Patent Application
20190062380
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 9, 2013, is named 7230-171WO_SL.txt and is 72,591 bytes in size.
The invention relates generally to the field of immunology. More particularly, the invention relates to fusion proteins, nucleic acids encoding fusion proteins, and methods of use thereof for vaccines and cell therapy.
Some current vaccine and cell therapy technologies are associated with the problem of properly activating immune cells for vaccination or immune therapy of patients as a treatment for cancer or infectious disease. There is thus a significant need for improved immune stimulators that provide potent activation of the immune response in patients suffering from cancer or an infectious disease, or who are at risk thereof.
Disclosed herein are compositions, cells, kits, and methods for inducing an immune response in a subject. The compositions can be used as immune stimulators to increase the response to a vaccine composition (in which case the immune stimulator is described as a vaccine adjuvant) or given to patients to improve ongoing immune responses such as those directed against cancer or an established infection. Representative cancers include melanoma, glioma, prostate, breast, and HPV-related cancers and representative infections include Human Immunodeficiency Virus (HIV), hepatitis C virus, human papilloma virus (HPV), malaria, biodefense related agents, and all other infectious agents that do not currently have effective safe vaccines. These compositions for immune stimulation can also be used in cell-based therapies (e.g. dendritic cell (DC) therapies) where cells are treated ex vivo and then delivered to the subject for preventing and treating disorders such as cancer and infection. The compositions, cells, kits and methods involve one or more nucleic acids that encode one or more LMP1 fusion proteins (also referred to as protein chimeras), and in a typical embodiment provide activation of immune responses by a combination of two or more LMP1 fusion proteins. By combining multiple fusion proteins composed of the LMP1 protein aggregation (transmembrane) domain and the cytoplasmic domain of toll-like receptors (TLR), proteins, Tumor Necrosis Factor SuperFamily Receptors (TNFSFR) proteins, pattern recognition receptor (PRR) proteins, or adapter proteins that are involved in innate and/or adaptive immune signaling pathways, introduction of the fusion proteins into immune cells such as dendritic cells or other antigen presenting cells (APCs) results in higher levels of activation. This technology can be used for traditional prophylactic or therapeutic vaccines against cancer and infectious diseases, as well as cell-based therapies such as dendritic cell therapy. In the experiments described herein, combinations of fusion proteins markedly enhanced immune responses and protection from infection, and combinations of one or more TLR agonists and a stimulator of the TNFSFR protein CD40 also induced a synergistic immune activation that protected experimental mice from tumor challenge. Also in the experiments described herein, a surprising result was that LMP1-IPS1 is effective at restricting HIV-1 replication. LMP1-IPS1 inhibits wild-type HIV-1 replication in cell culture and in primary human CD4+ T cells. Bystander cells expressing LMP1-IPS1 can inhibit viral replication, suggesting that LMP1-IPS1 can be given as a therapy in HIV-infected patients. LMP1-IPS1 can be given as a viral vector (i.e. lentiviral vector) targeting sites of HIV-1 infection, thereby reducing HIV-1 replication in tissue reservoirs that are normally resistant to antiretroviral drugs. The compositions, cells, kits and methods described herein address the problem of properly activating and maturing APCs such as dendritic cells for vaccination or immune therapy of patients as a treatment for cancer or infectious disease, and can also be used to develop prophylactic vaccines and other immune therapies dependent on immune activation.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, a “nucleic acid” or a “nucleic acid molecule” means a chain of two or more nucleotides such as RNA (ribonucleic acid) or DNA (deoxyribonucleic acid), and chemically-modified nucleotides. The nucleic acid molecule may be purified. A “purified” nucleic acid molecule is one that is substantially separated from other nucleic acid sequences in a cell or organism in which the nucleic acid naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants). The terms include, e.g., a recombinant nucleic acid molecule incorporated into a vector, a plasmid, a virus, or a genome of a prokaryote or eukaryote. Examples of purified nucleic acid molecules include cDNAs, fragments of genomic nucleic acid molecules, nucleic acid molecules produced by polymerase chain reaction (PCR), nucleic acid molecules formed by restriction enzyme treatment of genomic nucleic acid molecules, recombinant nucleic acid molecules, and chemically synthesized nucleic acid molecules.
By the term “LMP1 gene,” is meant a native Epstein Barr virus LMP1-encoding nucleic acid sequence, e.g., the native Epstein Barr virus LMP1 gene; a nucleic acid having sequences from which a LMP1 cDNA can be transcribed; and/or allelic variants and homologs of the foregoing. An exemplary nucleic acid sequence of LMP1 is GenBank Accession No. M58153.1. The term encompasses double-stranded DNA, single-stranded DNA, and RNA.
By the term “LMP1 protein,” is meant an expression product of a LMP1 gene or a protein that shares at least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity with the foregoing and displays a functional activity of a native LMP1 protein. A “functional activity” of a protein is any activity associated with the physiological function of the protein. LMP1 consists of an N-terminal transmembrane region linked to a C-terminal cell signaling region that is analogous to the CD40 receptor on immune cells (see
As used herein, “protein” and “polypeptide” are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation. The terms “fusion protein,” “chimeric protein,” and “chimera” are used interchangeably herein, and mean a protein made by translation of an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
When referring to a peptide, oligopeptide or protein, the terms “amino acid residue”, “amino acid” and “residue” are used interchangeably and, as used herein, mean an amino acid or amino acid mimetic joined covalently to at least one other amino acid or amino acid mimetic through an amide bond or amide bond mimetic.
When referring to a nucleic acid molecule, polypeptide, or infectious pathogen, the term “native” refers to a naturally-occurring (e.g., a wild-type (WT)) nucleic acid, polypeptide, or infectious pathogen.
As used herein, the term “antigen” or “immunogen” means a molecule that is specifically recognized and bound by an antibody.
The term “antibody” is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments, regions or derivatives thereof, provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques.
As used herein the term “adjuvant” means any material which enhances the humoral and/or cellular immune response.
As used herein, an “immune cell” refers to dendritic cells, macrophages, lymphocytes, mast cells, endothelial cells, lymphatic vessel cells and the like which can, when properly stimulated, serve as an antigen-presenting cell (APC) to initiate an immune response or as an effector cell of an immune response.
As used herein, the terms “displayed”, “presented”, or “surface exposed” are considered to be synonyms, and refer to antigens or other molecules that are present (e.g., accessible to immune site recognition) at the external surface of a structure such as a cell.
As used herein, “vaccine” includes all prophylactic and therapeutic vaccines.
By the phrase “immune response” is meant induction of antibody and/or immune cell-mediated responses specific against an antigen or antigens or allergen(s) or drug or biologic. The induction of an immune response depends on many factors, including the immunogenic constitution of the challenged organism, the chemical composition and configuration of the antigen or allergen or drug or biologic, and the manner and period of administration of the antigen or allergen or drug or biologic. An immune response has many facets, some of which are exhibited by the cells of the immune system (e.g., B-lymphocytes, T-lymphocytes, macrophages, and plasma cells). Immune system cells may participate in the immune response through interaction with an antigen or allergen or other cells of the immune system, the release of cytokines and reactivity to those cytokines. Immune responses are generally divided into two main categories—humoral and cell-mediated. The humoral component of the immune response includes production of antibodies specific for an antigen or allergen or drug or biologic. The cell-mediated component includes the generation of delayed-type hypersensitivity and cytotoxic effector cells against the antigen or allergen.
As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of the therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease, or the predisposition toward disease.
As used herein, the phrase “safe and effective amount” refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. By “therapeutically effective amount” is meant an amount of a composition of the present invention effective to yield the desired therapeutic response, for example, an amount effective to activate an immune response in an individual. The specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed.
The terms “patient” “subject” and “individual” are used interchangeably herein, and mean a mammalian subject (e.g., human, rodent, non-human primates, canine, bovine, ovine, equine, feline, etc.) who is to be treated, who has been treated, or who is being considered for treatment, and/or to obtain a biological sample from, with human patients being preferred. In some cases, the methods, kits, and compositions described herein find use in experimental animals, in veterinary applications for livestock, domesticated animals, and companion animals, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, as well as non-human primates.
Accordingly, described herein is a composition for inducing an immune response in a subject. The composition includes at least a first nucleic acid encoding at least a first fusion protein, the at least first fusion protein including a transmembrane domain of LMP1 to provide for clustering of the at least first fusion protein in cells without the need for any exogenous molecule and at least one signaling domain from at least one of: an immune activating receptor and/or an adaptor protein, the at least first nucleic acid in an amount sufficient to induce an immune response in a subject. The immune activating receptor can be, for example, a Toll-like receptor (TLR) or a TNF superfamily receptor (TNFSFR). The at least first fusion protein can include a signaling domain from a TLR and a signaling domain from a TNFSFR. In another embodiment, the at least first fusion protein can include a signaling domain from an immune activating receptor and/or a signaling domain from an adaptor protein. The immune activating receptor can be, for example, TNF Receptor SuperFamily (TNFRSF) proteins, preferably CD40, 4-1BB, RANK, TACT, OX40, CD27, GITR, LTβR, and BAFFR; Toll-Like Receptors (TLR) TLR1 to TLR13, preferably TLR7; integrins; FcγRIII; Dectin1; Dectin2; NOD1; NOD2; CD16; IL-2R; Type I/II interferon receptor; chemokine receptors such as CCR5 and CCR7; G-protein coupled receptors (GPCRs); TREM1; the B cell receptor (BCR) complex that includes CD79A, CD79B, and Ig-alpha, etc. The adaptor protein can be, for example, one of: IPS-1; MyD88; RIG-1; MDA5; CD3 zeta chain; MyD88ΔTIR; TRIF; TRAM; TIRAP; MAL; BTK; RTK; RAC1; SYK; NALP3 (NLRP3); NALP3ΔLRR; NALP1; CARDS; DAI; IPAG; STING; Zap70; and LAT. The at least first nucleic acid can further encode an antigen. The at least first nucleic acid can be included within a viral vector. The viral vector (including the nucleic acid sequence) can be within (encapsulated by) a recombinant virion. The composition can further include a second nucleic acid encoding a second fusion protein, the second fusion protein including a transmembrane domain of LMP1 and at least one signaling domain from at least one of: an immune activating receptor and an adaptor protein. The at least first and the second nucleic acid can be included within a viral vector. Typically, the at least first nucleic acid is an amount effective for activating and maturing immune cells (e.g., dendritic cells) when administered to a subject (e.g., a human). In a composition as described herein, the at least first nucleic acid can be in an amount effective for inducing expression of cytokines and protecting primary CD4+T cells from infection by a virus (e.g., human immunodeficiency virus (HIV)) when administered to a subject (e.g., a human). The at least first fusion protein can include a transmembrane domain of LMP1 and a signaling domain from IPS-1.
Also described herein is a vaccine formulation for preventing or treating a disease or condition in a subject including a composition as described herein and a pharmaceutically acceptable excipient. The disease or condition can be, for example, cancer or infection.
Further described herein is a vaccine adjuvant for enhancing an immune response to a vaccine. Typically the vaccine adjuvant includes a composition as described herein and an antigen or a nucleic acid encoding an antigen in an amount effective for enhancing an immune response to a vaccine and a pharmaceutically acceptable excipient. In one embodiment, the at least first nucleic acid can further encode the antigen.
Still further described herein is an immune cell transduced with a composition as described herein. In a typical embodiment, the immune cell is a dendritic cell. The immune cell can be within a vaccine formulation for preventing or treating a disease or condition (e.g., cancer or infection) in a subject.
Additionally described herein is A method of inducing an immune response against cancer or infection in a subject. The method includes administering a composition as described herein to the subject in a therapeutically effective amount for inducing an immune response against the cancer or infection in the subject. Administering the composition to the subject vaccinates the subject against cancer or infection. Inducing an immune response against the cancer or the infection in the subject can include at least one of: activating and maturing dendritic cells, and inducing expression of at least one cytokine in the subject. The cancer can be, for example, melanoma, glioma, prostate cancer, breast cancer, and the infection is selected from the group consisting of: HIV infection, hepatitis C infection or human papilloma virus infection. In one embodiment, the subject has an HIV infection, and inducing an immune response against the infection includes induction of type I interferon and protection of primary CD4+ T cells in the subject. In another embodiment, the subject has a cancerous tumor, and the at least first fusion protein includes a transmembrane domain of LMP1 and a signaling domain from IPS-1 or a signaling domain from MyD88. In this embodiment, inducing an immune response results in a decrease in growth of the cancerous tumor.
Yet further described herein is a kit for preventing or treating a disease or condition in a subject. A kit includes a composition, vaccine formulation, or vaccine adjuvant as described herein; instructions for use; and packaging.
Also described herein is a composition for inducing an immune response in a subject including a nucleic acid encoding a fusion protein. In the composition, the fusion protein includes a transmembrane domain of LMP1 and an amino acid sequence including two or more signaling motifs from immune activating receptors and/or adaptor proteins. The nucleic acid is in an amount sufficient to induce an immune response in a subject.
Although compositions, cells, kits, and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions, cells, kits, and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.
Described herein are LMP1 fusion proteins, nucleic acids encoding the fusion proteins, viral vectors containing the nucleic acids, and cells transduced with the nucleic acids for inducing an immune response in a subject. In our previous publications (S. Gupta et al., Journal of Leukocyte Biology 90:389-398, 2011; and S. Gupta et al., Retrovirology 8:39, 2011) and a prior application (R. S. Kornbluth and G. W. Stone, PCT WO 2011/119628, now US patent application pub. No. 2013/0039942 A1 and EP11760085.8), the invention of LMP1 fusion proteins is described. These were described as fusion proteins that combine an LMP1 transmembrane domain and a single (not more than one) signaling domain taken from protein receptors in the Tumor Necrosis Factor Receptor SuperFamily (TNFRSF). In the instant invention, LMP1 fusions are made with a plurality (two or more) of signaling domains. Additionally, the instant invention presents fusion proteins of LMP1 combined with a signaling domain that is not derived from a TNFRSF but instead is derived from other types of immune activating receptors such as Toll-Like Receptors (TLRs) and/or adaptor proteins that are not part of receptors at the cell surface but rather intracytoplasmic adaptor molecules that initiate signaling events downstream from cell surface receptors in the cell such as IPS-1. Collectively, the LMP1 fusion proteins of the instant invention contain signaling domains selected from the TNFRSFs, TLRs, and/or adaptor molecules such that two or more signaling domains are fused with portions of LMP1. As an exception, LMP1 itself has a C-terminal intracytoplasmic domain that is recognized as functionally similar to a TNFRSF signaling domain, specifically from the CD40 receptor, a member of the TNFRSFs. Consequently, fusion proteins of the instant invention are of the form LMP1 transmembrane domain (LMP1TM) operatively linked to signaling domain X and operatively linked to signaling domain Y to form LMP1TM-X-Y, where the linkage is normally provided by peptide bonds that make the fusion protein a single polypeptide strand. In cases where the entire LMP1 protein is used, the “X” portion is the signaling domain that naturally occurs in LMP1 followed by one or more “Y” signaling domains so that again the fusion proteins contains two or more signaling domains. In yet another embodiment, the instant invention describes LMP1 fusion proteins wherein a peptidic antigen (Ag) is included in the LMP fusion protein polypeptide. Such fusion proteins have the structure of Ag-LMP1TM-X-Y and may have sequences that include an internal ribosome entry site (IRES) between the sequence encoding the antigen and the sequence encoding the N-terminal transmembrane domain of LMP1. The unifying feature of all of these fusion proteins is the presence of the LMP1 N-terminal transmembrane domain which (1) anchors the fusion proteins into membranes and (2) aggregates them into clusters in the cell membrane. By clustering the signaling domains, LMP1 fusion proteins allow those signaling domains to engage downstream adaptor molecules that lead to altered gene expression in the nucleus. Prior to this invention, it was not known in the art that the same LMP1 transmembrane domain could activate more than one signaling domain when the LMP1 and two or more signaling domains are joined in tandem in a single polypeptide chain. As a modification of this concept, an LMP1 fusion protein was made using Interferon Promoter Stimulator-1 (IPS-1, also called MAVS, VISA, or Cardif). In this case, fusion to LMP1 provides for the aggregation of IPS-1 that is needed for cellular activation by the IPS-1 protein (F. Hou et al., Cell 146:448-461, 2011). This novel molecule, LMP1-IPS-1, has only one signaling domain but that signal domain is used in a way not previously known in the art.
Latent membrane protein-1 (LMP1) is a gene in the Epstein-Barr Virus (EBV). As shown in
In making LMP1 fusion proteins, the signaling domains comprise one or more cytoplasmic signaling domains of immune activating receptors (e.g., TLR cytoplasmic domains and/or TNFSFR cytoplasmic domains) and/or adaptor proteins. The immune activating receptors being contemplated in this invention include, for example, TNFRSFs (e.g., CD40, 4-1BB, RANK, TACI, OX40, CD27, GITR, LTβR, and BAFFR), TLR1 through TLR13 inclusive, integrins, FcγRIII, Dectin1, Dectin2, NOD1, NOD2, CD16, IL-2R, Type VII interferon receptor, chemokine receptors such as CCR5 and CCR7, GPCRs, TREM1, and the B cell receptor (BCR) complex that includes CD79A, CD79B, Ig-alpha, and Ig-beta. The adaptor proteins being contemplated in this invention include, for example, IPS-1, RIG-1, MDA5, CD3 zeta chain, MyD88, MyD88ΔTIR, TRIF, TRAM, TIRAP, MAL, BTK, RTK, RAC1, SYK, NALP3 (NLRP3), NALP3ΔLRR, NALP1, CARDS, DAI, IPAG, STING, Zap70, and LAT. These LMP1 fusion proteins can be encoded within recombinant DNA, RNA, or viral vectors for the treatment of subjects. In some formulations, an antigen will be encoded within the same vector as the LMP1-fusion, inducing antigen-specific immune activation. Based on the protein binding motifs within each of these cytoplasmic domains, an artificial cytoplasmic domain can be constructed that incorporates signaling domains from one or more immune activating receptors and/or one or more adapter proteins in combinations that give the greatest immune response. This artificial signaling domain is anticipated to induce greater immune activation, and more effective vaccines or immune therapy, compared to the wild-type signaling domains. An artificial cytoplasmic domain composed of intracellular signaling motifs from multiple TLRs, TNFSFR, or adapter molecules can, as a single gene product, mimic the synergy induced by multiple but separate LMP1 fusion proteins. Such a superactivating LMP1 construct can be used as a vaccine adjuvant and immune stimulator.
Several of the signaling domains contemplated in this invention are part of the inflammasome pathway. These include IPS-1, NALP2, NLRP3, and AIM2. A key function of the inflammasome pathway is to activate the proteases (e.g., caspase-1) that cleave the inactive precursor of interleukin-1-beta (pro-IL-1beta) into the active form of this cytokine (IL-1beta). The precursor protein, pro-IL-1-beta, is not normally present in immune cells but instead the transcription of its gene and the production of the inactive protein is initiated by activating TLRs and downstream adaptor molecules for the TLR pathway. Thus, the induction of the inflammasome and cleavage of pro-IL-1beta to IL-1beta may be accomplished by induction of IL-1beta gene using LMP1-TLR2, LMP1-MyD88, etc, together with caspase inflammasome induction with LMP1 fused to domains of proteins such as NALP2, NLRP3, or AIM2 which are involved in inflammasome induction. The LMP1 fusions described herein would also be effective even in the context where the APC does not express the natural receptor for the TLR agonist or TNFSFR agonist, since the LMP1 fusions already encode the receptor in a constitutively active state.
The below described preferred embodiments illustrate adaptations of these compositions, cells, kits, and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.
Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2001); and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, (1992) (with periodic updates). Immunology techniques are generally known in the art and are described in detail in methodology treatises such as Current Protocols in Immunology, ed. Coligan et al., Greene Publishing and Wiley-Interscience, New York, (1992) (with periodic updates); Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic Press, Burlington, Mass., (2007); Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howard and Matthew R. Kaser, CRC Press, Boca Raton, Fl, (2006); Medical Immunology, 6th ed., edited by Gabriel Virella, Informa Healthcare Press, London, England, (2007); and Harlow and Lane ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988). Conventional methods of gene transfer and gene therapy may also be adapted for use in the present invention. See, e.g., Gene Therapy: Principles and Applications, ed. T. Blackenstein, Springer Verlag, 1999; Gene Therapy Protocols (Methods in Molecular Medicine), ed. P. D. Robbins, Humana Press, 1997; Viral Vectors for Gene Therapy: Methods and Protocols, ed. Otto-Wilhelm Merten and Mohammed Al-Rubeai, Humana Press, 2011; and Nonviral Vectors for Gene Therapy: Methods and Protocols, ed. Mark A. Findeis, Humana Press, 2010.
Described herein are nucleic acids encoding LMP1 fusion proteins and compositions including such nucleic acids. The nucleic acids and compositions can be administered to a subject in order to induce an immune response in the subject (e.g., an immune response to a particular antigen) or to modulate an ongoing immune response in a subject (e.g., the naturally occurring but otherwise ineffective immune response to cancer). Nucleic acid molecules encoding LMP fusion proteins and antigens as described herein may be in the form of RNA (e.g., mRNA or synthetic chemically modified RNA) or in the form of DNA (e.g., cDNA, genomic DNA, and synthetic DNA). The DNA may be double-stranded or single-stranded, and if single-stranded, may be the coding (sense) strand or non-coding (anti-sense) template strand.
In one embodiment, a composition for inducing an immune response in a subject includes at least a first nucleic acid encoding at least a first fusion protein, the at least first fusion protein including a transmembrane domain of LMP1 and two or more signaling domains selected from the group of immune activating receptors and/or the group of adaptor proteins, the at least first nucleic acid in an amount sufficient to induce an immune response in a subject. Any suitable immune activating receptor can be used. For example, the immune activating receptor can be a TLR or a TNFSFR. Examples of immune activating receptors include: TLRs 1-13, RIG-1, MDA5, FcγRIII, Dectin1, Dectin2, NOD1, NOD2, CD16, IL-2R, TNFSFR (e.g., CD40, 4-1BB, RANK, TACI, OX40, CD27, GITR, LTβR, and BAFFR), Type I/II interferon receptor, TREM1, RTK, GPCR, Integrin, CCR7, CCR5, BCR (B cell receptor complex proteins). A fusion protein may include a signaling domain from a TLR and a signaling domain from a TNFSFR. Additionally or alternatively, a fusion protein may include a signaling domain from an immune activating receptor and a signaling domain from an adaptor protein. Any suitable adaptor protein may be used. Examples of adaptor proteins include CD3 zeta chain, MyD88, MyD88ΔTIR, IPS-1, TRIF, TRAM, TIRAP, MAL, BTK, RAC1, SYK, NALP3 (NLRP3), NALP3ΔLRR, NALP1, CARD9, DAI, IPAG, STING, Zap70, and LAT. The at least first nucleic acid may further encode an antigen.
Many vectors useful for introducing exogenous nucleic acids into target mammalian cells are available. The vectors may be episomal, e.g. plasmids, virus derived vectors such cytomegalovirus, adenovirus, adeno-associated virus (AAV), lentivirus etc., or may be integrated into the target cell genome, through homologous recombination or random integration, e.g. retrovirus derived vectors such MMLV, HIV-1, ALV, etc. In a typical embodiment, the at least first nucleic acid sequence is contained within a viral vector. In such an embodiment, recombinant virions (particles) containing the viral vector are administered to the subject. Viruses are naturally evolved vehicles which efficiently deliver their genes into host cells and therefore are desirable vector systems for the delivery of therapeutic nucleic acids. Preferred viral vectors exhibit low toxicity to the host cell and produce/deliver therapeutic quantities of the nucleic acid of interest (in some embodiments, in a tissue-specific manner). Retrovirus-based vectors, Lentivirus vectors, adenovirus based vectors, AAV-based vectors, rhabdovirus (e.g. VS V) vectors, poxvirus (e.g. vaccinia) vectors, alphavirus (e.g., Venezuelan equine encephalitis, Semliki Forest virus, Sindbis virus) vectors, and Herpesvirus (e.g., CMV) vectors are examples of viral vectors that may be used. Such recombinant virions may be pseudotyped.
In some embodiments, a composition can include a second nucleic acid encoding a second fusion protein as described herein. In such an embodiment, both the first and second nucleic acids can be contained within a single vector (e.g., a single viral vector) or multiple vectors (e.g., two, three, four, five, etc., viral vectors). A single composition can include two or more nucleic acids, each nucleic acid encoding a single LMP1 fusion protein, or can include two or more nucleic acids, one or more of which encodes multiple LMP1 fusion proteins.
Typically, the at least first nucleic acid (and the second nucleic acid if present) is in an amount effective for activating immune cells (e.g., dendritic cells) when administered to a subject. If a composition is being administered to a subject to prevent or treat a viral infection (e.g., HIV), the at least first nucleic acid (and the second nucleic acid if present) may be in an amount effective for inducing expression of cytokines and protecting primary CD4+ T cells from infection by a virus when administered to a subject. In one embodiment in which the composition is being used to prevent or treat HIV infection, the at least first fusion protein may include a transmembrane domain of LMP1 and a signaling domain from IPS-1. Similarly, if a composition is being administered to a subject to prevent or treat cancer, in one embodiment, the at least first fusion protein may include a transmembrane domain of LMP1 and a signaling domain from IPS-1, and is in an amount effective for preventing growth of a tumor or decreasing the size or growth of an existing tumor in a subject. In this embodiment of preventing or treating cancer, the composition may also include a nucleic acid encoding full-length LMP1 or a portion thereof (e.g., a composition including a nucleic acid encoding an LMP1 fusion protein and full-length LMP1, or a composition including a first nucleic acid encoding an LMP1 fusion protein and a second nucleic acid encoding full-length LMP1).
Also described herein is an LMP1 fusion protein that includes the LMP1 transmembrane domain fused to an amino acid sequence that acts as a cytoplasmic domain and that includes intracellular signaling motifs from two or more (e.g., 2, 3, 4, 5, 6, etc.) TLRs, TNFSFR, or adapter molecules, as well as nucleic acids encoding such fusion proteins. Such an artificial cytoplasmic domain composed of intracellular signaling motifs from multiple TLRs, TNFSFR, or adapter molecules can, as a single gene product, mimic the synergy induced by multiple LMP1 fusion proteins. Such a superactivating LMP1 construct (see
Vaccine formulations and vaccine adjuvants including nucleic acids encoding LMP1 fusion proteins are also described herein. A vaccine formulation for preventing or treating a disease or condition in a subject includes a pharmaceutically acceptable excipient and at least a first nucleic acid or protein encoded by such a nucleic acid for at least a first fusion protein that includes a transmembrane domain of LMP1 and at least one signaling domain from one or more of: an immune activating receptor and/or an adaptor protein. The at least first nucleic acid is in an amount sufficient to induce an immune response in a subject. In an embodiment in which the subject has a viral infection, typically, administration of the at least first nucleic acid induces anti-viral factors that directly reduce viral replication in an infected (e.g., chronically infected) subject. In one embodiment, the composition further includes a second nucleic acid encoding a second fusion protein that includes a transmembrane domain of LMP1 and at least one signaling domain from one or more of: an immune activating receptor and/or an adaptor protein. Typically, the vaccine formulation is administered to vaccinate a subject against cancer or infection. Similarly, a vaccine adjuvant for enhancing an immune response to a vaccine includes a pharmaceutically acceptable excipient and at least a first nucleic acid encoding at least a first fusion protein that includes a transmembrane domain of LMP1 and at least one signaling domain from one or more of: an immune activating receptor and/or an adaptor protein, plus an antigen, in an amount effective for enhancing an immune response to a vaccine (e.g., a conventional or currently available vaccine). Alternatively, the antigen may be encoded by a separate nucleic acid (a second or third nucleic acid). In such a vaccine adjuvant, a second nucleic acid encoding a second fusion protein may be included. In another embodiment, the antigen is administered to a subject in a separate composition. A vaccine formulation or adjuvant may include immune cells (e.g., dendritic cells) transduced with one or more nucleic acids encoding one or more LMP1 fusion protein as described herein.
Methods of inducing an immune response against cancer or infection in a subject are described herein. In one embodiment, the method includes administering a composition as described herein to the subject in a therapeutically effective amount for inducing an immune response against the cancer or infection in the subject. In another embodiment, the method includes administering immune cells transduced with one or more nucleic acids encoding one or more LMP1 fusion proteins as described herein to the subject in a therapeutically effective amount for inducing an immune response against the cancer or infection in the subject. In some embodiments, administering the composition to the subject vaccinates the subject against an infection or cancer or enhances the pre-existing immune response to cancer or an ongoing infection. Inducing an immune response against the cancer or the infection in the subject can include activating DCs, maturing DCs, and/or inducing expression of at least one cytokine in the subject. In a method of inducing an immune response against an HIV infection, administration of the composition induces type I interferon expression and/or activity, and protection of primary CD4+ T cells in the subject. In a method of inducing an immune response against cancer in a subject, administration of the composition prevents or decreases growth of cancer cells (e.g., a tumor, leukemia cells, etc.) in the subject. In methods of inducing an immune response against cancer and methods of inducing an immune response against an HIV infection, the at least first fusion protein may include a transmembrane domain of LMP1 and a signaling domain from IPS-1 or a signaling domain from MyD88. In such a method, the nucleic acid encoding the at least first fusion protein may also encode a full-length LMP1 protein.
An immune response may include the activation of immune cells such as dendritic cells (DCs). DCs are a part of the immune system that act as APCs. DCs process antigen material and present it on their cell surface using MHC molecules. Pattern recognition receptors, such as TLRs, assist the DCs in detecting viruses and bacteria. After a DC is activated, it migrates to the lymph nodes. DCs interact with other cells within the immune system such as T cells and B cells. Stimulated DCs produce IL-12. IL-12 helps naïve CD4+ T cells obtain a T helper cell type 1 (Th1) phenotype. Cytokines cause the development of T helper cell type 1 (Th1) and T helper cell type 2 (Th2) cells from naïve CD4+ T cells. The Th phenotypes each produce particular cytokines and can be identified by specific cell-surface markers.
An immune response may be mounted to an antigen or antigens from any pathogen as a result of vaccination against that antigen or antigens. In one embodiment, the antigen may be derived from, but not limited to, pathogenic bacterial, fungal, or viral organisms, including Streptococcus species, Candida species, Brucella species, Salmonella species, Shigella species, Pseudomonas species, Bordetella species, Clostridium species, Norwalk virus, Bacillus anthracia, Mycobacterium tuberculosis, HIV, Chlamydia species, human Papillomaviruses, Influenza virus, Parainfluenza viruses, Paramyxovirus species, Herpes virus, Cytomegalovirus, Varicella-Zoster virus, Epstein-Barr virus, Hepatitis viruses (including HAV, HBV, and HCV), flaviviruses (including dengue and West Nile virus), filoviruses (including Ebola), coronaviruses (including SARS and MERS), Plasmodium species, Trichomonas species, Leishmania species, Neisseria meningitides, sexually transmitted disease agents, viral encephalitis agents, protozoan disease agents, fungal disease agents, and bacterial disease agents. A subject may be treated for any infectious pathogen, including those listed herein.
In some embodiments, the antigen is derived from cancer cells. The vaccine may be used against any cancer or with any other therapy or intervention for cancer. Examples of cancers include HPV-induced cervical cancers (e.g., E7/E7 tumor associated antigens (TAA)), glioma, human melanoma (e.g., TRP-1, TRP-2, gp-100, MAGE-1, MAGE-3 and/or p53), breast cancer, and prostate cancer (e.g., TSA). Similarly for lung tumors, breast tumors, and leukemia, any suitable tumor associated antigen can be used, and many have been described. Many such TAA are common between various cancers (e.g., CEA, MUC-1, Her2, CD20). In addition, tumor cells may have uncategorized antigens such as those created by gene mutations (e.g., BRAF V600E in melanoma), gene transpositions (e.g., BCR-abl in chronic myelogenous leukemia), or simply tumor rejection antigens caused by overexpression of normal genes (e.g., telomerase).
The compositions, cells, kits, vaccines, vaccine adjuvants, and methods described herein solve the problem of properly activating and maturing DCs for therapeutic vaccination of DCs into patients as a treatment for cancer or chronic infections. They may also be used to develop prophylactic vaccines and other immune therapies dependent on immune activation.
In some embodiments, prior to or after administration of a composition or transduced immune cells as described herein to a subject, the subject's immune response is analyzed or measured. Any suitable biological sample can be tested for analyzing or measuring a subject's immune response. Examples of biological samples include blood, serum, plasma, urine, saliva and tissue. The sample may be tested using any suitable protocol or assay. Examples of suitable assays include enzyme-linked immunosorbent assays (ELISAs), Western blots, flow cytometry assays, immunofluorescence assays, qPCR, microarray analysis, etc.
Immune cells such as dendritic cells transduced with nucleic acids encoding LMP1 fusion proteins can be used as an effective reagent for cell therapy for a subject in need thereof (e.g., a human subject having cancer or chronic infection). Immune cells (e.g., human dendritic cells) transduced with a nucleic acid or composition as described herein are encompassed within the invention. LMP1 fusion proteins can costimulate the activation of the transduced DC while allowing the transduced DC to induce T cell memory with secretion of IL-12 cytokine. Nucleic acids encoding LMP fusion proteins as described herein may be transfected into immune cells such as DCs. Alternatively, a viral vector may be used to introduce such a nucleic acid into immune cells (e.g., infection of the cells by a recombinant virus). The DCs may also be transduced with an antigen (e.g., an antigen encoded by the nucleic acid encoding an LMP1 fusion protein, an antigen encoded by a separate nucleic acid). Administration of the DCs transduced with one or more nucleic acids encoding one or more LMP1 fusion proteins to a subject can induce an immune response against cancer or infection including secretion of IL-12 by the DCs and activation of Th1 cells. An example is provided in
Given that DC therapy often uses RNA encoding the antigen or immune stimulatory proteins for transfection, LMP1 fusion proteins can also be transfected as RNA. RNA is a safer method of transfection compared to DNA or viral vectors, given that RNA cannot integrate into the genome or be maintained. The RNA is degraded over time, and therefore LMP1 encoded as RNA would not persist in the patient after treatment.
The compositions, nucleic acids, and cells described herein may be administered in combination with any other standard cell (e.g., DC) therapy; such methods are known to the skilled artisan and described in Remington's Pharmaceutical Sciences by E. W. Martin.
Described herein are kits for inducing an immune response and preventing or treating a disease or condition in a subject and for preparing a vaccine formulation. In one embodiment, a kit for inducing an immune response in a subject includes a composition including at least a first nucleic acid encoding at least a first fusion protein, the at least first fusion protein including a transmembrane domain of LMP1 and at least one signaling domain from at least one of: an immune activating receptor and/or an adaptor protein. In the composition, the at least first nucleic acid is in an amount sufficient to induce an immune response in a subject. In another embodiment, the composition includes a second nucleic acid encoding a second fusion protein, the second fusion protein including a transmembrane domain of LMP1 and at least one signaling domain from at least one of: an immune activating receptor and/or an adaptor protein. In some embodiments, the at least first nucleic acid also encodes an antigen. A kit as described herein can include a vaccine formulation that includes one or more nucleic acids encoding one or more LMP fusion proteins or a formulation that includes the proteins themselves. Similarly, a kit can include a vaccine adjuvant as described herein. In a kit, the instructions generally include one or more of: a description of the composition; dosage schedule and administration for prevention or treatment (e.g., vaccination) of cancer or infectious disease; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. Generally, a kit as described herein also includes packaging. In some embodiments, the kit includes a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding cells or medicaments.
The compositions, vaccines and cells described above are preferably administered to a mammal (e.g., non-human primate, bovine, canine, rodent, human) in an effective amount, that is, an amount capable of producing a desirable result in a treated subject (e.g., delaying or preventing onset of a disease or disorder in the subject). Toxicity and therapeutic efficacy of the compositions utilized in methods described herein can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, dosage for any one animal depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently.
The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the pathology of the disease. A composition as described herein is typically administered at a dosage that activates and matures dendritic cells, as assayed using any assay that measures activation or maturation of dendritic cells, such as analyzing expression of one or more of: IL-1a, IL-1β, IFN-α, IFN-β, IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-12, IL-15, IL-16, IL-17, IL-18, and TNF-alpha.
Therapeutic compositions described herein can be administered to a subject by any suitable delivery vehicle (e.g., DNA plasmid, RNA, viral vector, recombinant virions, or purified protein) and route. The administration of a composition may include a therapeutically effective amount of a vaccine formulation or vaccine adjuvant. The composition may be provided in a dosage form that is suitable for local or systemic administration (e.g., parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, intracranially). In various embodiments, the composition may be provided in a dosage form that is suitable for oral administration or intranasal administration. The compositions and vaccines may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, (2000) and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, Marcel Dekker, New York (1988-1999)).
Compositions, vaccines, vaccine adjuvants, and cells as described herein may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.
Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added. The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the agent that activates immune cells (e.g., activates and matures dendritic cells), the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing agents.
As indicated above, the compositions described herein may be in a form suitable for sterile injection. To prepare such a composition, the suitable active therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamine) and, poly(lactic acid). Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be non-biodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or combinations thereof).
Formulations for oral use include a liquid containing the active ingredient(s) (e.g., a nucleic acid encoding an LMP1 fusion protein) in a mixture with non-toxic pharmaceutically acceptable excipients.
The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way.
The current technology uses TLR agonist chemicals and CD40 stimulation with agonistic antibodies. These methods rely on the presence of TLR and TNRSFR on the surface of immune cells. If those receptors are not present, or are downregulated, activation will be reduced. It would be preferable to have constitutive activation of the receptors independent of native expression by the cell. In the experiments described herein, combinations of LMP1-TLRs, LMP1 fused to intracellular signaling adapter molecules, and LMP1-TNFSFR were shown to induce activation of the immune response.
A model of LMP1 fusion protein technology is illustrated in
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Innate immune responses are key determinants of the outcome of HIV infection, influencing critical events in the earliest stages of infection. Innate antiviral immune defenses are triggered through the recognition of conserved pathogen associated molecular pattern (PAMP) motifs within viral products by intracellular pattern recognition receptor (PRR) proteins in infected cells. Type I interferons (IFNα and β) are induced directly in response to viral infection, resulting in an antiviral state for the cell. IFN-β promoter stimulator (IPS-1), also known as mitochondrial antiviral signaling protein (MAVS), virus-induced signaling adaptor (VISA), and CARD adaptor inducing IFN-β (Cardif), was recently identified as an adaptor linking RIG-I and Mda5 to the downstream signaling molecules, which has been found to have roles in anti-viral immune responses. LMP1 and chimeric LMP1-CD40 has been shown to increase maturation and activation of dendritic cells and macrophages and induce strong TH1 cytokine responses. In this study, the role of chimeric LMP1-IPS-1 fusion molecule in immune activation and anti-viral immune response was examined for the prevention of HIV-1 replication.
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LMP1-IPS-1 is a potent inducer of NF-κB and IFN-β responses. LMP1-IPS-1 causes activation and maturation of the RAW 264.7 macrophage cell line by increasing expression of surface markers such as CD80, CD86, CD40, IA/IE and CCR7. LMP1-IPS-1 induces very high levels of IL-6 from transfected RAW 264.7 cells. LMP1-IPS-1 prevents the replication of both HIV and VSV-G pseudotyped scSIV in TZM-bl cells as well as primary CD4+ T cells both in cis and in trans. LMP1-IPS-1 induces type 1 interferons, interferons stimulated genes (ISGs) and chemokines genes implicated in inhibition of HIV replication. In conclusion, these studies suggest that LMP1-IPS-1 is immunostimulatory and induces a strong anti-viral immune response for the prevention of HIV-1 infection of primary CD4+ T cells.
SEQ ID NO:1 is Epstein Barr Virus latent membrane protein 1 (without the cytoplasmic domain) fused to the cytoplasmic domain of murine TLR7.
ATGACCGCGGCGCAGCGGCAGAATCTGCAAGC
SEQ ID NO:7 is Epstein Barr Virus latent membrane protein 1 (without the cytoplasmic domain) fused to human MyD88.
ATGACATTTGCTGAGGACA
AGAGTGAAGTTCAGCAG
ATGGCGTCTCAAGGCACCAAACGATCTTACGAACAGATGGAGACTGA
TGGAGAACGCCAGAATGCCACTGAAATCAGAGCATCCGTCGGAAAAA
TGATTGGTGGAATTGGACGATTCTACATCCAAATGTGCACCGAACTC
AAACTCAGTGATTATGAGGGACGGTTGATCCAAAACAGCTTAACAAT
AGAGAGAATGGTGCTCTCTGCTTTTGACGAAAGGAGAAATAAATACC
TTGAAGAACATCCCAGTGCGGGGAAAGATCCTAAGAAAACTGGAGGA
CCTATATACAGGAGAGTAAACGGAAAGTGGATGAGAGAACTCATCCT
TTATGACAAAGAAGAAATAAGGCGAATCTGGCGCCAAGCTAATAATG
GTGACGATGCAACGGCTGGTCTGACTCACATGATGATCTGGCATTCC
AATTTGAATGATGCAACTTATCAGAGGACAAGAGCTCTTGTTCGCAC
CGGAATGGATCCCAGGATGTGCTCTCTGATGCAAGGTTCAACTCTCC
CTAGGAGGTCTGGAGCCGCAGGTGCTGCAGTCAAAGGAGTTGGAACA
ATGGTGATGGAATTGGTCAGAATGATCAAACGTGGGATCAATGATCG
GAACTTCTGGAGGGGTGAGAATGGACGAAAAACAAGAATTGCTTATG
AAAGAATGTGCAACATTCTCAAAGGGAAATTTCAAACTGCTGCACAA
AAAGCAATGATGGATCAAGTGAGAGAGAGCCGGAACCCAGGGAATGC
GAGGGTCGGTTGCTCACAAGTCCTGCCTGCCTGCCTGTGTGTATGGA
CCTGCCGTAGCCAGTGGGTACGACTTTGAAAGGGAGGGATACTCTCT
AGTCGGAATAGACCCTTTCAGACTGCTTCAAAACAGCCAAGTGTACA
GCCTAATCAGACCAAATGAGAATCCAGCACACAAGAGTCAACTGGTG
TGGATGGCATGCCATTCTGCCGCATTTGAAGATCTAAGAGTATTAAG
CTTCATCAAAGGGACGAAGGTGCTCCCAAGAGGGAAGCTTTCCACTA
GAGGAGTTCAAATTGCTTCCAATGAAAATATGGAGACTATGGAATCA
AGTACACTTGAACTGAGAAGCAGGTACTGGGCCATAAGGACCAGAAG
TGGAGGAAACACCAATCAACAGAGGGCATCTGCGGGCCAAATCAGCA
TACAACCTACGTTCTCAGTACAGAGAAATCTCCCTTTTGACAGAACA
ACCATTATGGCAGCATTCAATGGGAATACAGAGGGGAGAACATCTGA
CATGAGGACCGAAATCATAAGGATGATGGAAAGTGCAAGACCAGAAG
ATGTGTCTTTCCAGGGGCGGGGAGTCTTCGAGCTCTCGGACGAAAAG
GCAGCGAGCCCGATCGTGCCTTCCTTTGACATGAGTAATGAAGGATC
TTATTTCTTCGGAGACAATGCAGAGGAGTACGACAATTAAGCCCCTC
ATGTCTGCGGGAGACCCCCGCGTGGGATCCG
AATGAGATCTTACCCCCTGCGGCTCGACGGCAAGATCCCCAGGAGAT
GGAAGATTATCCCGGTCATAACACCGCTGCTCCAGTGCAGGAGACGC
TGCACGGGTGTCAGCCTGTCACACAGGAGGATGGTAAAGAGAGTCGC
ATCTCAGTGCAGGAGCGGCAGGTGACAGACAGCATAGCCTTGAGGCC
CCTGGTCTGA
ATGTCTGCGGGAGACCCCCGCG
CCAAAGGATAATGAGATCTTACCCCCTGCGGCTCGACGGCAAGATCC
CCAGGAGATGGAAGATTATCCCGGTCATAACACCGCTGCTCCAGTGC
AGGAGACGCTGCACGGGTGTCAGCCTGTCACACAGGAGGATGGTAAA
GAGAGTCGCATCTCAGTGCAGGAGCGGCAGGTGACAGACAGCATAGC
CTTGAGGCCCCTGGTCTGA
ATATTTACCATTTCTGTAAGGCCAAGATAAAGGGGTATCAGCGTCTA
ATATCACCAGACTGTTGCTATGATGCTTTTATTGTGTATGACACTAA
AGACCCAGCTGTGACCGAGTGGGTTTTGGCTGAGCTGGTGGCCAAAC
TGGAAGACCCAAGAGAGAAACATTTTAATTTATGTCTCGAGGAAAGG
GACTGGTTACCAGGGCAGCCAGTTCTGGAAAACCTTTCCCAGAGCAT
ACAGCTTAGCAAAAAGACAGTGTTTGTGATGACAGACAAGTATGCAA
AGACTGAAAATTTTAAGATAGCATTTTACTTGTCCCATCAGAGGCTC
ATGGATGAAAAAGTTGATGTGATTATCTTGATATTTCTTGAGAAGCC
CTTTCAGAAGTCCAAGTTCCTCCAGCTCCGGAAAAGGCTCTGTGGGA
GTTCTGTCCTTGAGTGGCCAACAAACCCGCAAGCTCACCCATACTTC
TGGCAGTGTCTAAAGAACGCCCTGGCCACAGACAATCATGTGGCCTA
TAGTCAGGTGTTCAAGGAAACGGTC
CACAGTGATGAACACCACCACG
ATGACTCCCTCCCGCACCCTCAACAAGCTACCGATGATTCTGGCCAT
GAATCTGACTCTAACTCCAACGAGGGCAGACACCACCTGCTCGTGAG
TGGAGCCGGCGACGGACCCCCACTCTGCTCTCAAAACCTAGGCGCAC
CTGGAGGTGGTCCTGACAATGGCCCACAGGACCCTGACAACACTGAT
GACAATGGCCCACAGGACCCTGACAACACTGATGACAATGGCCCACA
TGACCCGCTGCCTCAGGACCCTGACAACACTGATGACAATGGCCCAC
AGGACCCTGACAACACTGATGACAATGGCCCACATGACCCGCTGCCT
GACGGAAGAGGTTGAAAACAAAGGAGGTGACCAGGGCCCGCCTTTGA
TGACAGACGGAGGCGGCGGTCATAGTCATGATTCCGGCCATGGCGGC
GGTGATCCACACCTTCCTACGCTGCTTTTGGGTTCTTCTGGTTCCGG
TGGAGATGATGACGACCCCCACGGCCCAGTTCAGCTAAGCTACTATG
ACTAA
Any improvement may be made in part or all of the compositions, cells, kits, and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. Any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.
This application claims the benefit of U.S. Provisional Application Ser. No. 61/698,109 filed on Sep. 7, 2012, which is hereby incorporated by reference in its entirety, for all purposes, herein.
| Number | Date | Country | |
|---|---|---|---|
| 61698109 | Sep 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14424562 | Feb 2015 | US |
| Child | 16115171 | US |
