Patent Grant
9481714
The present invention relates generally to fusion proteins, and more specifically to fusion proteins for enhancing T cell-mediated immune response.
Molecular biology has enabled the production of subunit vaccines, in which the immunogen is a fragment or subunit of a parent protein or complex. The development of a stable vaccine that could elicit T cell sensitizing responses, and be flexible enough to incorporate sequences from many strains of an infectious agent would be desirable.
In one aspect, the invention relates to a fusion protein comprising:
In one embodiment of the invention, the APC-binding domain or the CD91 receptor-binding domain is a polypeptide comprising an amino acid sequence that is at least 90% identical to the sequence selected from the group consisting of SEQ ID NOs: 5, 9, 6, 7, and 8. Alternatively, the APC-binding domain is selected from the group consisting of receptor-associated protein-1 (RAP1) domain III, alpha-2-macroglobulin receptor-associated protein (A2M) HIV-Tat, and heat shock proteins (HSPs), and Pseudomonas exotoxin A (PE) binding domain I.
In another embodiment of the invention, the fusion protein is free of the amino acid sequence of Pseudomonas exotoxin A (PE) binding domain I.
In another embodiment of the invention, the fusion protein further comprises an endoplasmic reticulum retention sequence located at the C-terminus of the fusion protein.
In another embodiment of the invention, the endoplasmic reticulum retention sequence comprises the amino acid sequence of Lys-Asp-Glu-Leu (SEQ ID NO: 14). The ER retention sequence may comprise a sequence selected from the group consisting of SEQ ID NOs: 14, 16-19. Alternatively, the ER retention sequence may consist of a sequence selected from the group consisting of SEQ ID NOs: 16-19.
In another embodiment of the invention, the fusion protein is free of an endoplasmic reticulum retention sequence at C-terminus thereof if the antigen contains 10 or more epitopes.
In another embodiment of the invention, the protein transduction domain is the fusion polypeptide (bi).
In another embodiment of the invention, the protein transduction domain is the T cell-sensitizing signal-transducing peptide (bii).
In another embodiment of the invention, the fusion protein further comprises an additional linker between the protein transduction domain and the antigen, the additional linker comprising SEQ ID NO: 15.
In another embodiment of the invention, the protein transduction domain is the translocation peptide (biii).
In another embodiment of the invention, the fusion protein further comprises an additional linker between the APC-binding domain or the CD91 receptor-binding domain and the translocation peptide, the additional linker comprising SEQ ID NO: 15.
In another embodiment of the invention, the protein transduction domain comprises the sequence of SEQ ID NO: 30.
In another embodiment of the invention, the APC-binding domain comprises an amino acid sequence that is at least 95% identical to the sequence selected from the group consisting of SEQ ID NOs: 5, 9, 6, 7, and 8.
In another embodiment of the invention, the APC-binding domain or the CD91 receptor-binding domain is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 6, 7, and 8.
In another embodiment of the invention, the T cell sensitizing signal-transducing peptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or 2.
In another embodiment of the invention, the T cell sensitizing signal-transducing peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 2.
In another embodiment of the invention, the translocation peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 20, and 4.
In another embodiment of the invention, the translocation peptide has 34-61 amino acid residues in length.
In another embodiment of the invention, the protein transduction domain of the fusion protein as aforementioned possesses the following features: (i) the T cell-sensitizing signal-transducing peptide comprises the amino acid sequence of SEQ ID NO: 1 or 2, and (ii) the translocation peptide comprises the amino acid sequence that is at least 95% identical to SEQ ID NO: 3.
The T cell sensitizing signal-transducing peptide exhibits a characteristic of eliciting an antibody that recognizes and binds to the amino acid sequence of K1(X)2E3(X)4(X)5Y6P7P8P9Y10 (SEQ ID NO: 32) of CD28 receptor on T cells, wherein (X)2 is I or L; (X)4 is V, F or A, and (X)5 is M or L.
In another aspect, the invention relates to a fusion protein consisting of:
The antigen-presenting cell (APC) may be selected from the group consisting of dendritic cells, macrophages, B-cells and monocytes.
In one embodiment of the invention, the cell membrane of the APC comprises a CD91 receptor.
In another aspect, the invention relates to a vaccine composition comprising: (a) a therapeutically effective amount of a fusion protein as aforementioned; and (b) an adjuvant.
The adjuvant is either an antigen delivery agent or an immune potentiator. In one embodiment of the invention, the vaccine composition comprises an antigen delivery agent and is free of an immune potentiator.
Further in another aspect, the invention relates to a method for inducing enhanced pathogen antigen-specific T cell responses, comprising: administering a vaccine composition comprising a therapeutically effective amount of a fusion protein as aforementioned to a subject in need thereof, and thereby inducing enhanced pathogen antigen-specific T cell responses.
Further in another aspect, the invention relates to a method for killing a disease cell that presents an antigen via class I MHC molecules on the cell membrane of the disease cell, comprising: administering a vaccine composition comprising a therapeutically effective amount of a fusion protein as aforementioned to a subject in need thereof, and thereby killing the disease cell that that presents the antigen via class I MHC molecules on the cell membrane of the disease cell.
In one embodiment of the invention, the disease cell is a cancer cell.
Yet in another aspect the invention relates to a method for preventing, treating infection caused by a pathogen, and/or minimizing symptoms caused by the infection, comprising: administering a vaccine composition comprising a therapeutically effective amount of the fusion protein as aforementioned to a subject in need thereof, and thereby preventing, treating infection caused by the pathogen, and/or minimizing symptoms caused by the infection.
The pathogen may be at least one selected from the group consisting of Human Papillomavirus (HPV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Human Immuno-deficient Virus (HIV-1), flu virus, dengue virus, Hepatitis C virus (HCV), Hepatitis B virus (HEW) and Porcine Circovirus 2 (PCV2).
In one embodiment of the invention, the fusion protein as aforementioned is for use in enhancing an antigen-specific cytotoxic T cell response in a subject in need thereof. The fusion protein may also be for use in enhancing an antigen-specific CD4+ T cell response, or for use as an immunogenic enhancer for inducing an enhanced antigen-specific antibody titer response, in a subject in need thereof.
These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.
The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.
The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Various embodiments of the invention are now described in detail. Referring to the drawings, like numbers indicate like components throughout the views. As used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein and throughout the claims that follow, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. Moreover, titles or subtitles may be used in the specification for the convenience of a reader, which shall have no influence on the scope of the present invention. Additionally, some terms used in this specification are more specifically defined below.
The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.
As used herein, “around”, “about” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term “around”, “about” or “approximately” can be inferred if not expressly stated.
The term “an antigen-presenting cell (APC) or accessory cell” refers to a cell that displays foreign antigens complexed with major histocompatibility complexes (MHC's) on their surfaces. T-cells may recognize these complexes using their T-cell receptors (TCRs). These cells process antigens and present them to T-cells. Main types of professional antigen-presenting cell: dendritic cells (DCs), macrophages, monocytes, and certain B-cells.
The term “an antigen-presenting cell (APC)-binding domain” refers to a domain that can bind to an antigen-presenting cell (APC). The APC-binding domain may be a polypeptide comprising an amino acid sequence that is at least 90% identical to the sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, and 9. An APC-binding domain is a ligand that recognizes and binds to a receptor on APC.
Cluster of differentiation 91 (CD91) is a protein that forms a receptor in the membrane of cells and is involved in receptor-mediated endocytosis.
The term “a protein transduction domain” refers to a polypeptide or a fusion polypeptide having a function to sensitize T-cells and thus enhance antigen-specific T cell responses, and/or to guide or direct an antigen toward (i.e., to target to) class I major histocompatibility complex (MHC-I) pathway (i.e., a cytotoxic T cell pathway) of antigen presentation.
The term “to sensitize T cells” generally means that CD8+ and CD4+ T cells are sensitized and as a result, CD8+ (CTL) and CD4+ T cell responses to an antigen challenge are enhanced. An antigen-specific cell mediated immune response is measured by quantifying the production of antigen-specific induced γ-interferon in response to an antigen. For example, without a sensitization signal (i.e., without the protein transduction domain), an antigen alone may induce weak or no cell mediated immune response at all, i.e., weak or no production of antigen-specific γ-interferon from CD8+ and CD4+ T cells, while in the presence of a sensitization signal (the protein transduction domain), the antigen may induce an enhanced cell mediated immune response. Thus, the function of a sensitization signal (the protein transduction domain) is to sensitize CD4+ and CD8+ T cells in a host so that when the host is later challenged by an antigen, the antigen can induce an enhanced antigen-specific cell mediated immune response due to prior CD4+ and CD8+ T cell sensitization.
A protein transduction domain may be a peptide and/or polypeptide selected from the group consisting of:
A protein transduction domain may be “a fusion polypeptide”, in which the fusion polypeptide comprises a T cell sensitizing signal-transducing peptide, a linker, and to translocation peptide. For example, the fusion polypeptide may be the polypeptide “CD28convPEt”.
The term “CD28conv” refers to a CD28 conserved region, which is a “T cell sensitizing signal-transducing peptide”. It's an epitope for inducing CD28 agonist antibody.
The term “PEt” or “PEt Core” refers to a RE translocation domain core with 34 amino acid residues in length.
A linker is present between the “CD28conv” and the “PEt”. The orientation or arrangement of the fusion polypeptide “CD28convPEt” is important in that “CD28conv” (or the T cell sensitizing signal-transducing peptide) must be at the upstream to the PEt (or the translocation peptide), i.e., PEt must be at the C-terminus of the “CD28conv” to obtain enhanced T-cell responses. The “CD28convPEt” can raise much higher IgG titer (called CD28-specific agonist antibody) specific to CD28conv than the reversed orientation fusion peptide PEtCD28conv. The CD28-specific agonist antibody can sensitize both CD4+ and CD8+ T cells. The correct orientation fusion polypeptide CD28convPEt contains a linker (RXRXKR) between CD28conv and PEt domains. The linker contains an antigen presenting cell (APC)-specific protease (cathepsin L) cutting site Lys-Arg (KR). Therefore, the fusion protein RAP1-CD28convPEt-Antigen-K3 can be digested into the two fragments: RAP1-CD28conv and PEt-Antigen-K3. The RAP1-CD28conv fragment can be further digested in the lysosome and the epitope of CD28conv is then presented to the APC cell surface via MHC II pathway, which in turn elicits a humoral immune response producing CD28 agonist antibody. Thus, CD28 agonist antibody is produced by B cells. This CD28 agonist antibody can bind to CD28 on the T cell surface and pre-activate the T cells (CD4+ and CD8+ T cells).
A “T cell-sensitizing signal-transducing peptide” has 28-53 amino acid residues in length and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 31, in which Xaa is I or L; Xaa10 is V, F or A, Xaa11 is M or L, Xaa17 is L or I.
The T cell-sensitizing signal-transducing peptide comprises the critical region K1(I/L)2E3(V/P/A)4(M/L)5Y6P7P5P9Y10(SEQ ID NO: 32), wherein (X)2 is I or L; (X)4 is V, F or A, (X)5 is M or L.
A T cell sensitizing signal-transducing peptide (TDIYCKIEFMYPPPYLDNEKSNGTIIH; SEQ ID NO: 31, wherein X8 is I, X10 is F, X11 is M) specific for mice was used in the following examples.
A PE translocation peptide may comprise the amino acid sequence that is at least 90% identical to SEQ ID NO: 3 or 20 For example, the amino acid sequence of a PE translocation peptide may be a.a. 280-a.a, 313 (SEQ ID NO: 3), a.a. 268-a.a 313 (SEQ ID NO: 20), a.a. 253-a.a. 313, or a.a. 253-a.a. 364 (SEQ ID NO: 4) of PE. That is, the amino acid sequence of a PE translocation peptide may contain any region of the PE domain II (a.a. 253 to a.a. 364; SEQ ID NO: 4) as long as it comprises a.a. 280-a.a. 313 (SEQ ID NO 3) essential fragment.
An antigen may be a pathogenic protein, polypeptide or peptide that is responsible for a disease caused by the pathogen, or is capable of inducing an immunological response in a host infected by the pathogen, or tumor-associated antigen (TAA) which is a polypeptide specifically expressed in tumor cells. The antigen may be selected from a pathogen or cancer cells including, but not limited to, Human Papillomavirus (HPV), PRRSV, HIV-1, flu virus, dengue virus, Hepatitis C virus (HCV), Hepatitis B virus (HBV), Porcine Circovirus 2 (PCV2), non-small cell lung cancer, breast carcinoma, melanoma, lymphomas, colon carcinoma, hepatocellular carcinoma and any combination thereof. For example, HPV E7 protein (E7), HCV core protein (HCV core), HBV X protein (HBx) were selected as antigens for vaccine development. The antigen may be a fusion antigen from a fusion of two or more antigens selected from one or more pathogenic proteins. For example, a fusion antigen of PRRSSV ORF6 and ORF5 fragments, or a fusion of antigenic proteins from PRRSV and PCV2 pathogens.
The function of an endoplasmic reticulum retention sequence is to assist translocation of an antigen from an endocytotic compartment into ER and retains it in the lumen. It comprises the sequence Lys Asp Glu Leu (KDEL) or RDEL. An ER sequence may comprise, or consists essentially of, or consist of, the sequence of KKDLRDELKDEL (SEQ ID NO: 16) KKDELRDELKDEL (SEQ ID NO: 17) KKDELRVELKDEL (SEQ ID NO: 18).
Receptor-associated protein (RAP1) with a molecular weight of 39 kDa is an ER resident protein and molecular chaperone for LDL receptor-related protein. It has a high binding affinity to CD91 (Kd˜3 nM) and is composed by three functional-similar domains.
The invention relates to the discovery of induction and enhancement of T cell mediated immune responses by fusion proteins according to the invention. Using RAP1-CD28convPEt-E7-K3 an example, the strategy of RAP1-CD28convPEt-E7-K3 vaccine is focused primarily on stimulating the production and activation of T cells that can recognize HPV16 infected cells expressing the target antigen E7. By delivering antigens to dendritic cells, it can generate antigen-specific CD8+ T cells and CD4+ T cells. Type 1-helper CD4+ T cells particularly are able to efficiently stimulate and augment the immune response of cytotoxic CD8+ T cells. Together, these two arms of the adaptive immune system have the specificity and potency to kill HPV16-infected cells or HPV16-associated tumor cells at multiple sites in the body without inflicting significant damage on normal tissues.
The term “subject” refers to a human or a non-human animal.
The term “treating” or “treatment” refers to administration of an effective amount of the fusion protein to a subject in need thereof, who has cancer or infection, or a symptom or predisposition toward such a disease, with the purpose of cure, alleviate, relieve, remedy, ameliorate, or prevent the disease, the symptoms of it, or the predisposition towards it. Such a subject can be identified by a health care professional based on results from any suitable diagnostic method.
The term “an effective amount” refers to the amount of an active compound that is required to confer a therapeutic effect on the treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on rout of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.
Abbreviations: CD 28, Cluster of Differentiation 28.
Without intent to limit the scope of the invention, exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action.
This expression vector was constructed by using the plasmid RAP1-K3 shown in
The fragment CD28convPEt contains cathepsin L and furin protease cutting sites.
The sequence of PE268-313 is pletftrhrqprgweqleqcgypvqrlvalylaarlswnqvdqvir (SEQ ID NO: 20). The whole sequence of CD28convPEt is as follows: tdiyfckiefmypppyldneksngtiihrarykrgweqleqcgypvqrlvalylaarlswnqvdqvirgs (SEQ ID NO: 30), the sequence underlined represents a linker sequence containing Cathepsin L and furin protease cutting sites.
E. coli BL21 cells harboring a protein expression vector were cultured in Luria Bertani broth containing 25 μg/ml kanamycin at 37° C. When the culture reached an early log phase, (A600=0.1 to 0.4), isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added at a final concentration of 0.5 to 2 mM for induction. Cells were harvested after 4 hours IPTG induction and disrupted by sonication. The overexpressed protein-containing inclusion bodies were isolated and solubilized in 8M urea/TN buffer (8M urea, 50 mM Tris, 50 mM NaCl, pH 8.0).
The refolding of the fusion protein RAP1-PE268-313-E7-K3 was performed by dialysis against 50× volume of TNZ buffer (50 mM Tris 50 mM NaCl and 0.01 mM ZnCl2, pH 8.0) at 4° C. overnight. The refolded proteins were subject to SDS-PAGE analyses under reduced (with dithiothreitol; +DTT) and non-reduced (without dithiothreitol; −DTT) conditions (
The fusion proteins PE407-E7-K3 and RAP1-PE268-313-E7-K3 were expressed as described above and protein refolding examined by SDS-PAGE (
The effects of the fusion proteins PE407-E7-K3 and RAP1-CD28convPEt-E7-K3 with or without an immune potentiator on tumor size and survival rate were examined. Mice were challenged with a higher dose of TC-01 cells (3×104) via s.c. injection. Seven days after the challenge, mice were vaccinated via s.c. with placebo, PE407-E7-K3 (100 μg/dose) or RAP1-CD28convPEt-E7-K3 (100 μg/dose) with the immune potentiator GPI-0100 or the protein absorbent aluminum phosphate once per week for 3 weeks (
In contrast, the potency of PE407-E7-K3 in suppressing the tumor growth depended on the adjuvant. When combined with the absorbent aluminum phosphate, PE407-E7-K3 became less potent than that when combined with the immune potentiator GPI-0100 (
On the other hand, mice administrated with RAP1-CD28convPEt-E7-K3 in combination with the immune potentiator GPI-0100 or the absorbent aluminum phosphate had a better survival rate than the groups vaccinated with PE407-E7-K3 in combination with GPI-0100 or aluminum phosphate (
The immunogenicities of various vaccines were tested. Briefly, mice were divided into the following groups: HPV16 E7, HPV18 E7, HCV core, HBV HBx, PCV2 ORF2 and PRRSV (
The immunization schedule, vaccines and dose are illustrated in
The stimulated splenocytes were washed with FACScan buffer and the cell surface markers CD8a, CD4, and CD3 were stained with phycoerythrin-conjugated monoclonal rat anti-mouse CD8a, AF700-conjugated monoclonal rat anti-mouse CD4 and AF647-conjugated monoclonal rat anti-mouse CD3 antibodies. The cells were then permeabilized and fixed by Cytofix/Cytoperm kit according to the manufacturer's instructions (BD Pharmingen) Intracellular IFN-γ was stained with AF488-conjugated rat anti-mouse IFN-γ to measure the immune response and cytokine levels. Flow cytometry analyses were performed using Gallios flow cytometry with Kaluza analysis software (Beckman Coulter).
The following PRRSV vaccines were tested for immunogenicities: PE407-PRRSV-K3, RAP1-PE268-313-PRRSV-K3 or RAP1-CD28convPEt-PRRSV-K3 vaccine. Each vaccine contained a mixture of four different fusion proteins, and each fusion protein contained a different antigen that is selected from the group consisting of DGD, M12, PQAB and RSAB (
The amino acid sequence of “DGD” (SEQ ID NO: 26) is as follows: RHHFTPSERQLCLSSIQTAFNQGAGTCILSDSGRISYTVEFSLPTHHTVRLIRVTAPPS ALDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVS LTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVRHHFTPSERQLCLSSIQTAFNQGA GTCILSDSGRISYTVEFSLPTHHTVRLIRVTAPPSA. DGD represents a fusion antigen of PRRSV ORF7 a.a. 64-a.a. 123 (boldface), linker (underlined) and ORF7 a.a. 64-a.a. 123 (boldface).
The term “M12” represents a antigen of PRRSV ORE1b a.a. 1046-a.a. 1210. Its amino acid sequence (SEQ ID NO: 27) is as follows:
The amino acid sequence of “PQAB” (SEQ ID NO: 28) is as follows: GSSLDDFCYDSTAPQKVLLAFSITYASNDSSSHLQLIYNLTLCELNGTDWLANKFDWA. PQAB represents a fusion antigen of PRRSV American strain ORF6 a.a. 2-a.a. 26 and ORE5 a.a. 31-a.a. 63 (underlined).
The amino acid sequence of RSAB is MGSLDDFCNDSTAAQKLVLAFSITYTPIFVAGGSSSTYQYIYNLTICELNGTDWLSNHFDWA (SEQ ID NO: 29). The term “RSAB” represents a fusion antigen of PRRSV European strain ORF6 a.a. 2-28 and ORF5 a.a. 31-64 (underlined).
The fragment of RAP1 domain 3 of the fusion protein RAP1-CD28convPEt-E7-K3 is replaced by A2M minimum (SEQ ID NO: 6), HIV-Tat minimum (SEQ ID NO: 7) or HSPs minimum (SEQ ID NO: 8) to generate the fusion proteins A2M-CD28convPEt-E7-K3, Tat-CD28convPEt-E7-K3 and HSP-CD28convPEt-E7-K3 vaccines, respectively. The TC-1 tumor suppression activity and cell mediated immune responses enhanced by these vaccines are examined using similar methods as described above. Table 1 shows SEQ ID NOs. of the components of various fusion proteins. Table 2 shows the fusion proteins tested for the effects on T cell-mediated immune responses in animals and the sequences of antigens.
In the immunogenicity assays, antigen-specific cell-mediated immune responses induced by various vaccines were evaluated by measuring the numbers of CD3+/CD4+/IFNγ+ and CD3+/CD8+/IFNγ+ T cells in the splenocytes. The results indicated that the vaccine RAP1-CD28convPEt-antigen-K3 can induce strong T cell responses.
The vaccine RAP1-CD28convPEt-antigen-K3 is superior to PE407-antigen-K3 in eliciting T cell-mediated immunogenicity. For example,
A fusion protein comprising RAP1 domain III, the sensitizing signal CD28conv alone without the translocation peptide PEt, antigen and an ER retention signal is sufficient in eliciting a strong antigen-specific T cell mediated immune responses when the antigen chosen comprises ten or greater than 10 epitopes.
It was unexpected that the ER retention signal is not essential for the fusion protein of the invention to elicit a strong cell-mediated immunogenicity. In other words, without the ER retention sequence, the fusion protein of the invention can still elicit strong T-cell responses.
In contrast, U.S. Pat. Nos. 7,378,100B2 and 7,335,361 show that the ER retention signal K3 is indispensable for PE-related fusion proteins (PE407-antigen-K3) to elicit T cell responses.
It was also discovered that a fusion protein comprising RAP1 domain III, the translocation peptide PE218-313 (without the sensitizing signal CD28conv), antigen and an ER retention signal is superior to a PE-related fusion protein without containing the RAP1 domain III.
The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching. The embodiments and examples were chosen and described in order to explain the principles of the invention and their practical application so as to enable others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.
Some references, which may include patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is “prior art” to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.
The present application claims the priority to U.S. Provisional Application Ser. No. 61/733,879, filed Dec. 5, 2012, which is herein incorporated by reference in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5686281 | Roberts | Nov 1997 | A |
| 7314632 | Fitzgerald | Jan 2008 | B1 |
| 7335361 | Liao et al. | Feb 2008 | B2 |
| 7378100 | Chang et al. | May 2008 | B2 |
| 7465455 | Chang et al. | Dec 2008 | B2 |
| 20090155297 | Mrsny | Jun 2009 | A1 |
| Number | Date | Country |
|---|---|---|
| 2078726 | Jul 2009 | EP |
| WO 94 02610 | Feb 1994 | WO |
| WO 2004087196 | Oct 2004 | WO |
| WO 2008036682 | Mar 2008 | WO |
| WO 2011161260 | Dec 2011 | WO |
| Entry |
|---|
| Ahn et al., CD28 isoform 2 [Bos taurus], Accession No. AAL40878.1 (2002). |
| D. Craig Hooper et al. “Rabies ribonucleocapsid as an oral immunogen and immunological enhancer” Proc. Natl. Acad. Sci. USA, vol. 91, pp. 10908-10912. Nov. 1994, Immunology. |
| Derek T O'Hagan,“New Generation Vaccine” Adjuvants Encyclopedia of Life Sciences & 2007. John Wiley & Sons, Ltd., pp 1-7. |
| Number | Date | Country | |
|---|---|---|---|
| 20140154285 A1 | Jun 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61733879 | Dec 2012 | US |
