INDUCED IMMUNOLOGICAL RESPONSE TO CANCEROUS CELLS USING VECTORS CONTAINING VIRAL GENES

Abstract

A method for treating cancer in mammals using an expression vector to transfect cancer cells in vivo and in situ. The vector includes one or more immunogenic exogenous polypeptides that are expressed by the cancer cells upon transfection. The body's immune response is triggered and directed to attack the transfected cancer cells. Once the immune response to the exogenous peptide on the cancer cells is initiated, an immune response to the other cancer associated or cancer specific antigens on or in the cancer cells takes over and eliminates all cancer cells, transfected or not.

Claims

1. A method of treating cancer in a mammal comprising transfecting tumors in vivo and in situ with a vector (plasmid or viral) that expresses an exogenous immunogenic polypeptide of viral, fungal, bacterial, non-human mammal or non-animal plant origin.

2. The method of treating cancer in a mammal of claim 1, wherein the immunogenic polypeptide is one or more polypeptides derived from the measles virus selected from the group consisting of Hemagglutinin (H), Neurominidase (N), Nucleoprotein (Np), Fusion (F), Hemagglutinin noose epitope (HNE; aa 379-410), MeV (aa 89-165) (DAG1597), MeV Hemagglutinin Mosaic (DAG1598), MeV (aa 399-525) (DAG1599), MeV Fusion Mosaic protein (DAG1600), MeV Hemagglutinin Mosaic (aa 1-30, 115-150, 379-410) (DAG2298), MeV Fusion protein (aa 399-525) (DAG3566), MeV Hemagglutinin Mosaic (aa 106-114, 519-550) (DAG3567), MeV (aa 399-525) (DAG4038), MeV Hemagglutinin Fusion Protein (aa 399-525) (DAG4317), MeV Nucleocapsid (aa 89-165) (DAG4318), and Measles Active Nucleocapsid (DAG-P2799).

3. The method of treating cancer in a mammal of claim 1, wherein the immunogenic polypeptide is one or more polypeptides derived from the Epstein Barr virus selected from the group consisting of EBV Mosaic EBNA1 protein [GST] (DAG1577), EBV NA1 (aa 1-90, 408-498) [GST] (DAG2362), EBV gp350/220, EBV P18 Mosaic protein (DAG1582), EBV BFRF3 [GST] (DAG1848), EBV BMRF1 [GST] (DAG1849), EBV EBNA1 [GST] (DAG1850), EBV Early Antigen (aa 306-390) (DAG2003), EBV Glycoprotein H(DI-II), gL, Gp 42 (aa 25-137)(Ectodomain) [His] (DAG2016), EBV Glycoprotein H(DI-III), and gL, Gp 42 (aa 31-223)(Ectodomain) [His] (DAG2017).

4. A method of treating cancer in a mammal comprising transfecting tumor cells in vivo using a vector including and expressing an exogenous immunogenic polypeptide of viral, fungal, non-human mammal or non-animal plant origin that also includes an immunostimulatory moiety such that both the immunogenic polypeptide and the immunostimulatory entity are co-expressed at the same time in the same transfected cells.

Description

DETAILED DESCRIPTION

[0017] The invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting.

[0018] The disclosed subject matter is described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the various embodiments of the subject disclosure. It may be evident, however, that the disclosed subject matter may be practiced without these specific details. In other instances, well-known structures and devices are shown in block diagram form in order to facilitate describing the various embodiments herein. Various embodiments of the disclosure could also include permutations of the various elements recited in the claims as if each dependent claim was a multiple dependent claim incorporating the limitations of each of the preceding dependent claims as well as the independent claims. Such permutations are expressly within the scope of this disclosure.

[0019] Unless otherwise indicated, all numbers expressing quantities of ingredients, dimensions, reaction conditions and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. The term “a” or “an” as used herein means “at least one” unless specified otherwise. In this specification and the claims, the use of the singular includes the plural unless specifically stated otherwise. In addition, use of “or” means “and/or” unless stated otherwise. Moreover, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one unit unless specifically stated otherwise.

[0020] Various embodiments of the disclosure could also include permutations of the various elements recited in the claims as if each dependent claim was a multiple dependent claim incorporating the limitations of each of the preceding dependent claims as well as the independent claims. Such permutations are expressly within the scope of this disclosure.

[0021] Disclosed are therapeutic methods for use in stimulating anticancer immunity in cancer patients. Vectors such as plasmids are constructed to express immunogenic polypeptides such as viral proteins, immunogenic regions of viral proteins, or any other non-human immunogenic protein. These vectors are injected intratumorally, i.e. directly into a tumor. The vectors transform cancerous cells, causing them to express the immunogenic polypeptides. These peptides are then presented on the exterior of the cancerous cells, thereby generating an immune response.

[0022] The vector expressed polypeptides may be immunogenic proteins or immunogenic fragments of proteins commonly found in viruses that humans or other mammals are commonly vaccinated for, such as for example measles, mumps and/or rubella. As a result, the body's natural response, heightened by the vaccination, is utilized to attack the cancerous cells. In addition, the use of viral peptides also triggers the antiviral cytotoxic immunological responses, further increasing the immune systems response time, strength and efficacy. The use of a body's natural immunological response to treat cancer also avoids the undesirable side effects of other anticancer therapies.

[0023] Suitable polypeptides from the measles virus include: Hemagglutinin (H), Neurominidase (N), Nucleoprotein (Np), Fusion (F), Hemagglutinin noose epitope (HNE; aa 379-410), MeV (aa 89-165) (DAG1597), MeV Hemagglutinin Mosaic (DAG1598), MeV (aa 399-525) (DAG1599), MeV Fusion Mosaic protein (DAG1600), MeV Hemagglutinin Mosaic (aa 1-30, 115-150, 379-410) (DAG2298), MeV Fusion protein (aa 399-525) (DAG3566), MeV Hemagglutinin Mosaic (aa 106-114, 519-550) (DAG3567), MeV (aa 399-525) (DAG4038), MeV Hemagglutinin Fusion Protein (aa 399-525) (DAG4317), MeV Nucleocapsid (aa 89-165) (DAG4318), and Measles Active Nucleocapsid (DAG-P2799).

[0024] Suitable polypeptides from the Epstein Barr virus include: EBV Mosaic EBNA1 protein [GST] (DAG1577), EBV NA1 (aa 1-90, 408-498) [GST] (DAG2362), EBV gp350/220, EBV P18 Mosaic protein (DAG1582), EBV BFRF3 [GST] (DAG1848), EBV BMRF1 [GST] (DAG1849), EBV EBNA1 [GST] (DAG1850), EBV Early Antigen (aa 306-390) (DAG2003), EBV Glycoprotein H(DI-II), gL, Gp 42 (aa 25-137)(Ectodomain) [His] (DAG2016), EBV Glycoprotein H(DI-III), and gL, Gp 42 (aa 31-223)(Ectodomain) [His] (DAG2017).

[0025] Suitable polypeptides from the Varicella Zoster virus include: VZV Glycoprotein E [GST] (DAG518), VZV Glycoprotein (DAG3110), VZV Glycoprotein E (aa 48-135) [GST] (DAG3633), VZV Envelope glycoprotein E (aa 48-135) (DAG-P2161), VZV Active Glycoprotein E (aa 48-135) (DAG-P2598), and VZV ORF9 (DAG-P2622).

[0026] Suitable polypeptides from the Rubella virus include: RuV E1 glycoprotein, RuV Envelope Protein 1 (DAG3134), RuV Nucleocapsid (full length) (DAG-P2986), RuV Capsid (aa 1-123) (DAG1979), RuV E1 protein (aa 157-276) [His] (DAG2000), RuV E2 Protein (aa 31-105) [His] (DAG2002), RuV E1 Mosaic (aa 157-176) (DAG3583), RuV Capsid C [GST] (DAG4369), RuV E1 protein (E1) [GST] (DAG472), RuV E1 Mosaic [His] (DAG488), RuV Capsid (DAG494), and RuV E1 Mosaic (DAG507).

[0027] Other suitable viral poly peptides include: Mumps Fusion (F) and Hemagglutinin/neurominadase (HN), Vaccinia virus H3L (an intracellular mature virion envelope protein) and virus B18R protein, Lymphocytic choriomeningitis virus (LCMV) cytotoxic T lymphocyte (CTL) epitope (NP118-126), Human Cytomegalovirus (HCMV) fusion protein gB, and two highly conserved anti-parallel (3-strands on the pre-fusion viral F protein of the Human respiratory syncytial virus (HRSV).

[0028] The immune response generated by these polypeptides will initially be directed against the viral antigen expressed on the surface of the tumor. However, the immune system cells, e.g. T cells, dendritic cells, NK cells and others, that infiltrate will begin to recognize the many different tumor associated and tumor specific antigens that make the cancer cell different. This is known as epitope spreading and is well documented. Epitope spreading allows these primed immune cells to travel throughout the body and recognize and destroy distant metastatic cancer cells that do not express the viral antigens thus eliminating all cancer cells and tumors throughout the body.

[0029] One advantage of using viral antigens over bacterial or endogenous (even tumor specific or associated antigens) is that the immune response to viruses is predominantly a cytotoxic T lymphocyte (CTL) response comprised of CD8+ and CD4+ and other effector cells which are the cells that are most effective at inducing cytolysis of tumor cells. This is superior to other regimens such as bacterial antigen expressing systems which primarily induce a humoral or B cell response. While the hum oral response may be somewhat effective, it is hypothesized that an anti-tumor response that is predominantly CTL mediated will be significantly more effective.

[0030] In some embodiments, viral fusion proteins that add a transmembrane tail may be incorporated into the expression vector. Those skilled in the art will appreciate that these viral fusion proteins may be anchored to the surface of the cancer cell and the therefore will be identified by memory T cells as a result of a previous vaccination. These viral fusion proteins may also migrate to a cancerous cell's membrane without the assistance of the major histocompatibility complex (MHC) which is compromised in some cancer cell types.

[0031] In other embodiments, the viral polypeptide incorporated into the expression vector is a viral protein known to be found on the membrane of an infected cell. Such polypeptides may use a host transport mechanisms that the virus is known to employ as a infection strategy.

[0032] In other embodiments, the immunogenic protein that is incorporated in the expression vector may be of bacterial origin. This may include modified immunogens such as tetanus toxoid. The obvious utility of this being that many people are already vaccinated against it.

[0033] In other embodiments the polypeptide is an antigenic portion of an MHC molecule from a non-human mammal. It is known that disparate MHC matching causes a robust immune response and therefore these polypeptides will elicit a robust immune response.

[0034] In other embodiments the polypeptide is from a non-mammalian animal source that is known to induce a potent immune response. These may be derived from species known to induce a cytotoxic adaptive immune response.

[0035] In other embodiments the polypeptide is from a non-animal source that is known to induce a potent immune response. These may be derived from plant or fungal species known to induce a cytotoxic adaptive immune response.

[0036] The expression of the immunogenic polypeptide, whether of viral origin or another source as described above, in tumor cells greatly increases tumor cell immunogenicity, which in turn promotes activation of both innate and adaptive antitumor immunity. A clinically meaningful response against tumor cells, such as tumor regression/prevention of recurrence results from in vivo priming and activation of the immune system.

[0037] The disclosed anticancer vector can also be used in conjunction with monoclonal antibodies to checkpoint inhibitory molecules such as CTLA-4, PD-1, PD-L1, PD-L2, LAG3, TIM3, TIGIT; antibodies to costimulatory molecules such as CD40, OX40; antibodies capable of regulating T regs such as anti-GITR and pan anti-BCL-2; or cytokines such as IL-2, TNF-α, IFN-γ, IFN-β, and TLR agonists administered in normal fashion for as the currently are for anti-cancer therapy.

[0038] The anticancer vector coding for the antigenic polypeptide can also comprise additional nucleic acid sequences that express immunologic molecules such as cytokines IL-2, IL-12, IL-18, CD-40L, CTLA-4 ligand and MHC genes. In this embodiment both the antigenic polypeptide and the costimulatory molecule are engineered into the same plasmid. Thus, a single vector plasmid will lead to expression of the antigenic polypeptide which will induce a priming response and the costimulatory molecule will overcome the inhibition that tumor cells exhibit.

[0039] Anti-Cancer Vectors. A wide variety of vectors, for example plasmids, may be utilized to deliver viral polypeptide genes into cancerous cells in accordance with principles of the invention. The vectors may include promoter and enhancer regions to increase expression of the exogenous polypeptides.

[0040] Delivery of Anticancer Vector into a Solid Tumor In Vivo. The anticancer vectors are injected directly into a tumor where they enter cancerous cells.

[0041] Whereas, the present invention has been described in relation to the drawings attached hereto, other and further modifications, apart from those shown or suggested herein, may be made within the spirit and scope of this invention. Those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. Descriptions of the embodiments shown in the drawings should not be construed as limiting or defining the ordinary and plain meanings of the terms of the claims unless such is explicitly indicated. The claims should be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.