinary tumors such cervix, uterus, ovarian cancer, vaginal cancer, testicular cancer, prostate cancer or penile cancer, bone tumors, vascular tumors, or cancers of the lip, nasopharynx, pharynx and oral cavity, esophagus, rectum, gall bladder, biliary tree, larynx, lung and bronchus, bladder, kidney, brain and other parts of the nervous system, thyroid, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma and leukemia.
Examples of antigens that may be delivered alone or in combination to immune cells for antigen presentation using the present invention include tumor proteins, e.g., mutated oncogenes; viral proteins associated with tumors; and tumor mucins and glycolipids. The antigens may be viral proteins associated with tumors would be those from the classes of viruses noted above. Certain antigens may be characteristic of tumors (one subset being proteins not usually expressed by a tumor precursor cell), or may be a protein which is normally expressed in a tumor precursor cell, but having a mutation characteristic of a tumor. Other antigens include mutant variant(s) of the normal protein having an altered activity or subcellular distribution, e.g., mutations of genes giving rise to tumor antigens.
Specific non-limiting examples of tumor antigens include: CEA, prostate specific antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12, MUC (Mucin) (e.g., MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides, ras, myc, tyrosinase, MART (melanoma antigen), Pmel 17(gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), Prostate Ca psm, PRAME (melanoma antigen), β-catenin, MUM-1-B (melanoma ubiquitous mutated gene product), GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10, c-ERB2 (Her2/neu), EBNA (Epstein-Barr Virus nuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and E7, p53, lung resistance protein (LRP), Bcl-2, and Ki-67. In addition, the immunogenic molecule can be an autoantigen involved in the initiation and/or propagation of an autoimmune disease, the pathology of which is largely due to the activity of antibodies specific for a molecule expressed by the relevant target organ, tissue, or cells, e.g., SLE or MG. In such diseases, it can be desirable to direct an ongoing antibody-mediated (i.e., a Th2-type) immune response to the relevant autoantigen towards a cellular (i.e., a Th1-type) immune response. Alternatively, it can be desirable to prevent onset of or decrease the level of a Th2 response to the autoantigen in a subject not having, but who is suspected of being susceptible to, the relevant autoimmune disease by prophylactically inducing a Th1 response to the appropriate autoantigen. Autoantigens of interest include, without limitation: (a) with respect to SLE, the Smith protein, RNP ribonucleoprotein, and the SS-A and SS-B proteins; and (b) with respect to MG, the acetylcholine receptor. Examples of other miscellaneous antigens involved in one or more types of autoimmune response include, e.g., endogenous hormones such as luteinizing hormone, follicular stimulating hormone, testosterone, growth hormone, prolactin, and other hormones.
Antigens involved in autoimmune diseases, allergy, and graft rejection can be used in the compositions and methods of the invention. For example, an antigen involved in any one or more of the following autoimmune diseases or disorders can be used in the present invention: diabetes, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome, including keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease, Graves opthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis. Examples of antigens involved in autoimmune disease include glutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and the thyroid stimulating hormone (TSH) receptor. Examples of antigens involved in allergy include pollen antigens such as Japanese cedar pollen antigens, ragweed pollen antigens, rye grass pollen antigens, animal derived antigens such as dust mite antigens and feline antigens, histocompatiblity antigens, and penicillin and other therapeutic drugs. Examples of antigens involved in graft rejection include antigenic components of the graft to be transplanted into the graft recipient such as heart, lung, liver, pancreas, kidney, and neural graft components. The antigen may be an altered peptide ligand useful in treating an autoimmune disease.
As used herein, the term “epitope(s)” refer to a peptide or protein antigen that includes a primary, secondary or tertiary structure similar to an epitope located within any of a number of pathogen polypeptides encoded by the pathogen DNA or RNA. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against such polypeptides will also bind to, react with, or otherwise recognize, the peptide or protein antigen. Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art. The identification of pathogen epitopes, and/or their functional equivalents, suitable for use in vaccines is part of the present invention. Once isolated and identified, one may readily obtain functional equivalents. For example, one may employ the methods of Hopp, as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, for example, Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acid sequence of these “epitopic core sequences” may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.
As used herein, the term “promoter” describes a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence (i.e., ORF) to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. A listing of promoters and/or enhancers that may be used with the present invention is described in, e.g., U.S. Pat. No. 6,410,241, relevant descriptions and tables incorporated herein by reference.
As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations, in vivo, ex vivo or in vitro. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of expressing a heterologous gene encoded by a vector as delivered using the rAb protein vector of the present invention. A host cell can, and has been, used as a recipient for vectors. A host cell may be “transfected” or “transformed,” which refers to a process by which the exogenous nucleic acid expressing an antigen, as disclosed herein, is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.
The preparation of vaccine compositions that includes the nucleic acids that encode antigens of the invention as the active ingredient, may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to infection can also be prepared. The preparation may be emulsified, encapsulated in liposomes. The active immunogenic ingredients are often mixed with carriers which are pharmaceutically acceptable and compatible with the active ingredient.
The term “pharmaceutically acceptable carrier” refers to a carrier that does not cause an allergic reaction or other untoward effect in subjects to whom it is administered. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants that may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Other examples of adjuvants include DDA (dimethyldioctadecylammonium bromide), Freund's complete and incomplete adjuvants and QuilA. In addition, immune modulating substances such as lymphokines (e.g., IFN-γ, IL-2 and IL-12) or synthetic IFN-γ inducers such as poly I:C can be used in combination with adjuvants described herein.
Pharmaceutical products that may include a naked polynucleotide with a single or multiple copies of the specific nucleotide sequences that bind to specific DNA-binding sites of the apolipoproteins present on plasma lipoproteins as described in the current invention. The polynucleotide may encode a biologically active peptide, antisense RNA, or ribozyme and will be provided in a physiologically acceptable administrable form. Another pharmaceutical product that may spring from the current invention may include a highly purified plasma lipoprotein fraction, isolated according to the methodology, described herein from either the patients blood or other source, and a polynucleotide containing single or multiple copies of the specific nucleotide sequences that bind to specific DNA-binding sites of the apolipoproteins present on plasma lipoproteins, prebound to the purified lipoprotein fraction in a physiologically acceptable, administrable form.
Yet another pharmaceutical product may include a highly purified plasma lipoprotein fraction which contains recombinant apolipoprotein fragments containing single or multiple copies of specific DNA-binding motifs, prebound to a polynucleotide containing single or multiple copies of the specific nucleotide sequences, in a physiologically acceptable administrable form. Yet another pharmaceutical product may include a highly purified plasma lipoprotein fraction which contains recombinant apolipoprotein fragments containing single or multiple copies of specific DNA-binding motifs, prebound to a polynucleotide containing single or multiple copies of the specific nucleotide sequences, in a physiologically acceptable administrable form.
The dosage to be administered depends to a great extent on the body weight and physical condition of the subject being treated as well as the route of administration and frequency of treatment. A pharmaceutical composition that includes the naked polynucleotide prebound to a highly purified lipoprotein fraction may be administered in amounts ranging from 1 μg to 1 mg polynucleotide and 1 μg to 100 mg protein.
Administration of the therapeutic virus particle to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of the vector. It is anticipated that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described gene therapy.
Where clinical application of a gene therapy is contemplated, it will be necessary to prepare the complex as a pharmaceutical composition appropriate for the intended application. Generally this will entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals. One also will generally desire to employ appropriate salts and buffers to render the complex stable and allow for complex uptake by target cells.
Aqueous compositions of the present invention may include an effective amount of the compound, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions can also be referred to as inocula. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. The compositions of the present invention may include classic pharmaceutical preparations. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
Disease States. Depending on the particular disease to be treated, administration of therapeutic compositions according to the present invention will be via any common route so long as the target tissue is available via that route in order to maximize the delivery of antigen to a site for maximum (or in some cases minimum) immune response. Administration will generally be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Other areas for delivery include: oral, nasal, buccal, rectal, vaginal or topical. Topical administration would be particularly advantageous for treatment of skin cancers. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
Vaccine or treatment compositions of the invention may be administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories, and in some cases, oral formulations or formulations suitable for distribution as aerosols. In the case of the oral formulations, the manipulation of T-cell subsets employing adjuvants, antigen packaging, or the addition of individual cytokines to various formulation that result in improved oral vaccines with optimized immune responses. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25-70%.
The antigen encoding nucleic acids of the invention may be formulated into the vaccine or treatment compositions as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
Vaccine or treatment compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered depends on the subject to be treated, including, e.g., capacity of the subject's immune system to synthesize antibodies, and the degree of protection or treatment desired. Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a range from about 0.1 mg to 1000 mg, such as in the range from about 1 mg to 300 mg, and preferably in the range from about 10 mg to 50 mg. Suitable regiments for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and may be peculiar to each subject. It will be apparent to those of skill in the art that the therapeutically effective amount of nucleic acid molecule or fusion polypeptides of this invention will depend, inter alia, upon the administration schedule, the unit dose of antigen administered, whether the nucleic acid molecule or fusion polypeptide is administered in combination with other therapeutic agents, the immune status and health of the recipient, and the therapeutic activity of the particular nucleic acid molecule or fusion polypeptide.
The compositions can be given in a single dose schedule or in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may include, e.g., 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. Periodic boosters at intervals of 1-5 years, usually 3 years, are desirable to maintain the desired levels of protective immunity. The course of the immunization can be followed by in vitro proliferation assays of peripheral blood lymphocytes (PBLs) co-cultured with ESAT6 or ST-CF, and by measuring the levels of IFN-γ released from the primed lymphocytes. The assays may be performed using conventional labels, such as radionuclides, enzymes, fluorescent labels and the like. These techniques are known to one skilled in the art and can be found in U.S. Pat. Nos. 3,791,932, 4,174,384 and 3,949,064, relevant portions incorporated by reference.
The modular rAb carrier and/or conjugated rAb carrier-(cohesion/dockerin and/or dockerin-cohesin)-antigen complex (rAb-DC/DC-antigen vaccine) may be provided in one or more “unit doses” depending on whether the nucleic acid vectors are used, the final purified proteins, or the final vaccine form is used. Unit dose is defined as containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. The subject to be treated may also be evaluated, in particular, the state of the subject's immune system and the protection desired. A unit dose need not be administered as a single injection but may include continuous infusion over a set period of time. Unit dose of the present invention may conveniently may be described in terms of DNA/kg (or protein/Kg) body weight, with ranges between about 0.05, 0.10, 0.15, 0.20, 0.25, 0.5, 1, 10, 50, 100, 1,000 or more mg/DNA or protein/kg body weight are administered. Likewise the amount of rAb-DC/DC-antigen vaccine delivered can vary from about 0.2 to about 8.0 mg/kg body weight. Thus, in particular embodiments, 0.4 mg, 0.5 mg, 0.8 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 4.0 mg, 5.0 mg, 5.5 mg, 6.0 mg, 6.5 mg, 7.0 mg and 7.5 mg of the vaccine may be delivered to an individual in vivo. The dosage of rAb-DC/DC-antigen vaccine to be administered depends to a great extent on the weight and physical condition of the subject being treated as well as the route of administration and the frequency of treatment. A pharmaceutical composition that includes a naked polynucleotide prebound to a liposomal or viral delivery vector may be administered in amounts ranging from 1 μg to 1 mg polynucleotide to 1 μg to 100 mg protein. Thus, particular compositions may include between about 1 μg, 5 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg or 1,000 μg polynucleotide or protein that is bound independently to 1 μg, 5 μg, 10 μg, 20 μg, 3.0 μg, 40 μg 50 μg, 60 μg, 70 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 1.5 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg vector.
The present invention was tested in an in vitro cellular system that measures immune stimulation of human Flu-specific T cells by dendritic cells to which Flu antigen has been targeted. The results shown herein demonstrate the specific expansion of such antigen specific cells at doses of the antigen which are by themselves ineffective in this system.
The present invention may also be used to make a modular rAb carrier that is, e.g., a recombinant humanized mAb (directed to a specific human dendritic cell receptor) complexed with protective antigens from Ricin, Anthrax toxin, and Staphylococcus B enterotoxin. The potential market for this entity is vaccination of all military personel and stored vaccine held in reserve to administer to large population centers in response to any biothreat related to these agents. The invention has broad application to the design of vaccines in general, both for human and animal use. Industries of interest is pharmaceutical and biotechnology
One commercial application of the invention is a recombinant humanized mAb (directed to the specific human dendritic cell receptor DCIR) fused through the Ab heavy chain to antigens known or suspected to encode protective antigens. These include as examples for vaccination against various agents-hemagglutinins from Influenza H5N1; HIV gag from attenuated toxins from Ricin, Anthrax toxin, and Staphylococcus B enterotoxin; ‘strings’ of antigenic peptides from melanona anigens, etc. The potential market for this entity is preventative or therapeutic vaccination of at risk or infected people. The invention has broad application for vaccination against many diseases and cancers, both for human and animal use. Industries of interest are pharmaceutical and biotechnology. In addition, this invention has implications beyond anti-DCIR application since it describes a method to identify particularly favorable sequences to enhance secretion of recombinant antibodies.
The application of anti-DCIR combining regions for making engineered recombinant monoclonal antibodies fused to antigens as potent therapeutic or preventative vaccination agents. Use of different V-region sequences against the same combining specificity to find those most compatible with efficient expression of a H chain C-terminal linked antigen or other protein sequence.
One type of therapeutic (in this case, vaccination) entity envisioned is a humanized DC-targeting mAb-antigen fusion protein, where the antibody variable region specificity is directed against an internalizing human dendritic cell receptor. The present state-of-the art is to engineer the fusion of the desired antigen to the C-terminus of the mAb H chain. This paradigm obviously allows different antigens (A1, A2, A3) to be engineered to the same proven targeting mAb backbone (Y in the figure below), thus extending the utility of the one mAb to immunizing against different pathogenic agents. This concept can be further extended by engineering, e.g., the A1, A2, A3 coding regions end-to-end fused to the IgGFc C-terminal coding region.
The present invention disclosed a new paradigm for linking the antigen to the targeting mAb that extends the concept for the first time to multiple antigens targeted in a complex simultaneously with the same engineered humanized mAb (MATCHMAB).
The MATCHMAB is based on using cellulosome-assembly cohesin-dockerin sequences to form modular non-covalent targeting mAb-antigen complexes. The relatively small and specific cohesin-dockerin protein-protein interaction domains can allow simple customized formulation of targeting mAb-antigen complexes. Thus, a single manufactured humanized mAb (in the above notation: Y.C1.C2.C3.Cn) can be use as the basis of delivering multiple antigens in various, yet strictly defined, combinations.
Example of sequence encoding C1.C2.C3.Cn is taken from the public sequence >gi|50656899|gb|AAT79550.1| of cellulosomal anchoring scaffoldin B precursor (Bacteroides cellulosolvens). Below with blue showing the leader secretion sequence and yellow and grey highlighting various cohesin domains. Red regions are linkers spacing some of the cohesin domains.
The cohesin domains (C) interact with small domains (e.g., 56 residues) called dockerins (D). These are Ca++ containing structures with two-fold symmetry and they can bind to a cognate cohesin with various affinities (e.g., 6E6 M, 2E7M). Affinities between dockerin and multiple cohesins (as found on scaffoldins) can be much higher (e.g., >E9 M). The interaction is non-covalent and is well defined (by structure analysis) for at least one C-D pair. Dockerins are designed to be domains linked to different domain (enzyme in nature), and cohesions are designed to function in linear arrays (either directly end-to-end, or joined by flexible PT-rich linkers of various sizes (e.g., 12, 17, 25, 28, 36). It is known that a particular dockerin can have specificity for a particular cohesin (e.g., a C-D pair from one bacterial species may not be interchangeable with a C-D pair from a different species). This feature makes it is possible to ensure the specific and precise interaction of various D-antigen fusion proteins with an engineered mAb containing cohesin domains of various specificities.
In practice, this invention includes adapting C-D pairs known from the literature, newly gleamed from nature, or developed with new specificities using phage display technology. The latter technology can also be used to enhance (‘mature’) the affinity of a C-D interaction, should this be desired. Also, engineering cysteine residues at opposing faces of the C-D interaction (based on modeling from the published C-D structures) could be used to make a covalent bond between C-D to strengthen the interaction. Furthermore, the dimeric nature of the mAb (and therefore the linked C-domains) can be used to advantage for affinity enhancement purposes. In this embodiment, e.g., the D-antigen fusion protein is engineered either with a second identical dockerin domain (D-antigen-D, or D-D-antigen), or with a homodimerization domain. This configuration, provided the linkers between domains were not constraining, will result in the preferred simultaneous binding of both D domains to the same mAb, with greatly enhanced stability compared to the single interaction.
Based on the crystal structure of the cohesin-dockerin complex (e.g., see PNAS 2003, 13809-13814, Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex. Ana L. Carvalho *, Fernando M. V. Dias, José A. M. Prates, Tibor Nagy, Harry J. Gilbert, Gideon J. Davies, Luis M. A. Ferreira, Maria J. Romão and Carlos M. G. A. Fonte), it is apparent that one embodiment is an antigen-dockerin fusion proteins (i.e., antigen fused to the N-terminus of a dockerin). However, both from the structure and from the nature of cohesin domain organization within scaffolding, it is apparent that cohesions can be fused end-to-end, even without spacer sequences. Furthermore, it is apparent that well-described techniques are available to engineer miniaturized versions of the cohesin and dockerin domains (see for example, Proc. Natl. Acad. Sci. USA Vol. 94, pp. 10080-10085, September 1997. Structural mimicry of a native protein by a minimized binding domain. Melissa A. Starovasnik, Andrew C. Braisted, And James A. Wells).
It is recognized herein that the linker sequences have a propensity for O-linked glycosylation resulting from ST richness. Also, both the C and D domains can have potential N-linked sites. These features can be advantageous in enhancing the solubility of the mammalian cell-expressed engineered mAb through decoration with carbohydrates. Of course, the consequences of glycosylation of the C domains needs to be check by function (binding to the cognate D), and if needed rectified by site directed mutagenesis. An attractive feature of this invention is that D-A can be expressed in whatever system is known to be best. For example, the tumor antigen MART1 is a membrane protein and is best prepared in high yield via E. coli inclusion bodies. Schema using antigens directly fused to the mAb are restricted to antigens that are compatible with mammalian-cell expression.
Another embodiment of the invention is the use of the D-C interaction to make bi-specific mAbs joined tail-to-tail.
Example 2 shows that particular cohesin and dockerin domains can be successfully and efficiently secreted from mammalian cells as fusion proteins while maintaining the specific and high affinity cohesin-dockerin protein-protein interaction. While the extensive cohesin-dockerin literature teaches the expectation that such fusion proteins should have this functionality, it does not describe production of such fusion proteins in mammalian secretion systems. The state of scientific knowledge does not allow the prediction of the discovery since the rules (other than features such as signal peptide) for successful secretion are not fully established. Furthermore, the cohesin linker regions are known to be glycosylated in their native bacteria, and the cohesin and dockerin domains contain predicted glycosylation sites. While this may actually favor secretion from mammalian cells, it is unclear if ‘unnatural’ glyosylation will perturb the cohesis-dockerin interaction.
While cohesin-dockerin interaction for various commercial applications has been published, the present invention is based on a previously unrealized utility for this interaction built around assembling specific protein complexes unrelated to the envisioned controlled assembly enzyme applications.
The invention includes the use of all cohesin-dockerin sequences from diverse cellulose degrading microbes, but describes the application of specific cohesin and dockerin and linker sequences from the microbe Clostridium thermocellum. For example, the sequence described in Table 1 encodes the H chain of a human IgG4 linked at the C-terminal codon to a Clostridium thermocellum dockerin sequence (called rAb.doc). Other embodiments of rAb.doc proteins are described similarly in Table 2 and these are engineered by simply transferring the dockerin coding region as a DNA fragment to vectors encoding the different H chain entities.
TABLE 1 shows the nucleic acid and amino acid sequences for rAB-pIRES2(hIgG4H-Dockerin) or C52. DNA (entire coding region) and amino acid sequence (the predicted secreted product) of human IgG4H.doc fusion protein is shown below. The dockerin domain (taken from Clostridium thermocellum celD is highlighted in yellow and the H chain and dockerin joining sequence is underlined. The highly predicted N-linked glycosylation site within the dockerin domain is highlighted in red.
TABLE 2 shows the nucleic acid and amino acid sequences for rAB-pIRES2(mAnti-DCIR2C9H-LV-hIgG4H-C-Dockerin) or C82. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The dockerin domain is highlighted in yellow and the H chain and dockerin joining sequence is underlined. The IgG variable region is highlighted in blue. The highly predicted N-linked glycosylation site within the dockerin domain is highlighted in red.
TABLE 3 shows the nucleic acid and amino acid sequences for rAB-(mAnti -ASGPR—49C11—7H-SLAML-V-hIgG4H-C-Dockerin) or C153. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The dockerin domain is highlighted in yellow and the H chain and dockerin joining sequence is underlined. The IgG variable region is highlighted in blue. The highly predicted N-linked glycosylation site within the dockerin domain is highlighted in red.
TABLE 4 shows the nucleic acid and amino acid sequences for rAB-pIRES2(mAnti-DC-SIGNL16E7H-LV-hIgG4H-C-Dockerin) or C92. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The dockerin domain is highlighted in yellow and the H chain and dockerin joining sequence is underlined. The IgG variable region is highlighted in blue. The highly predicted N-linked glycosylation site within the dockerin domain is highlighted in red.
Mammalian expression plasmids encoding such rAb.doc IgG H chain proteins are created using standard molecular biology techniques and can be based on commercially available expression plasmid vectors such as pIRES2-DsRed2 (BD Biosciences). To produce secreted rAb.doc, mammalian cells are co-transfected with this expression plasmid and an expression plasmid encoding a complimentary IgG L chain (exemplified in Table 3). Standard protocols (such as the FreeStyle™ 293 Expression System, Invitrogen) are used as for mammalian cells, transfection reagents, and culture media. Transfected cells are cultured for 3-7 days and the culture supernatant is harvested by centrifugation, clarified by filtration, and the rAB.doc protein purified by Protein G affinity chromatography using protocols from the column manufacturer (GE Pharmacia).
TABLE 5 shows the nucleic acid and amino acid sequences for rAB-pIRES2(mAnti-DC-SIGNL16E7K-LV-hIgGK-C) or C73. DNA (entire coding region) and amino acid sequence (the predicted secreted product) of IgG Kappa protein fusing the V region from the mAnti-DC-SIGNL16E7 hybridoma (highlighted in blue) to a human C region (highlighted in yellow).
The invention embodies the unanticipated presence and use of this glycosylation site that likely confers onto mammalian cell-secreted dockerin fusion proteins desirable solubility and pharmacokinetic properties well known to be associated with glycosylation.
The invention embodies the property of the dockerin domain to be efficiently and functionally expressed in the context of fusion proteins other than hIgG4 and its close derivatives. For example, Table 6 shows the sequence of a rAb.doc entity based on a mouse IgG2b H chain fusion protein.
TABLE 6 shows the nucleic acid and amino acid sequences for rAB-pCMV(mIgG2bH-Dockerin) or C19. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown. The dockerin domain is highlighted in yellow and the H chain and dockerin joining sequence is underlined. The highly predicted N-linked glycosylation site within the dockerin domain is highlighted in red.
The use of the rAb.doc invention detailed above is the assembly of rAb-antigen or toxin or activator or enzyme complexes via the specificity and tenacity of the dockerin-cohesin interaction. Table 5 shows one embodiment of the invention in the form of a cohesin.alkaline phosphatase fusion protein (coh.AP). Also described are additional embodiments such as an alkaline phosphatase fusion protein containing two cohesion domains (coh.coh.AP) and other proteins are examples of the generality of the invention such as the single cohesin domain fused to other sequences such as the mature sequence of human prostate specific antigen (coh.hPSA) and to the HA1 domain of influenza A HA5 (coh.Flu HA5-1).
TABLE 7 shows the nucleic acid and amino acid sequences for Mam-pCDM8(Cohesin-SLAML-AP-6×His) or C16. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The cohesin domain is highlighted in yellow and the cohesin and alkaline phosphatase joining sequence is underlined. The highly predicted (G-score >0.5, NetOGlyc 3.1 Server—Technical University of Denmark) O-linked glycosylation sites within the cohesin domain and the linker distal to the cohesin domain are highlighted in red. Residues highlighted grey are a C-terminal His tag to facilitate purification via metal affinity chromatography.
TABLE 8 shows the nucleic acid and amino acid sequences for Mam-pCDM8(Cohesin-Cohesin-SLAML-AP-6×His) or C17. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The cohesin domain is highlighted in yellow and the cohesin and alkaline phosphatase joining sequence is underlined. The highly predicted O-linked glycosylation sites within the linker distal to the cohesin domains are highlighted in red as is a single highly predicted N-linked glycosylation site (NPT). Residues highlighted grey are a C-terminal His tag to facilitate purification via metal affinity chromatography.
TABLE 9 shows the nucleic acid and amino acid sequences for Mam-pCDM8(SLAML-Cohesin-hPSA) or C149. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The cohesin domain is highlighted in yellow and the cohesin and hPSA joining sequence is underlined. The highly predicted O-linked glycosylation sites within the linker distal to the cohesin domains and a single highly predicted N-linked glycosylation site within the cohesin domain are highlighted in red.
TABLE 10 shows the nucleic acid and amino acid sequences for Mam-pCDM8(SLAML-Cohesin-FluHA5-1-6×His) or C24. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The cohesin domain is highlighted in yellow and the cohesin and Flu HA5-1 joining sequence is underlined. The highly predicted O-linked glycosylation sites within the linker distal to the cohesin domains and a single highly predicted N-linked glycosylation site within the cohesin domain are highlighted in red. Residues highlighted grey are a C-terminal His tag to facilitate purification via metal affinity chromatography.
Similar to the above mentioned rAb.doc constructs, the invention embodies the efficient secretion from mammalian cells of functional cohesin fusion proteins (called herein coh.fusions). It was not obvious that cohesin domains could be so successfully secreted while retaining dockerin-binding function.
The invention embodies the application of assembly of specific protein complexes based on the cohesin.dockerin interaction. Specific antibody.antigen complexes can also be assembled using the established interaction of protein A or protein G IgFc binding domains. The invention embodies unique properties of the cohesin.dockerin interaction that result in greatly superior complex formation compared to the e.g., protein G interaction with IgG. In
This example shows the use of such coh.doc complexes in settings containing serum (e.g., tissue culture media and in vivo administration).
The invention also embodies a particular utility of the coh.doc interaction that permits a production process that ensures complete complex formation and that can be concomitant with a purification process for the coh.fusion protein entity. This invention is exemplified in
A well described feature of cohesin domains is their compatibility with the standard E. coli bacterial expression system. The invention embodies the novel use of expression of dockerin fusion proteins in mammalian secretion systems, and it also encompasses the formation of coh.doc complexes where the different components (i.e., coh and doc) are expressed in different systems. This is a great advantage since it affords the possibility of using the most favorable expression system for each component. For example, coh.Flu M1 expression constructs failed to efficiently direct the synthesis of secreted product from transfected mammalian cells. However, coh.Flu M1 was very efficiently expressed as a soluble protein in E. coli. Table 6 shows the sequence of the coh.Flu M1 used in this example.
TABLE 11 shows the nucleic and amino acid sequence for E coli-pET28(Cohesin-FluM1-6×His) or C32 is shown below. In the amino acid sequence the cohesin domain is highlighted in yellow and the point of fusion between cohesion and influenza A M1 protein is underlined. Residues highlighted grey are a C-terminal His tag to facilitate purification via metal affinity chromatography.
E coli-pET28(Cohesin-FluM1-6×His) or C32.
The invention embodies the use of the dockerin.cohesin interaction to assemble ordered and specific complexes for various therapeutic or vaccination purposes. An example is the use of rAb.doc with binding specificity to an internalizing human Dendritic Cell (DC) receptor complexed with coh.Flu M1 protein.
While one type of embodiment of the invention is a vaccine composed of an Anti-DC-rAb.doc:coh.antigen complex, it is envisioned that in some cases a preferred DC-targeting vaccine will be Anti-DC-rAb.antigen where antigen is likely a string of protective antigens. Identification of such antigens in efficacious combinations compatible with efficient expression in production systems is extremely problematic. One embodiment of the invention affords a method to streamline testing of antigen epitope combinations for the development of such vaccines. Specifically, the invention teaches a method to screen likely antigen epitopes alone and in combinations for efficacy as a prelude to addressing production of the desired Anti-DC-rAb.antigen. For example, TABLE 13 shows the sequences of exemplative cohesin.peptide constructs which can be readily expressed via E. coli systems. Using techniques similar to those described in
TABLE 12 shows the amino acid sequence of the melanoma-associated antigen gp100. Well known HLA-A201-restricted dominant peptides are shaded and detailed below the sequence. Peptide sequences labeled M are variants with enhanced affinity for HLA-A201. C180 is an E. coli expression construct that encodes the sequence shown below in which the cohesin domain is shaded blue and the gp100 peptide is shaded grey. Underlined residues bounding the peptide are native to gp100. C-terminal His tags are to facilitate purification via metal affinity chromatography.
Shown below is the gp100 sequence and the associated peptides referred to above.
The HLA-A0201 restricted peptide sequences are:
C180 is E. coli-pET28(Cohesin-hgp100-PeptideA-6×His):
TABLE 13 shows the amino acid sequence of the melanoma antigen MART-1. Well known HLA-A201-restricted dominant peptides are shaded and detailed below the sequence. M peptides show peptide sequence variants with enhanced affinity for HLA-A201. C181 is an E. coli expression construct that encodes the sequence shown below in which the cohesin domain is shaded yellow and the MART-1 peptide is shaded grey. Underlined residues bounding the peptide are native to MART-1. C172 and C174 are two constructs directing the expression of anti-DC_rAb.MART-1 peptide and a matching control rAb.MART-1 peptide H chain. Only the sequences appended to the C-terminal residue are shown. C-terminal His tags are to facilitate purification via metal affinity chromatography.
MART-1 is:
The HLA-A0201 restricted peptides sequences are:
C181 is E. coli-pET28(Cohesin-hMART-1-PeptideB-6×His)
C186 is E. coli-pET28(Cohesin-Flex-hMART-1-PeptideA-6×His)
C172 is rAB-pIRES2(mAnti-ASGPR—49C11—7H-LV-hIgG4H-hMART-1-PeptideA)
C174 is rAB-pIRES2(hIgG4H-hMART-1-PeptideA)
This Example shows the successful use of cohesin and dockerin fusion proteins secreted from mammalian cells. If both fusion partners are rAbs with different specificities (i.e., rAb1.doc and rAb2.coh), then simple mixing results in rAb1.doc:rAb2.coh which is a bi-specific antibody. Bispecific antibodies have many potential therapeutic and technical applications. The invention provides a simple and predictable means to assemble such entities through the doc:coh interaction. Alternately, if rAb1.doc:rAb1.coh were assembled such entities represent controlled cross-linked mAbs with potentially unique biological properties.
Cohesin.dockerin modules exist in diverse cellulose degrading species. While they have sequence similarities, they can have specificities that do not cross between species. This affords an opportunity to build novel scaffolds composed of cohesins with different specificities and use this scaffold to assemble high order complexes in a spatially and numerically controlled manner. Others have described the core technology for using this notion for biotechnology applications (see Fierobe, H.-P., Mechaly, A., Tardif, C., Belaich, A., Lamed, R., Shoham, Y., Belaich, J.-P., and Bayer, E. A. (2001) Design and production of active cellulosome chimeras: Selective incorporation of dockerin-containing enzymes into defined functional complexes. J. Biol. Chem. 276, 21257-21261.). The invention embodies the specific use of this technology for applications related to manufacture of rAb.(doc:coh.fusion)n complexes where n represents >1 pairings of doc:coh interactions with unique specificities. Thus, the invention envisions the assembly (by simple mixing of components) of spatially ordered complexes between rAb.doc1.doc2.doc3.etc. and coh1.fusionA, coh2.fusionB, coh3.fusion3, etc. The coh.fusion proteins could represent different antigens, or combinations of antigens and activating agents like cytokines.
By extension multiple coh:doc specificities could also be used to make bivalent rAbs with higher order antigen specificities. Cellulose degrading bacteria and similar organisms also use cellulose binding domains (CBD) to organize the degradation machinery. The structure of a CBD from Clostridium thermocellum shows that the N and C-termini are in close proximity and are not an integral part of the CBD functional structure. In fact CBD typically occurs linked to other domains such as coh.CBD.coh in cipA. The invention encompasses the use of entities such as coh.CBD.coh to assemble spatially and numerically ordered complexes mimicking antibodies and multi subunit receptors. For example, a IgG kappa chain v region fused to doc1 and a IgG H chain V region linked to doc2 can assemble with coh1.CBD.coh2 to yield VL.doc1:coh1.CBD.coh2:VH.doc2 to yield an entity with affinity and binding specificity analogous to the original mAb. Such entities should be e.g., very useful screening tools for refining mAb specificities through mutagenesis procedures, particularly since the VL and VH component could be mutated independently and combined by mixing in various combinations. As described above, this technology can be readily extended to multiple controlled coh:V.doc combinations potentially yielding binding entities with extremely high specificities and affinities. An extension of this would be using e.g., coh1.coh2.CBD.coh3 as a template for assembly of cytoR1.doc+cytoR2.doc+cytoR3.doc (where cytoR represents the ectodomain of one subunit of a complex cytokine receptor). Such entities will have utility for blocking cytokine interactions for therapy and in biotechnology for measuring cytokines in complex supernatants.
Currently 1.2 million Americans develop cancer each year and about 500,000 die from the disease, because most cancers cannot be cured once they have metastasized. To develop a new treatment for metastatic cancer, genetic engineering has been used to modify a powerful bacterial toxin, Pseudomonas exotoxin A (PE), so that instead of killing normal cells it selectively kills cancer cells. PE is a three domain protein composed of 613 amino acids. Anti-cancer agents are produced by deleting its binding domain (aa 1-252) and replacing it with the Fv fragment of an antibody or with a growth factor that binds to antigens present on cancer cells. These agents are termed recombinant immunotoxins (RITs). RITs have been made that target Ley present on colon, breast, lung and other epithelial cancers (B3(Fv)-PE38), that target the EGF receptor overexpressed on glioblastomas (TGF-alpha-PE38), that target mutant EGF receptors present on glioblastomas (MR-1(Fv)-PE38 KDEL), and that target the IL-2 receptor present on many T and B cell leukemias and lymphomas LMB-2 or anti-Tac(Fv)-PE38 and that target CD22 on B cell malignancies and that target BL22 or RFB4(dsFv)-PE38 ovarian cancers and mesotheliomas (SS1P). These agents are produced in E. coli because large amounts can be readily purified from this source and because the toxin itself would kill mammalian cells expressing it. When administered to mice with the appropriate human cancer xenograft, all these RITs produce complete tumor regressions. Most of these agents are now in clinical trials in humans and several have produced complete and partial remissions in humans with cancer.
An ideal immunotoxin should be very active so that only small amounts need to be given to cause tumor regressions, stable so it remains functional during the 5-10 hours required to reach the interior of a tumor, and non immunogenic so it can be given repeatedly. Initially, recombinant immunotoxins contained amino acids 253-613 of PE (domains II and III). It has been determined that amino acids 364-395 can be deleted without loss of activity. Increased stability can be addressed by linking the toxin to a whole antibody, which are well known to have long half-lives and the technology in the invention provides this solution.
While the rAb.Doc:Coh.toxin technology can be applied to known cancer antigens, it can also be tested to kill intra-tumoral DC that are suspected to foster escape of the tumor from immune surveillance. In this latter case, anti-DC toxin therapy could be doubly advantageous since build up of immunity against the administered toxin itself should be suppressed (that is because DC themselves are key to the initiation of this immune response via uptake and processing of the antigen. In this therapy, the DCs that uptake the antigen die and cannot mount the anti-toxin response).
Frankel (Clinical Cancer Research, 8, 942-944, 2002) describes issues hindering the wider application of immunotoxins. These include production problems which often require refolding of E. coli inclusion body expressed material where misfolding contaminants are problematic. Also, affinity of the immunotoxin for its target is often difficult to obtain in sufficient strength. The technology basis of this invention addresses both these issues—firstly, we found that cohesin.PE38 fusion protein is expressed in E. coli as a soluble protein that can be purified in a fully functional state (with both cohesin and toxin activities in tact) by simple biochemical means without complex refolding. Secondly, high affinity monoclonal antibodies against target antigens can be routinely obtained by one practiced in the art. What is difficult is engineering the antibody variable regions in a form that is fused with toxin and fully functional for target binding. The usual means (e.g., sFv forms) of engineering invarably lead to significant loss of affinity against the target compared to the initial monoclonal antibody. The rAb.Doc:Coh.toxin technology circumvents this issue affording a means to preserve both the high affinity binding sites of the initial mAb (note that humanization of mouse mAb V regions while maintaining high and specific binding activity is routine to one practiced in the art), as well as the beneficial properties of long half-life and non-antigenicity of a full recombinant hIgG context.
Furthermore since the cohesin.toxin is produced independently, one formulation of the toxin can be conjugated to any number of separately produced targeting rAb.Doc proteins by simple mixing of the component prior to injection of the patient. This greatly simplifies manufacturing as well as research development time. The technology described in the invention can be readily applied to any toxin and any rAb specificity.
Details of the rAb.Doc:Coh.toxin technology. pRB 391 (from Dr. Pastan) Pastan, Chief of the Laboratory of Molecular Biology, Division of Basic Sciences. NCI, NIH) was used as a template for PCR with primers PE38-N3 (cacggtcaccgtctccaaagcttccggagctagcGAGGGCGGCAGCCTGGCCGCGCT (SEQ ID NO.:39)) and PE38-C3 (GGCCGGCTCCTGCGAAGGGAGCCGGCCGGTCGCGGCCGCTTACTTCAGGTCCTCGC GCGGCGGTTTGCCG (SEQ ID NO.:40)).
Cloning was into the previously established construct C21 or E. coli-pET28(Cohesin-6×His) to generate a fusion protein encoding Cohesin-PE38 corresponding to the amino acid sequence shown below (grey residues are cohesin; yellow residues are PE38, separated by a linker sequence native to the cohesin domain).
Expression and purification of recombinant Coh.PE38 protein—E. coli cells from each 1 L fermentation were resuspended in 25 ml ice-cold 50 mM Tris, 1 mM EDTA pH 8.0 with 0.1 ml of protease inhibitor Cocktail II (Calbiochem). The cells were sonicated on ice 2×5 min at setting 18 (Fisher Sonic Dismembrator 60) with a 5 min rest period and then spun at 17,000 r.p.m. (Sorvall SA-600) for 20 min at 4° C. The supernatant was passed through 1 ml ANX Sepharose column equilibrated in 50 mM Tris, 1 mM EDTA pH 8.0 and eluted with a 0-1 M NaCl gradient in Buffer B. Fractions containing Cohesin.PE38 sere identified by SDS.PAGE and pooled fractions were further purified by purification via anti-cohesin mAb affinity chromatography with elution by 0.1 M glycine pH 2.7.
Selective killing of human DC by rAb.Doc targeted Coh.PE38-Human DC were prepared from blood monocytes by culture for 6 days in with GM-CSF and IL-4. The DCs were then cultured with either Coh.PE38 alone, anti-DC-SIGN/L 16E7 rAb.Doc alone, anti-DCIR 24A5.Doc alone, or the rAb.Docs together with Coh.PE38 (1.25 ug/ml of agents were added). After 48 hr the cells were stained with a reagent (7-AAD) that detects apoptotic cells and analyzed by FACS scoring forward versus side scatter and 7-AAD fluorescence.
Using Cohesin-Dockerin Technology to make Multivalent Antibodies. A Cohesin domain was engineered in-frame with the C-terminus of a rAb H chain using PCR based on C17 (Mam-pCDM8(Cohesin-Cohesin-SLAML-AP-6×His)) as template. The resulting secreted H chain sequence is shown below (the cohesin domain is highlighted in grey and the C-terminal H chain residue is in bold):
This expression construct was co-transfected with the appropriate rAb L chain into 293F cells and expression of secreted rAb was appraised by anti-hIgGFc ELISA at day 3.
Thus both Cohesin and Dockerin domains are readily expressed as rAb fusion proteins. This property is essential for the use of (rAb1.Coh:rAb2.Doc) complexes as bivalent antibodies (i.e., having two different combining specificities in one protein). Bivalent antibodies have many desirable features suited to industrial, analytic, and therapeutic applications. They are, however, difficult to develop and molecular tools used to engineer them typically adulterate desirable features of high affinity and specificity inherent to the parent monoclonal antibodies. The (rAb1.Coh:rAb2.Doc) technology circumvents this obstacle and is, moreover, extensible to higher (than 2) valency of combining power by incorporating multiple Cohesin or Dockerin strings with pair wise specificities as described elsewhere in this application. Furthermore, this technology can be extended to using, e.g., a cytokine to provide the additional valency (i.e., rAb1.Doc:Coh.cytokine).
For example, a fusion protein between a Cohesin domain and IL-21 was engineered as an expression construct and the Coh.IL-21 protein was efficiently secreted from transiently transfected 293F cells and easily purified by sequential Q Sepharose and anti-Cohesin affinity chromatography. The sequence of the secreted product is shown below with the cohesin domain shown in grey and the IL-21 domain in yellow. This product was fully functional as determined by it's efficacy in sustaining proliferation of human B cells.
Mam-pCDM8(SLAML-Cohesin-hIL-21)
Thus rAb.Doc:Coh.IL-21 can deliver concomitant proliferation and activation signals to a B cell (i.e., if the rAb itself has activation properties). This notion can be extended to any rAb with biological properties directed to a particular cell type and any cytokine with activity directed to the same cell type.
It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
This application claims priority to U.S. Provisional Application Ser. No. 60/888,029, filed Feb. 2, 2007, the contents of which is incorporated by reference herein in its entirety.
This invention was made with U.S. Government support under Contract No. 1U19AI057234-0100003 awarded by the NIH. The government has certain rights in this invention.
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