WO2019133647A1

WO2019133647A1 - Combination of a fusion protein vaccine with anti-ox40 antibody for use in eliciting antigen-specific immune responses

Info

Publication number: WO2019133647A1
Application number: PCT/US2018/067576
Authority: WO
Inventors: Chia-Mao Wu; Yin-Ching LIN
Original assignee: Thevax Genetics Vaccine Co., Ltd.; Healthbanks Biotech Usa Inc.
Priority date: 2017-12-29
Filing date: 2018-12-26
Publication date: 2019-07-04

Abstract

A pharmaceutical composition comprising a mixture of a vaccine composition and anti-OX40 antibody is disclosed. The vaccine comprises a fusion protein and a saponin-base adjuvant or a Toll-like receptor (TLR) agonist adjuvant. The fusion protein comprises an antigen-presenting cell-binding domain or a CD91 receptor-binding domain, a protein transduction domain, an antigen, and optionally an endoplasmic reticulum retention signal. The protein transduction domain is selected from (I) a fusion polypeptide, comprising a T cell sensitizing signal-transducing peptide, a translocation peptide and a linker, (II) a T cell-sensitizing signal-transducing peptide, or (III) a translocation peptide of 34-112 amino acid residues in length.

Description

COMBINATION OF A FUSION PROTEIN VACCINE WITH ANTI-OX40 ANTIBODY FOR USE IN ELICITING ANTIGEN-SPECIFIC IMMUNE RESPONSES

FIELD OF THE INVENTION

The present invention relates generally to a pharmaceutical formulation, and more specifically to a fusion protein vaccine in combination with anti-OX40 antibody for use in treatment of infections or tumors.

BACKGROUND OF THE INVENTION

There is a need for exploring the potential of using anti-OX40 antibody with an antigen-specific fusion protein in a vaccine.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a pharmaceutical composition comprising a mixture of:

(A) a vaccine composition, comprising a therapeutically effective amount of a fusion protein and a saponin-base adjuvant or a Toll-like receptor (TLR) agonist adjuvant, wherein the saponin-base adjuvant is selected from the group consisting of GPI-0100 and QS-21, and the Toll-like receptor (TLR) agonist adjuvant is selected from the group consisting of monophosphoryl lipid A (MPL) and CpG oligonucleotide; and

(B) an anti-OX40 antibody;

wherein the fusion protein comprises:

(a) an antigen-presenting cell (APC)-binding domain or a CD91 receptor-binding domain, located at the N-terminus of the fusion protein;

(b) a protein transduction domain, located at the C-terminus of the APC-binding domain or the CD91 receptor-binding domain, the protein transduction domain being selected from the group consisting of:

(I) a fusion polypeptide comprising:

(l) a T cell sensitizing signal-transducing peptide consisting of 28-53 amino acid residues in length, comprising the amino acid sequence of SEQ ID NO: 31 , in which Xaa⁸ is I or L; Xaa¹⁰ is V, F or A, Xaa¹¹ is M or L, Xaa¹⁷ is L or I, being located at the N-terminus of the fusion polypeptide;

(2) a translocation peptide consisting of 34-1 12 amino acid residues in length, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, 4, 20 or 41 ; and (3) a linker, comprising SEQ ID NO: 15 linking the T cell sensitizing signal- transducing peptide and the translocation peptide;

(II) a T cell-sensitizing signal-transducing peptide consisting of 28-53 amino acid residues in length, comprising the amino acid sequence of SEQ ID NO: 31, in which Xaa⁸ is I or L; Xaa¹⁰ is V, F or A, Xaa¹¹ is M or L, Xaa¹⁷ is L or I; and

(III) a Pse domonas exotoxin A (PE) translocation peptide of 34-1 12 amino acid residues in length, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, 4, 20 or 41;

(c) an antigen of a pathogen or a tumor antigen; and

(d) optionally an endoplasmic reticulum retention signal, located at the C -terminus of the fusion protein.

In one embodiment, the fusion protein comprises the endoplasmic reticulum retention signal. In another embodiment, the antigen is selected from the group consisting of human

papillomavirus (HPV) E7 protein, Hepatitis B virus (HBV) HBx protein, Hepatitis C virus (HCV) core antigen, Flu virus M2 antigen, and a tumor associated antigen.

In another embodiment, the APC-binding domain or the CD91 receptor-binding domain is a Pseudomonas exotoxin A (PE) binding domain I and the protein transduction domain is the

Pseudomonas exotoxin A (PE) translocation peptide.

In another embodiment, the GPI-0100 is the sole adjuvant.

In another embodiment, 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.

In another embodiment, the protein transduction domain is the fusion polypeptide comprising:

(1) the T cell sensitizing signal-transducing peptide;

(2) the translocation peptide; and

(3) the linker.

In another embodiment, the CpG oligonucleotide is the sole adjuvant.

In another embodiment, the protein transduction domain comprises the amino acid sequence of SEQ ID NO: 30.

In another embodiment, the pathogen is at least one selected f om 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 (HBV) and Porcine Circovirus 2 (PCV2). In another embodiment, the tumor associated antigen is selected from the group consisting of SSX2, MAGE- A3, NY-ESO-1, iLRP, WT12-281, RNF43, and CEA-NE3.

In another embodiment, the protein transduction domain is the Pseudomonas exotoxin A (PE) translocation peptide, and the fusion protein further comprises a nuclear export signal comprising the amino acid sequence of SEQ ID NO: 44, located between the antigen and the endoplasmic reticulum retention signal, or between the translocation peptide and the antigen.

In another embodiment, the antigen comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 21 or 22, and

(1) 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 SEQ ID NO: 9, and the protein transduction domain is the Pseudomonas exotoxin A (PE) translocation peptide; or (ii) 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 SEQ ID NO: 5; and the protein transduction domain comprises the sequence of SEQ ID NO: 30.

In another aspect, the invention relates to use of a pharmaceutical composition according to the invention in the manufacture of a medicament for inducing an enhanced cell-mediated immune response or an enhanced pathogen antigen or tumor antigen specific T cell response in a subject in need thereof.

Further in another aspect, the invention relates to use of a pharmaceutical composition according to the invention in the manufacture of a medicament for inhibiting growth of cancer resulting from human papillomavirus (HPV) and increasing cancer patient survival in a subject in need thereof, wherein the antigen is human papillomavirus (HPV) E7 protein in a subject in need thereof.

Further in another aspect, the invention relates to a method for preparation of a pharmaceutical composition according to the invention, comprising the steps of:

( 1 ) providing a vaccine composition according to the invention;

(2) providing the anti-OX40 antibody;

(3) admixing the vaccine composition and the anti-OX40 antibody together to form the mixture for at least 8 hours before use of the pharmaceutical composition for vaccination in a subject in need thereof.

In one embodiment, the mixture is formed by admixing the vaccine composition and the anti-

OX40 antibody together to form the mixture for at least 8 to 16, 9 to 16, 10 to 16, 1 1 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16 hours, or to form the mixture for at least 8, 9, 10, 1 1, 12, 13, 14, 15, or 16 hours, before use of the medicament or the pharmaceutical composition for vaccination in a subject in need thereof. In another embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 54 or 55.

In another embodiment, the T cell sensitizing signal-transducing peptide comprises the amino acid sequence of SEQ ID NO: 1 or 2.

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, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an immunization schedule for mice vaccinated with various compositions as indicated in FIG. 1B to test the immunogenicity elicited by a mixture of PEK+GPI-0100 (TVGV-1 ) and OX40 agonistic antibody.

FIG. IB shows the results of ELISA analyses of humoral immunogenicity in mice of FIG. 1A. TVGV-1 stands for PEK+GPI-0100. The term "aOX40" stands for agonistic antibodies to OX40.

FIG. 1C shows CD4⁺ immune cell populations in the splenocytes from mice of FIGs. 1A-B. The splenocytes were analyzed by flow cytometry.

FIG. ID shows CD8⁺ immune cell populations in the splenocytes from mice of FIGs. 1A-B. FIG. 2 A shows an immunization schedule for TC- 1 tumor-bearing mice vaccinated with various compositions as indicated in FIG. 2B to examine the effects of the combination of PEK+GPI-0100 (TVGV-1) and OX40 agonistic antibody on tumor growth and survival rate in tumor-bearing mice.

FIG. 2B shows the survival rate in each group of the TC-1 tumor mice of FIG. 2A after vaccinations. Mice were considered and counted as dead if they had a tumor size larger than 2000 mm³ or had a tumor with an average diameter greater than 20 mm.

FIG. 2C shows the size of the tumors in each group of the TC-1 tumor mice of FIG. 2B. P value = 0.039 (calculated between the group of TVGV-1 mixed with OX-40 agonist antibody and the group of TVGV-1 mixed with IgG). A difference is statistically significant if a p value is less than 0.05.

FIG. 3 A shows an immunization schedule for TC-1 tumor-bearing mice. The mice were sacrificed a week after the last vaccination and the tumors were removed to examine tumor- infiltrating lymphocytes (TILs).

FIG. 3B-C show percentage of double positive in CD3+ T cells in splenocytes from mice of FIG. 3A. The splenocyte IFNγ-5 was color stained and analyzed by flow cytometry.

FIG. 3D-E show the number of double-positive in CD3+ T cells in tumor tissue cell supernatants from mice of FIG. 3A.

FIG. 3F-G show the number of double-positive in CD3+ T cells in tumor tissue total cells from mice of FIG. 3 A.

FIG. 4A shows an immunization schedule for TC- 1 tumor-bearing mice vaccinated with various compositions as indicated in FIG. 4B to examine the effects of the combination of RAP1- CD28convPEt-E7-K3+CpG1826 (TVGV-2) and OX40 agonistic antibody on tumor growth and survival rate in tumor-bearing mice.

FIG. 4B shows the survival rate in each group of the TC-1 tumor mice of FIG. 4A after vaccinations. Mice were considered and counted as dead if they had a tumor size larger than 2000 mm³ or had a tumor with an average diameter greater than 20 mm.

FIG. 4C shows the size of the tumors in each group of the TC-1 tumor mice of FIGs. 4A-B. P value = 0.037 (calculated between the group of TVGV-2 mixed with OX-40 agonist antibody and the group of TVGV-2 mixed with IgG).

FIGs. 4D-E show percentage of double positive in CD3+ T cells in splenocytes from mice of FIG. 4A. The splenocyte IFNγ-5 was color stained and analyzed by flow cytometry.

FIG. 4F shows percentage of double positive in CD3+ T cells in tumor tissue from mice of FIG. 4A.

FIG. SA shows an immunization schedule for TC-1 tumor-bearing mice vaccinated with various compositions as indicated in FIG. 5B to examine the effects of the combination of RAP1- CD28convPEt-E7-K3+CpG1826 (TVGV-2) and aCD40 or aOX40 agonistic antibody on tumor growth and survival rate in tumor-bearing mice.

FIG. 5B shows the average area of the tumors in each treatment group of the TC-1 tumor mice of FIGs. 5 A. P value = 0.006 (calculated between the group of TVGV-2 mixed with OX-40 agonist antibody and the group of TVGV-2 mixed with IgG); P value = 0.002 (calculated between the group of TVGV-2 mixed with OX-40 agonist antibody and the group of TVGV-2 mixed with CD-40 agonist antibody).

DETAILED DESCRIPTION OF THE INVENTION

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.

DEFINITIONS

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.

Immunogenic proteins such as fusion proteins for use as immunogenic enhancers for inducing antigen-specific T cell responses are disclosed in the U.S. Patent No. 20140154285 Al and 20140154280 A 1 , each of which is incorporated herein by reference in its entirety.

The saponin derivative GPI- 100 is a semi-synthetic triterpenoid glycoside. It is derived from QS-7, one of the purified components of Quil A, a saponin adjuvant extracted from the bark of the Molina tree Quillaia saponaria. A Toll like receptor (TLR) 4 ligand, particularly an agonist such as a lipid A derivative particularly monophosphoryl lipid A or more particularly 3 Deacylated monophosphoryl lipid A (3 D-MPL). 3D-MPL is sold under the name MPL by GlaxoSmithKline Biologicals N.A. and is referred throughout the document as MPL or 3 D-MPL.

QUIL-A® adjuvant contains the water-extractable fraction of saponins from the South-American tree, Quillaja saponaria Molina. QUIL-A®, and fractions thereof are described in U.S. Pat. No. 5,057,540 and "Saponins as vaccine adjuvants", Kensil, C. R., Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2): 1-55; and EP 0 362 279 Bl.

QS-21 is a natural saponin derived from the bark of Quillaja saponaria Molina. It is a HPLC purified non-toxic fraction of Qutl A and is disclosed in U.S. patent No. 5,057,540.

CD 134 (OX40) is a member of the tumor necrosis factor receptor superfamily. It acts as a costimulatory receptor on T cells.

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 T cell mediated immune response due to prior CD4+ and CD8+ T cell sensitization.

A protein transduction domain may be "a fusion polypeptide", in which the fusion polypeptide comprises a T cell sensitizing signal-transducing peptide, a tinker, and a translocation peptide. For example, the fusion polypeptide may be the polypeptide "CD28convPE ".

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 "PE " or "PE_tCore" refers to a PE translocation domain core with 34 amino acid residues in length.

A linker is present between the "CD28conv" and the "PE«". The orientation or arrangement of the fusion polypeptide "CD28convPE_t " is important in that "CD28conv" (or the T cell sensitizing signal-transducing peptide) must be at the upstream to the PE_t ( or the translocation peptide), i.e., PE_t must be at the C-terminus of the "CD28conv" to obtain enhanced T-cell responses. The "CD28convPE " can raise much higher IgG titer (called CD28-specific agonist antibody) specific to CD28conv than the reversed orientation fusion peptide PE_tCD28conv. The CD28-specific agonist antibody can sensitize both CD4+ and CD8+ T cells. The correct orientation fusion polypeptide CD28convPE_t contains a linker (R'X²R³X⁴K⁵R⁶) between CD28conv and PE_t domains. The linker contains an antigen presenting cell (APC)-specific protease (cathepsin L) cutting site Lys-Arg (KR). Therefore, the fusion protein RAPl-CD28convPE_t-Antigen-K3 can be digested into the two fragments: RAPl-CD28conv and PE_t-Antigen-K3. The RAPl-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).

US Patent No. 9481714 disclose a fusion protein RAP l-CD28convPE_t- Antigen (without the ER retention signal K3) can still elicit strong T-cell responses.

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 X⁸ is I or L; X'°is V, F or A, X" is M or L, X¹⁷ is L or I.

The T cell-sensitizing signal-transducing peptide comprises the critical region Κ¹Χ²Ε³Χ⁴Χ⁵Υ⁶Ρ⁷Ρ⁸Ρ⁹Υ¹⁰(SEQ ID NO: 32) , wherein X² is I or L; X⁴ is V, F or A, X⁵ is M or L. A T cell sensitizing signal-transducing peptide (TDIYFCKIEFMYPPPYLDNEKSNGTIIH; SEQ ID NO: 31, wherein X⁸ is I, X¹⁰ is F, X¹¹ is M, X¹⁷ is L) specific for mice was illustrated in the U.S. Patent No. 9481714.

A PE translocation peptide may comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, 4, 20 or 41. 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 (SEQ ID NO: 41), or a.a. 253 - a.a. 364 (SEQ ID NO: 4) of full length PE (SEQ ID NO: 10). 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), Porcine reproductive and respiratory syndrome virus (PRRSV), Human immunodeficiency virus- 1 (HIV- 1), flu virus, Dengue virus, Hepatitis C virus (HCV), Hepatitis B virus (HBV), Porcine Circovirus 2 (PCV2), Classical Swine Fever Virus (CSFV), Foot- and-mouth disease virus (FMDV), Newcastle disease virus (NDV), Transmissible gastroenteritis virus (TGEV), Porcine epidemic diarrhea virus (PEDV), Influenza virus, Pseudorabies virus, Parvovirus, Pseudorabies virus, Swine vesicular disease virus (SVDV), Poxvirus, Rotavirus, Mycoplasma pneumonia, Herpes virus, infectious bronchitis, or infectious bursal disease virus, 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 PRRSV ORF6 and ORF5 fragments, or a fusion of antigenic proteins from PRRSV and PCV2 pathogens.

The function of an endoplasmic reticulum retention signal 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 (K.DEL) or RDEL. An ER retention 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), or KDELKDELKDEL (SEQ ID NO: 19). Receptor-associated protein (RAP!) 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 PE₄₀₇ (or PE1-407; SEQ ID NO. 40) is described in prior patent (US 7,335,361 B2) as ΡΕ(ΔΙΙΙ).

A nuclear export signal (NES) refers to a short amino acid sequence of 4 hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. The NES is recognized and bound by exportins. The most common spacing of the hydrophobic residues to be L¹X²X³K⁴L⁵X⁶X⁷L^,X⁹L^,0X¹¹ (SEQ ID NO. 44), where **L" is leucine, "K" is lysine and "χ^{2 3}·⁶·⁷·^{9 11}" is any naturally occurring amino acid. For example, an artificial NES may comprise the sequence Leu Gin Lys Lys Leu Glu Glu Leu Glu Leu Ala (LQKKLEELELA; SEQ ID NO: 45).

The term "NESK" refers to a fusion peptide of a NES and an ER retention signal (i.e., a NES fused to an ER retention signal). It is an artificial peptide possessing the function of a nuclear export signal (NES) and an ER retention sequence. Thus, it can export an antigen from the cell nucleus to the cytoplasm through the nuclear pore complex and assist translocation of an antigen from the cytoplasm to ER and retain the antigen in the lumen of the ER. For example, the amino acid sequence of NESK may be LQKKLEELELAKDEL (SEQ ID NO: 43). A fusion protein comprising NESK has been disclosed in US Patent No. 9339536.

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; TVGV-1 , PE₄₀₇-E7-K3 (PEK) + GPI-0100;

TVGV-2, RAPl-CD28convPE_t-E7-K3 + CpG1826.

EXAMPLES

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 theory or scheme of action.

General materials and methods

Immunogenic protein preparation:

The immunogenic proteins were expressed in E. coli expression system as described in U.S. Patent No. 9,775,898. They may be antigen itself only fused to the C-terminus of RAP1- CD28convPE_t fusion protein to generate RAP 1 -CD28convPE_t-( Antigen) fusion protein (without the ER retention signal K3). US Patent No. 9481714 discloses a fusion protein RAPl-CD28convPE,- Antigen (without the ER retention signal K3) can still elicit strong T-cell responses.

The immunogenic protein may be an antigen and an ER retention signal (K3) fused to the C- terminus of Pseudomonas exotoxin A domains I and II (i.e., PE₄₀₇) to generate PEto7-(antigen)-K3 fusion protein, or an antigen and an ER retention signal fused to the C-terminus of RAP1- CD28convPE_t fusion protein to generate RAPl-CD28convPE_t-(Antigen)-K3 fusion protein. The antigen HPV E7 antigen was used to construct two fusion protein PE₄₀₇-E7-K3 (SEQ ID NO: 54) and RAPl-CD28convPE_t-E7-K3 (SEQ ID NO: 55) for illustrations of the invention.

Humoral immunity studies:

Animals were vaccinated and serum samples collected. The serum samples from each animal at each collection time point were diluted for 10000 times in blocking buffer. The level of HPV 16 E7 specific IgG was detected by ELISA method (coating E7 pet32a 1 μg/well).

Splenocyte sample preparation and flow cytometric analysis:

Spleen tissues were isolated from mice and processed into single cell suspensions using PP micro centrifuge sample pestle. Splenocytes were seeded on 6-well plates at 2x10⁷/2ml, stimulated with or without HPV16E7-peptide and cultured overnight in a C0₂-containing incubator. The cells were then treated with the protein transport inhibitor monensin (Cat. no. 00-4505-51) and brefeldin (Cat. no. 00-4506-51) for 4 hours at 37°C. Afterward, cells were harvested, washed twice with PBS and stained for the surface marker CD3, CD4 and CD8 for 30 minutes at 4°C. After washing, the cells were fixed for 30 minutes at the room temperature using intracellular (IC) fixation buffer (Cat. no. 00-8222-49), then washed in permeabilization buffer (Cat. no. 00-8333-56) and stained with IFNγ antibody.

Events of 1.5 million cells were acquired on BACK.MAN COULTER® GALLIOS™. Flow data were analyzed using Kaluza software (vl.2). Populations were first gated on CD3+ T cells, then gated on viable mononuclear cell using forward and side scatter, and subsequently sub-gated on either CD4+/IFNγ+ double-positive cells or CD8+/IFNγ+ double-positive cells.

Tumor tissue sample preparation and Flow Cytometric Analysis:

Tumor tissues were isolated from mice and processed into single cell suspensions using a mouse tumor dissociation kit (Cat. no. 130-096-730). Cells were seeded on 10 cm-petri dishes at 1x10⁷ cells/10 ml, stimulated with or without HPV16E7 -peptide and cultured overnight in a CO₂- containing incubator. The cells were then treated with the protein transport inhibitor monensin and brefeldin for 4 hours at 37°C, harvested, washed twice with PBS, and stained for the surface marker CD3, CD4, COS, or isotype control rat IgG for 30 minutes at 4°C. After washing, the cells were fixed for 30 min at the room temperature using IC fixation buffer, washed in permeabilization buffer and stained with IFNγ antibody or TNFa antibody.

For large tumors, tissue samples were analyzed for individual mice. For small tumors, tissue samples were pooled within the groups in order to have a sufficient number of tumor tissue cells for analyses. Events of 1.5 million cells were acquired on the BACKMAN COULTER® GALLIOS™. Flow data were analyzed using Kaluza software (vl.2). Populations were first gated on viable lymphocytes using forward and side scatter, subsequently gated on CD3+ T cells, and then sub-gated on desired double-positive T cells (e.g. CD4+/IFNγ+, CD8+/ ΙΡΝγ+, CD4+/TNFa+ or CD8+/TNFα+).

The following antibodies for staining were used: CD8-PE (CD8 monoclonal antibody, R- phycoerythrin); CD3-AF647 (ALEXA FLUOR® 647 anti-mouse CD3 antibody); CD4-AF700 (ALEXA FLUOR® 700 anti-mouse CD4 antibody); AF488 (ALEXA FLUOR® 488 Dye) for staining IFNγ; PE/Cy7 anti-mouse TNF-α antibody for staining Tumor necrosis factor-a.

Example 1

Immunogenicity analysis of the mixture of PEK vaccine and OX40 antibody: The E7 immunogenic proteins comprises the antigen HPV E7. The exemplified E7 fusion proteins are PE₄₀₇-E7-K3, and RAPl-CD28convPE_t-E7-K3. Each fusion protein were combined with the adjuvant GPI-0100 (Hawaii Biotech) or CpG1826 (Cat. No. tlrl-1826, InvivoGen), and anti- OX40 antibody (Cat. No. 1 19408, Biolegend) and the immunogenicity of each mixture was tested in mice.

Female mice C57BL/6 at 5 weeks old of age were purchased from BioLASCO Taiwan Co., Ltd.

5 mice/cage with a 12-hour day/12-hour night light cycle. Given free access to food and water, the mice were housed for one week and maintained under standard conditions prior to experimentation. Mice were vaccinated once per week for 3 weeks with vaccine formulations as indicated in Table 1 and FIG. 1A. All mice were sacrificed 7 days after the last immunization, and the spleens were harvested. Splenocytes were isolated and analyzed by flow cytometric analysis.

Table 1 shows PE₄₀₇-E7-K3 (PEK) vaccination groups, adjuvant GPI-0100, OX40 antibody doses. FIG. IB shows the results of the ELISA analysis of the humoral immune response. TVGV-1 stands for PE₄₀₇-E7-K3 (PEK) plus GPI-0100. Rat IgG served as OX40 control antibody. The result indicated that the combination of PEK plus GPI-0100 with anti-OX40 antibody elicited the greatest humoral immune response (FIG. IB) and E7-specific CD4 and CD8 T-cell responses (FIGs. 1C-D).

Table 1

Example 2

Combination of PEK/GPI-0100 with anti-OX40 antibody Studies on TC-1 tumor animal model

The effects of the composition of PEK+GPI-0100 (TVGV-1) and OX40 agonistic antibody on tumor growth and survival rate in tumor-bearing mice were examined. Mice were injected s.c. with 5x10⁴ TC-1 tumor cells in 200μ1 PBS prior to vaccinations. FIG. 2 A shows an immunization schedule for TC-1 tumor-bearing mice. Table 2 shows the dose of PE*o7-E7-K3 (PEK), adjuvant GPI-0100 (also abbreviated as "GPI"), OX40 antibody (also abbreviated as "aOX40") in each group. Rat IgG served as OX40 control antibody. Mice were considered and counted as dead if they had a tumor size larger than 2000 mm³ or had a tumor with an average diameter greater than 20 mm.

The results indicated that the combination of PEK plus GPI-0100 with anti-OX40 antibody showed the greatest increase in tumor mouse survival. It was unexpected to find that the antigen- specific fusion protein vaccine TVGV-1 (PEK+GPI-0100) and anti-OX40 antibody being mixed together before injection produced a much better outcome in increasing tumor mouse survival than the antigen-specific fusion protein vaccine TVGV-1 (PEK+GPI-0100) and the agonistic anti-OX40 antibody not being mixed together before injection but being injected separately (FIG. 2B). The combination of PEK plus GPI-0100 with anti-OX40 antibody in a mixture also showed the greatest decrease in tumor size (FIG. 2C).

Table 2

Example 3

The effect of the combination of PEK+GPI-0100 with anti-OX40 antibody

on tumor-infiltrating lymphocytes (TILs)

Mice were injected s.c. with 1x10³ TC-1 tumor cells in 200μ1 PBS prior to vaccinations to examine tumor growth and cell-mediated immunities. One week after completion of the vaccinations, mice were sacrificed (FIG. 3A), tumors removed, and the relationship between TILs and tumor cells were studied via flow cytometric analyses as described above. Table 3 shows the vaccination groups and doses.

The combination of PEK plus GPI-0100 with anti-OX40 antibody elicited the greatest number of CD8+/IFNγ+ double-positive T cells, but not the CD4+ IFNγ+ T cells in the splenocytes (FIG. 3C) and the tumor cell supernatant (FIGs. 3D-E). This indicated that PEK plus GPI-0100 with anti-OX40 antibody induced more cytotoxic T-cells to infiltrate into the tumor tissues. Mixing the antigen- specific fusion protein TVGV-1 (PEK+GPI-0100) and anti-OX40 antibody together before injection produced a better outcome in increasing cytotoxic CD8+ T cells than being injected separately (FIGs. 3C-E). The combination of PEK plus GPI-0100 with anti-OX40 antibody also elicited the greatest number of CD8+/IFNγ+ double positive T cells, but not the CD4+/IFNγ+ T cells in the total tumor cells (FIGs. 3F-G).

Table 3

Example 4

Combination of RAPl-CD28convPE_t-E7-K3+CpG1826 with anti-OX40 antibody Studies on

TC-1 tumor animal model

The effects of the composition of RAP l-CD28convPE_t-E7-K3+CpG 1826 (TVGV-2) and OX40 agonistic antibody on tumor growth and survival rate in tumor-bearing mice were examined. Mice were injected s.c. with 5x10⁴ TC-1 tumor cells in 200 μl PBS prior to vaccinations. FIG. 4A shows an immunization schedule for TC-1 tumor-bearing mice. The amount per dose for antigenic fusion protein is 100 μg, the adjuvant CpG1826 20 μg, the OX 40 agonistic antibody 100 μg. Rat IgG served as OX40 control antibody. The fusion protein vaccine TVGV-2 and OX40 monoclonal antibody were mixed before the injection. After sacrifice, spleen and tumor tissues were separately collected and analyzed by flow cytometric analysis as described above.

The combination of RAPl-CD28convPE_t -E7-K3+CpG1826 with anti-OX40 antibody showed the greatest increase in tumor mouse survival and the greatest decrease in tumor size (FIGs. 4B-C). The combination of RAPl-CD28convPE_t -E7-K3/CpG 1826 with anti-OX40 antibody in a mixture also showed the greatest increase in CD4+/IFNγ+ and CD8+/IFNγ+ double-positive T cells in splenocytes (FIG. 4D-E).

The combination of RAPl-CD28convPE_t -E7-K3+CpG1826 with anti-OX40 antibody induced the highest percentage of CD8+/IFNγ+ double-positive T cells in the dissociated tumor tissues (FIG. 4F).

Example 5

Comparison of RAP l-CD28convPE_t-E7-K3+CpG 1826 combined with anti-OX40 antibody or anti-CD40 antibody studies on TC-1 tumor animal model

The effects of the composition of RAPl-CD28convPE_t-E7-K3+CpG oligonucleotide (TVGV-2) and anti-CD40 antibody on tumor growth and survival rate in tumor-bearing mice were examined.

Mice were injected s.c. with 5x10⁴ TC-1 tumor cells in 200 μl PBS prior to vaccinations. FIG. 5 A shows an immunization schedule for TC-1 tumor-bearing mice. The amount per dose for the antigenic fusion protein RAPl-CD28convPE_t -E7-K3 was 50 μg, the adjuvant CpG1826 20 μg, the anti-CD40 antibody 100 μg or the anti-OX 40 agonistic antibody 100 μg (200 μl). The fusion protein vaccine TVGV-2 and monoclonal antibody were mixed before the injection.

The combination of RAP 1 -CD28convPE_t -E7-K3+CpG 1826 (TVGV-2) with anti-OX40 antibody showed the best effect in decreasing the tumor size (FIGs. 5B, 4B-C) and was better than the combination with anti-CD40 antibody. In contrast, the combination of fusion protein vaccine TVGV- 2 with anti-CD40 antibody has no additional effect or synergistic effect on the improvement of tumor mice survival and tumor size.

Table 4 shows the SEQ ID NOs. of the components of various fusion proteins.

Table 5 shows the fusion proteins tested for the effects on T cell-mediated immune responses in animals and the sequences of antigens.

Table 4

Table 5

The embodiments and examples were chosen and described in order to explain the principles of the invention and their practical application 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. 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.

Claims

CLAIMS What is claimed is:

1. A pharmaceutical composition comprising a mixture of:

(B) an anti-OX40 antibody;

wherein the fusion protein comprises:

(I) a fusion polypeptide comprising:

(1) a T cell sensitizing signal-transducing peptide consisting of 28-53 amino acid residues in length, comprising the amino acid sequence of SEQ ID NO: 31 , in which Xaa⁸ is I or L; Xaa¹⁰ is V, F or A, Xaa¹¹ is M or L, Xaa¹⁷ is L or I, being located at the N-terminus of the fusion polypeptide;

(2) a translocation peptide consisting of 34-112 amino acid residues in length, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, 4, 20 or 41 ; and

(3) a linker, comprising SEQ ID NO: 15 linking the T cell sensitizing signal- transducing peptide and the translocation peptide;

(II) a T cell-sensitizing signal-transducing peptide consisting of 28-53 amino acid residues in length, comprising the amino acid sequence of SEQ ID NO: 31 , in which Xaa⁸ is I or L; Xaa¹⁰ is V, F or A, Xaa¹¹ is M or L, Xaa¹⁷ is L or I; and

(III) a Pseudomonas exotoxin A (PE) translocation peptide of 34-1 12 amino acid residues in length, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, 4, 20 or 41;

(c) an antigen of a pathogen or a tumor antigen; and (d) optionally an endoplasmic reticulum retention signal, located at the C -terminus of the usion protein.

2. The composition of claim 1, wherein the fusion protein comprises the endoplasmic reticulum

retention signal.

3. The composition of claim 1 or 2, wherein the antigen is selected from the group consisting of human papillomavirus (HPV) E7 protein, Hepatitis B virus (HBV) HBx protein, Hepatitis C virus (HCV) core antigen, Flu virus M2 antigen, and a tumor associated antigen.

4. The composition of claim 2, wherein the APC-binding domain or the CD91 receptor-binding domain is a Pseudomonas exotoxin A (PE) binding domain I and the protein transduction domain is the Pseudomonas exotoxin A (PE) translocation peptide.

5. The composition of claim 4, wherein the GPI-0100 is the sole adjuvant.

6. The composition as claimed in any one of claims 1 to 3, wherein 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.

7. The composition as claimed in any one of claims 1 to 3 and 6, wherein the protein transduction domain is the fusion polypeptide comprising:

(1) the T cell sensitizing signal-transducing peptide;

(2) the translocation peptide; and

(3) the linker.

8. The composition of claim 7, wherein the CpG oligonucleotide is the sole adjuvant.

9. The composition of claim 7, wherein the protein transduction domain comprises the amino acid sequence of SEQ ID NO: 30.

10. The composition of claim 1 , wherein the pathogen is 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 (HBV) and Porcine Circovirus 2 (PCV2).

11. The composition of claim 3, wherein the tumor associated antigen is selected from the group consisting of SSX2, MAGE-A3, NY-ESO-1, iLRP, WT12-281, RNF43, and CEA-NE3.

12. The composition of claim 2, wherein the protein transduction domain is the Pseudomonas

exotoxin A (PE) translocation peptide, and the fusion protein further comprises a nuclear export signal comprising the amino acid sequence of SEQ ID NO: 44, located between the antigen and the endoplasmic reticulum retention signal, or between the translocation peptide and the antigen.

13. The composition of claim 1, wherein the antigen comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 21 or 22; and

(i) 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 SEQ ID NO: 9, and the protein transduction domain is the Pseudomonas exotoxin A (PE) translocation peptide; or

(ii) 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 SEQ ID NO: 5; and the protein transduction domain comprises the sequence of SEQ ID NO: 30.

14. Use of a pharmaceutical composition as claimed in any one of claims 1 to 13 in the manufacture of a medicament for inducing an enhanced antigen specific T cell mediated immune response in a subject in need thereof.

15. The use of claim 14, wherein the pharmaceutical composition is prepared by admixing the

vaccine composition and the anti-OX40 antibody together to form the mixture for at least 8 hours before the use of the medicament for vaccination in the subject in need thereof.

16. A method for preparation of the pharmaceutical composition of claim 1 , comprising the steps of:

(1) providing the vaccine composition;

(2) providing the anti-OX40 antibody;

17. The pharmaceutical composition of claim 1, wherein the mixture is formed by admixing the vaccine composition and the anti-OX40 antibody together for at least 8 hours before use of the pharmaceutical composition for vaccination in a subject in need thereof.

18. The pharmaceutical composition of claim 17, or the use of claim 15, or the method for

preparation of claim 16, wherein the mixture is formed for at least 10 to 16 hours before the use of the medicament or the pharmaceutical composition for vaccination in the subject in need thereof.

19. The composition of claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 54 or 55.

20. The composition as claimed in any one of claims 1 to 4, wherein the T cell sensitizing signal- transducing peptide comprises the amino acid sequence of SEQ ID NO: 1 or 2.