WO2024149832A1 - Sarna (vrp) modifié recombinant pour vaccin contre le cancer - Google Patents
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- C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
- C12N2740/13034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C12N2770/00011—Details
- C12N2770/36011—Togaviridae
- C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
- C12N2770/36141—Use of virus, viral particle or viral elements as a vector
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- C12N2820/00—Vectors comprising a special origin of replication system
- C12N2820/60—Vectors comprising a special origin of replication system from viruses
Definitions
- a vaccine comprises a nucleic acid molecule that produces a cancer protective response in a patient.
- An embodiment provides the vaccine may be delivered by a self-amplifying RNA (saRNA), e.g. by an alphavirus replicon (VRP) and in a preferred embodiment, an alphavirus vector replicon particle vaccine is administered to the patient.
- saRNA self-amplifying RNA
- VRP alphavirus replicon
- a saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA) and a nucleic acid encoding IL-12.
- TAA tumor-associated antigen
- the invention thus relates also to compositions comprising a saRNA, e.g. a VRP encoding a IL-12, and a tumor-associated antigen (TAA).
- the present invention also relates to vaccination methods, in particular homologous prime-boost vaccination regimes, employing two viral vector compositions. More particularly, the invention relates to a recombinant VRP (saRNA) for use in a homologous prime-boost vaccination regime.
- saRNA recombinant VRP
- the invention also relates to products, methods and uses thereof, e.g., suitable to induce a protective immune response in a subject.
- a vaccine is one of the most efficacious, safe and economical strategies for preventing disease and controlling the spread of disease.
- Conventional vaccines are a form of immunoprophylaxis given before disease occurrence to afford immunoprotection by generating a strong host immunological memory against a specific antigen.
- the primary aim of vaccination is to activate the adaptive specific immune response, primarily to generate B and T lymphocytes against specific antigen(s) associated with the disease or the disease agent.
- cancer vaccines aim to generate immune responses against cancer tumor- associated antigens.
- Cancers can be immunogenic and can activate host immune responses capable of controlling the disease and causing tumor regression. However, cancer at the same time can be specifically and nonspecifically immunosuppressive and can evade the host's immune system.
- Many protein/glycoprotein tumor-associated antigens have been identified and linked to certain types of cancer. Her-2-neu, PSA, PSMA, MAGE-3, MAGE-1, gp100, TRP-2, tyrosinase, MART-1, ⁇ -HCG, CEA, Ras; B- catenin, gp43, GAGE-1, BAGE-1, MUC-1,2,3, and HSP-70 are just a few examples. Natural and recombinant cancer protein antigen vaccines are subunit vaccines.
- these subunit vaccines contain defined immunogenic antigens at standardized levels.
- the key problem with such vaccines is finding the right adjuvant and delivery system.
- purification of natural or recombinant tumor antigens is tedious and not always logistically practical.
- Protein cancer vaccines require culturing tumor cells, purifying tumor antigens, or producing specific peptides or recombinant proteins.
- vaccines that are made solely from tumor protein/peptides pose intrinsic problems in that they can be limited in the ability to be directed into the correct antigen presentation pathways or may not be recognized by the host due to host major histocompatibility complex (MHC) polymorphisms.
- MHC major histocompatibility complex
- Vaccines which include nucleic acid encoding the tumor antigens rather than vaccines comprising the antigen itself, address some of these problems. To date these approaches have shown the most promise in pre-clinical and clinical testing. Amongst the current technologies being applied to cancer vaccination, two particular systems have shown significant potential for application in this field. The first is delivery of TAAs using viral vectors, including but not limited to adenoviral, adeno associated virus, retroviral, poxviruses, flaviviruses, picornaviruses, herpesviruses and alphaviruses (see WO 99/51263).
- viral vectors including but not limited to adenoviral, adeno associated virus, retroviral, poxviruses, flaviviruses, picornaviruses, herpesviruses and alphaviruses.
- the second is vaccination with tumor cell protein or RNA using ex vivo derived dendritic cells as the delivery vehicle for transfer and expression of the TAAs into the host (Heiser et al., 2002. J. Clin. Inv. 109:409-417 and Kumamoto et al., 2002. Nature Biotech.20:64-69).
- naked DNA, RNA, viral and bacterial vectors have been tested for their ability to induce cancer specific responses against a tumor antigen. Attempts to augment the immune responses elicited to naked nucleic acid vectors include the use of self- replicating viral vectors delivered in the form of naked RNA or DNA (Ying et al., 1999, Nature Medicine, 5:823-827).
- Alphaviral vector delivery systems have been identified as attractive vaccine vectors for a number of reasons including: high expression of heterologous gene sequences, the derivation of non-replicating (alpha)virus replicon particles (ARP) with good safety profiles, an RNA genome which replicates in the cytoplasm of the target cell and negates the chance of genomic integration of the vector, and finally the demonstration that certain alphaviral vectors are intrinsically targeted for replication in dendritic cells and thus can generate strong and comprehensive immune responses to a multitude of vaccine antigens (reviewed in Rayner, Dryga and Kamrud, 2002, Rev. Med. Virol.12:279-296).
- the Alphavirus genus includes a variety of viruses, all of which are members of the Togaviridae family.
- the alphaviruses include Eastern Equine Encephalitis Virus (EEE), Venezuelan Equine Encephalitis Virus (VEE), Everglades Virus, Mucambo Virus, Pixuna Virus, Western Equine Encephalitis Virus (WEE), Sindbis Virus, Semliki Forest Virus, Middleburg Virus, Chikungunya Virus, O'nyong-nyong Virus, Ross River Virus, Barmah Forest Virus, Getah Virus, Sagiyama Virus, Bebaru Virus, Mayaro Virus, Una Virus, Aura Virus, Whataroa Virus, Babanki Virus, Kyzylagach Virus, Highlands J Virus, Fort Morgan Virus, Ndumu Virus, and Buggy Creek Virus.
- EEE Eastern Equine Encephalitis Virus
- VEE Venezuelan Equine Encephalitis Virus
- WEE Western Equine Encephalitis Virus
- Sindbis Virus Semliki Forest Virus, Middleburg Virus
- the viral genome is a single-stranded, messenger-sense RNA, modified at the 5′-end with a methylated cap and at the 3′-end with a variable-length poly (A) tract.
- the capsid is surrounded by a lipid envelope covered with a regular array of transmembrane protein spikes, each of which consists of a heterodimeric complex of two glycoproteins, usually E1 and E2. See Pedersen et al., J. Virol 14:40 (1974).
- Sindbis and Semliki Forest viruses are considered the prototypical alphaviruses and have been studied extensively. See Schlesinger, The Togaviridae and Flaviviridae, Plenum Publishing Corp., New York (1986).
- the VEE virus has also been extensively studied. See, e.g., U.S. Pat. No.5,185,440, and other references cited herein. The studies of these viruses have led to the development of techniques for vaccination against the alphavirus diseases and against other diseases through the use of alphavirus vectors for the introduction of foreign DNA encoding antigens of interest. See U.S. Pat. No. 5,185,440 to Davis et al., and PCT Publication WO 92/10578.
- Sindbis virus constructs express a truncated form of the influenza hemagglutinin protein.
- Another approach is the use of infective, propagation-defective alphavirus particles, as described in U.S. Pat. No.6,190,666 to Garoff et al., U.S. Pat. Nos.5,792,462 and 6,156,558 to Johnston et al., U.S. Published Application No. 2002/0015945 A1 (Polo et al.), U.S. Published Application No.2001/0016199 (Johnston et al.), Frolov et al., 1996, Proc. Natl. Acad. Sci.
- SFV Semliki Forest Virus
- VRP vaccines are capable of boosting adaptive antigen-specific immunity and augmenting anti-tumor responses (de Mare A et al., Gene Ther. 2008;15(6):393–403; Lambeck AJ et al., Vaccine. 2010; 28(26):4275–82; Riezebos-Brilman A et al., Gene Ther. 2007;14(24):1695–704).
- VRPs have been widely used and have demonstrated anti-tumor immunity against a variety of foreign and self- antigen targets expressed in cancer.
- VEE VRP vaccines to induce immunity against a variety of melanoma differentiation antigens (MDAs) including Tyr, gp100 and TRP-2.
- MDAs melanoma differentiation antigens
- VRP-TRP2 vaccination was the most potent to activate TRP-2 specific cellular and humoral immunity that resulted in a strong prophylactic and therapeutic effect on B16 murine melanoma tumors (Avogadri et al., Plos One 2010; 5(9):e12670).
- TAAs tumor associated antigens
- VEE VRP encoding rat ErB2 (VRP-neu) vaccination in combination with chemotherapy, resulted in significantly delayed tumor progression in mice, due to VRP- neu vaccination-enhanced infiltration of antigen specific T cells in the tumors (Eralp et al., Breast Cancer Res 2004; 6(4):R275-R283).
- VRP-DCs VRP-induced DCs
- VRP-DCs VRP-transduced DCs
- a single VRP-DCs vaccination was able to promote the regression of large established tumors in mice. This antitumorigenic effect was completely abrogated upon depletion of CD4 + T cells suggesting that VRP-DC vaccines induced immunity against established tumors is dependent on CD4 + T cell and B cell responses (Moran et al., Vaccine 2007; 25(36):6604-6612.
- VRP vaccines in combination with different immunomodulatory molecules have been also explored.
- An in vivo study has shown that the immunogenicity and efficacy of VRP-TRP2 was improved in combination with either antagonist anti-CTLA or agonist anti-GITR immunomodulatory monoclonal antibodies (mAbs), due to enhanced TRP-2-specific cellular and humoral responses (Avogadri et al., Cancer Immunol Res. 2014; 2(5):448-458).
- VRP-CEA vaccine was used to elicit immune response against carcinoembryonic antigen (CEA), which is often overexpressed in colorectal cancer
- CEA carcinoembryonic antigen
- Tumor-bearing mice were vaccinated with VRP-CEA in a combination with IL-12 encoding VRP (VRP-IL12).
- This combined vaccination elicited a strong CEA-specific B- cell and T cell responses, and prolonged survival compared to the single injection of these vectors (Osada et al, Cancer Immunol Immunother. 2012; 61(11):1941-51).
- SFV-IL12 SFV-IL12 saRNA electroporation
- IL-12 is a type 1 cytokine that has also been investigated as a monotherapy treatment for cancer, but early clinical trials found dose-limiting toxicity (see, e.g., Nguyen et al.
- the tumor microenvironment is composed of a large variety of cell types, from immune cell infiltrates to cancer cells, extracellular matrix, endothelial cells, and other cellular components and factors that influence tumor progression. This complex and entangled equilibrium changes not only from patient to patient, but within lesions in the same subject (Jiménez-Sánchez et al. (2017) Cell 170(5): 927-938). Stratification of tumors based on Tumor Infiltrating Lymphocytes (TIL) and Programmed Death Ligand 1 (PD- L1) expression emphasizes the importance of an inflammatory environment to achieve objective responses against cancer (Teng et al. (2015) Cancer Res.75(11): 2139-45).
- TIL Tumor Infiltrating Lymphocytes
- PD- L1 Programmed Death Ligand 1
- the present invention relates to a self-amplifying RNA (saRNA) for use in the treatment of tumors, comprising a nucleic acid encoding a tumor-associated antigen (TAA); and a nucleic acid encoding IL-12, wherein the intratumoral administration of the saRNA increases an inflammatory response in a tumor, reduces the growth rate and/or size of the tumor, and/or increases overall survival of the subject as compared to a non- intratumoral injection of said saRNA or an injection of a saRNA that does not comprise a nucleic acid encoding IL-12; and wherein the saRNA is administered intratumorally.
- saRNA self-amplifying RNA
- the invention also provides methods of use and/or treatment with the saRNA in which one or more saRNAs of the invention is administered intratumorally, intravenously, or intraperitoneally to a subject having tumors.
- the saRNAs of the invention are used to prepare a medicament to increase the immune response of a subject to a tumor.
- the saRNAs of the invention are used to prepare a medicament for intratumoral injection to increase the immune response of the subject to the injected tumor; in some embodiments, injection of the medicament into the tumor may decrease the size and/or growth rate of the injected tumor and may also decrease the size and/or growth rate of other tumors that were also present in the subject but that were not intratumorally injected with the medicament (i.e., with the saRNAs).
- the subject has peritoneal tumors and the medicament is for intraperitoneal injection, whereby an immune response to peritoneal tumors is stimulated or enhanced.
- the invention provides an intravenously or intratumorally administered saRNA comprising a nucleic acid encoding a TAA and a nucleic acid encoding IL-12, optionally in combination with CD40L.
- the invention provides an intratumorally and/or intravenously administered saRNA comprising nucleic acids encoding a TAA and IL-12; in other embodiments, the invention provides a combination of saRNAs, one of which encodes IL-12, and at least one of which also encodes a TAA. This combination of saRNAs is administered to a subject so that for some period of time, they are present in the subject together.
- Figure 1A, 1B, and 1C show the expression of model antigen Ovalbumin (OVA) in two different murine cancer cell lines (4T1 and CT26) as well as murine fibroblast cell line (A31) in different time points after infection with VEEV-VRP-BFP-OVA (VRP-BN001) or SFV-VRP-BFP-OVA (VRP-BN010).
- VEEV-VRP-BFP-OVA VRP-BN001
- SFV-VRP-BFP-OVA VRP-BN010
- Cells were seeded in 12-well plates and incubated overnight (O/N). Next day they were either treated with mock (TNE Buffer) or infected with 30 TU/cell (cancer cells) or 10TU-30TU-100TU/cell (fibroblasts) of VEEV-VRP- OVA-BFP and SFV-VRP-BFP-OVA.
- FIG. 1A and 1B Concentration (ng/ml) of OVA in the supernatants of 4T1 cells and CT26 cells infected with 30TU/cell of VRP-BN001 and VRP-BN010 is shown.
- Figure 2A and 2B show the effect of VEEV-VRP-OVA-BFP and SFV-VRP-BFP-OVA infections on the viability of different murine cell lines. Cells were seeded in 96-well plates and incubated O/N. Next day some cells were left without any treatment and kept in fresh, regular culture media (+10% FCS).
- Figure 3A and 3B show Ovalbumin (OVA) mRNA expression in the tumor and tumor draining lymph node (TdLN) of 4T1 tumor-bearing mice 6 hours after intratumoral (IT) administration of VEEV-VRP-OVA-BFP.
- 4T1 tumor cells were subcutaneously (SC) injected into the right flank of Balb/c mice.
- FIG. 4A and 4B show the biodistribution of VEEV-VRP-OVA-BFP in the tumor and tumor draining lymph node (TdLN) of B16.F10 tumor-bearing mice 6 hours after IT immunization.
- FIG. 4A and 4B Representative images of the sections stained with DAPI, FITC labelled CD45.2 antibody, BFP binding unlabelled antiTagRFP antibody + AF647-labelled 2 nd antibody or merged images, respectively, are displayed for tumor and TdLN. White arrows indicate BFP + cells on the images.
- Figure 5A shows that IT injection with VRP-BN005 (VRP-Gp70) induces shrinkage or complete rejection of CT26 colon carcinomas.
- Balb/c mice were inoculated subcutaneously with 5x10 5 CT26.WT (wild type) cells.12 days later, mice were grouped and injected IT with either saline or 1x10 8 TU VRP-Gp70.
- mice received additional (“boost”) IT immunizations at days 4 and 7 (vertical dotted lines). Tumor growth was monitored at regular intervals. Number of cured mice is indicated in the lower right corner.
- Figure 5B and 5C show that IT injection of VRP- BN005 (VRP-Gp70) induces peripheral antigen-specific CD8 + T cell responses. Mice were treated as explained in Figure 5A. Two days after the last IT immunization, blood was withdrawn and subjected to peptide restimulation.
- Figure 5B Percentage of CD8 + T cells among alive counterparts and
- Figure 6A shows that repetitive IT injection with VRP-BN005 (VRP-Gp70) results in temporary tumor growth control in B16.F10 melanoma-bearing mice.
- mice received 5x10 5 B16.F10 melanoma cells on the right flank via subcutaneous injection.7 days later, mice were grouped and injected IT either with saline or 1x10 8 TU VRP-Gp70. This day was accepted as day 0. Mice received additional (“boost”) IT immunizations at days 5 and 8 (vertical dotted lines). Tumor growth was monitored at regular intervals.
- Figure 6B and 6C indicate that IT injection of VRP-BN005 (VRP-Gp70) induces peripheral antigen-specific CD8 + T cell responses. Mice were treated as explained in Figure 6A. Four days after the last IT immunization, blood was withdrawn and subjected to peptide restimulation.
- Figure 7A-7C indicate rejection of tumors after rechallenging by previously cured mice upon IT VRP-BN005 (VRP-Gp70) treatment. Na ⁇ ve Balb/c mice and Balb/c mice that were cured of CT26.WT tumors were rechallenged with 5x10 5 CT26.WT tumor cells in the opposite flank where the primary tumor was placed, via subcutaneous injection. Tumor growth was measured at regular intervals.
- FIG. 7A Tumor free survival of mice upon time is shown.
- Figure 7B Percentage of CD8 + T cells among alive counterparts and
- FIGS. 8A-8E show that IT VRP-BN005 (VRP-Gp70) injection resulted in long-term protection in mice against tumor re-challenge via inducing antigen-specific CD8 + T cell memory response.
- Na ⁇ ve and cured Balb/c mice were rechallenged with subcutaneous CT26.WT injection as explained in Figure 8A-8E.
- Thirty-three days after the rechallenge mice were sacrificed. Spleen, tumor draining lymph node (TdLN), non-draining lymph node (non-dLN) from the injection site of both na ⁇ ve and cured mice, and skin from cured mice were harvested. Cells were isolated, stained and analyzed using flow cytometry.
- TdLN tumor draining lymph node
- non-dLN non-draining lymph node
- Antigen-specific CD8 + T cells were identified via AH-1 pentamer staining.
- Figure 8A Percentage of CD44 + AH-1 + cells
- Figure 8B Percentage of CD127 + CD62L + central memory T cells (TCM)
- Figure 8C Percentage of CD127 + CD62L- effector memory T cells (TEM) among live CD8 + T cells in TdLN, non-dLN, spleen and skin are displayed.
- Figure 8D and 8E Percentage of CD127 + CD62L- CD69 + CD103- and CD127 + CD62L- CD69 + CD103 + tissue resident memory T cells (TRM) among live CD8 + T cells in the skin of cured mice are shown. Data are expressed as Mean ⁇ SEM.
- n 2-5 mice per group.
- Figure 9A-9D indicate that IT VRP-BN005 (VRP-Gp70) injection induces CD4 + T cell memory response.
- Na ⁇ ve and cured Balb/c mice were rechallenged with subcutaneous CT26.WT injection as explained in Figure 8. Thirty-three days after the rechallenge, mice were sacrificed. Spleen, tumor draining lymph node (TdLN), non-draining lymph node (non-dLN) from the injection site of both na ⁇ ve and cured mice, and skin from cured mice were harvested. Cells were isolated, stained and analyzed using flow cytometry.
- FIG. 9A Percentage of CD127 + CD62L + central memory T cells (TCM)
- Figure 9B Percentage of CD127 + CD62L- effector memory T cells (TEM) among live CD4 + T cells in TdLN, non- dLN, spleen and skin are shown.
- FIGS. 10A and 10B show the anti-tumorigenic effect and alterations in the number/percentage of lymphocytes in the tumor microenvironment (TME) of B16.F10 melanoma-bearing mice induced by a single IT injection of VRP-BN005 (VRP-Gp70).
- TME tumor microenvironment
- C57BL/6 mice received 5x10 5 B16.F10 cells on the right flank via subcutaneous injection. Seven days later when tumors measured above 65 mm 3 in size, mice were grouped and intratumorally injected with either TNE buffer or 1x10 8 TUVRP-Gp70. One day and seven days after immunization, mice were sacrificed.
- Tumors were harvested, digested with Collagenase/DNAse, cells were stained and analyzed by flow cytometry.
- Figure 10A Tumor weights on day one and day seven are shown.
- FIGS 11A-11H show that VRP encoding IL-12 (VRP-IL12; VRP-BN006) induces activation of immune cells stronger than VRP (VRP-BN015) in vitro.1x10 6 splenocytes (from C57BL/6 wild type mouse) were infected with 10, 30 and 100 TU/cell VRP-BN015 or VRP-BN006.
- Recombinant IL-12p70 (rIL12p70) was used at a final concentration of 200 ng/ml.
- unstimulated cells were included in the experiment. 18h after infection cells were harvested, stained with fluorochrome labelled antibodies and analysed by flow cytometry to assess activation and cytotoxicity of CD8 + T cells and NK cells.
- FIG. 11A Percentage of CD69 + , Granzyme B + or IFN ⁇ + cells among NK cells is shown.
- Figure 11B Percentage of CD69 + , Granzyme B + or IFN ⁇ + cells among CD8 + cells is shown.
- Figure 11C-H Concentration (pg/ml) of IL-12p70, IFN ⁇ , IL-10, IL-6, GM-CSF and TNF ⁇ , respectively, is indicated. Data are expressed as Mean ⁇ SEM.
- Figures 12A-12F indicates that repetitive IT VRP-BN006 (VRP-IL12) injection induces a stronger anti-tumorigenic response and prolonged survival in B16.F10 tumor bearing mice compared to VRP (VRP-BN015) injections, without causing any IL-12 related cytotoxicity.
- C57BL/6 mice received 5x10 5 B16.F10 cells subcutaneously.6 days later, mice were grouped and received 1x10 8 TU VRP or increasing titers (1x10 6 TU, 1x10 7 , 1x10 8 TU) of VRP-IL12 via intratumoral (IT) injections. Control mice were IT treated with TNE buffer.
- FIG. 12A-E Tumor mean in diameter (mm) is shown for the mice treated with TNE buffer, 1x10 8 TU VRP-BN015, 1x10 6 TU VRP-BN006, 1x10 7 TU VRP- BN006, 1x10 8 TU VRP-BN006, respectively.
- Figures 13A-13D show that repetitive IT injection of VRP-BN005 (VRP-Gp70) and VRP- BN006 (VRP-IL12) combination elicits a more potent anti-tumorigenic immune response and enhanced the survival compared to the IT injection of VRP-BN015 (VRP) and VRP- BN005 (VRP-Gp70) combination in B16.F10 melanoma bearing mice.
- C57BL/6 mice received 5x10 5 B16.F10 cells subcutaneously.
- mice 8 days later, mice were grouped and immunized IT with VRP-BN015 (VRP) plus VRP-BN005 (VRP-Gp70) combination or VRP-BN005 (VRP-Gp70) plus VRP-BN006 (VRP-IL12) combination.
- the total VRP titer of these combinations was 1x10 8 TU.
- Control mice were IT administered with the TNE buffer. This day was accepted as day 0.
- Boost immunizations were repeated on days 5 and after prime immunization (vertical dotted lines). Tumor growth was measured at regular intervals.
- FIG. 13A-C Tumor mean in diameter (mm) is shown for the mice treated with TNE buffer, VRP-BN015 (VRP) and VRP-BN005 (VRP- Gp70) combination (in total 1x10 8 TU), VRP-BN005 (VRP-Gp70) and VRP-BN006 (VRP- IL12) combination (in total 1x10 8 TU) on days 0,5 and 8.
- FIGS 14A-14F show that repetitive IT VRP-BN006 (VRP-IL12) injection on a weekly schedule can induce a potent antitumorigenic immune response in B16.F10 tumor bearing mice similarly to VRP-IL12 IT injections with shorter intervals.
- C57BL/6 mice received 5x10 5 B16.F10 cells subcutaneously.7 days later, mice were grouped and IT immunized with 1x10 8 TU VRP or VRP-IL12. This day was accepted as day 0.
- VRP and VRP-IL12 boost immunizations were repeated either on days 5 and 8 or on days 7 and 14 after prime immunization (vertical dotted lines).
- Control mice were IT treated with TNE buffer on days 0, 5 and 8. Tumor growth was measured at regular intervals.
- FIG 14A- C Tumor mean in diameter (mm) is shown for the mice treated with TNE buffer, 1x10 8 VRP-BN015 or 1x10 8 TU VRP-BN006 on days 0,5 and 8.
- Figure 14D, E Tumor mean in diameter (mm) is shown for the mice treated with 1x10 8 VRP-BN015 or 1x10 8 TU VRP- BN006 on days 0,7 and 14.
- SEQ ID NO: 1 depict the BRP-OVA nucleotide sequence
- SEQ ID NO: 2 depict the BFP-OVA amino acid sequence
- SEQ ID NO: 3 depict the murine gp70 nucleotide sequence
- SEQ ID NO: 4 depict the murine gp70 amino acid sequence
- SEQ ID NO: 5 depicts the murine IL-12 nucleic acid sequence
- SEQ ID NO: 6 depicts the murine IL-12 amino acid sequence DETAILED DESCRIPTION OF THE INVENTION
- the saRNAs and methods of the present invention increase and enhance multiple aspects of a subject’s immune response to one or more tumors.
- the present invention demonstrates that when a saRNA comprising a nucleic acid encoding at least one tumor-associated antigen (TAA) and a nucleic acid encoding IL-12 is administered intratumorally to a subject, there is an increased anti-tumor effect realized in the subject.
- this anti-tumor effect includes, for example, a decrease in tumor size/volume, a decrease in tumor growth rate, increased overall survival rate, an enhanced CD8+ T cell response to the TAA, and enhanced inflammatory responses such as increased cytokine production in the tumor and even in some embodiments systemically in the subject, as compared to an administration of a saRNA by itself.
- a saRNA encoding IL-12 when administered in combination with a saRNA encoding TAA further increases the effectiveness of and/or enhances the immune response and therefore the treatment of a subject having tumors.
- Recombinant modified saRNA as used herein refers to an saRNA comprising at least one polynucleotide encoding a heterologous gene, such as, for example, a tumor- associated antigen (TAA).
- TAA tumor- associated antigen
- a combination comprising saRNA encoding TAA and a saRNA encoding IL-12 are present in the subject at the same time, even though they may be administered to the subject at different times and/or by different routes of administration.
- saRNAs in a combination treatment may be administered together or may be administered to the subject at separate times, so long as both are present together in the subject for a period of time (such as, for example, at least several hours, at least 12 hours, at least 24 hours, or at least 2 or more days).
- the IL-12 is encoded by the same saRNA; that is, in some embodiments, a saRNA of the invention comprises a nucleic acid encoding a TAA, and a nucleic acid encoding IL-12..
- a saRNA encoding TAA and IL-12 is injected intratumorally or intravenously into a subject having tumors.
- TAA and IL-12 are encoded by separate saRNAs, at least one of the saRNAs encodes at least one TAA, and in some embodiments both saRNAs encode a TAA.
- a saRNA encoding a TAA, and IL-12 is injected into a subject to provide the combination of saRNA-encoded TAA and IL-12. That is, in some embodiments, the IL-12 and TAA are all encoded by the same saRNA, which can be administered to a subject to stimulate an immune response.
- the invention also provides saRNAs for preparing a medicament for intratumoral or intravenous injection for the treatment of tumors and/or to increase an immune response in a subject to a tumor. In some embodiments, this medicament comprises a saRNA encoding a TAA and IL-12; and optionally the same TAA or a different TAA.
- the medicament comprises a saRNA encoding at least one TAA and IL- 12; in these embodiments, the nucleic acids encoding each of the TAA, and IL-12 may be adjacent to each other in the saRNA or may be separated by nucleic acids encoding one or more other genes, or may be inserted into different locations in the saRNA.
- TAA tumor-associated antigen
- IL-12 administered intratumorally increases and enhances the immune response of a subject to the antigen. In this manner, the invention provides improved treatment of a subject having at least one tumor, including for example a human cancer patient.
- saRNAs and combinations thereof of the present invention caused increased inflammation in the tumor when injected intratumorally.
- data presented in the working examples herein showed that subjects cured of tumors following treatment with saRNA encoding TAA and IL-12 were more likely to reject tumors when subsequently challenged with newly implanted tumors.
- the present invention includes a method for enhancing the immune response, reducing tumor size, and/or increasing survival in a subject having a cancerous tumor, the method comprising intratumorally administering to the subject a recombinant modified saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA) and IL-12, wherein the intratumoral administration of the saRNA increases and/or enhances an inflammatory response in the tumor, decreases the size of the tumor, and/or decreases the growth rate of the tumor, and/or increases overall survival of the subject as compared to the result expected from injection of saRNA alone.
- TAA tumor-associated antigen
- this method further comprises intratumorally administering to the subject a saRNA comprising a nucleic acid encoding a TAA that is the same or different from the TAA encoded by another saRNA administered to said subject.
- a saRNA comprising a nucleic acid encoding a TAA that is the same or different from the TAA encoded by another saRNA administered to said subject.
- the TAA may be encoded by either the saRNA that also encodes IL-12 or the saRNA that encodes TAA only.
- the present invention includes a method for increasing and/or enhancing the immune response, reducing tumor size, and/or increasing survival in a subject having a tumor, the method comprising intratumorally administering to the subject a recombinant modified saRNA comprising a nucleic acid encoding a tumor- associated antigen (TAA) and a second nucleic acid encoding IL-12, wherein the intratumoral administration of the saRNA increases and/or enhances an inflammatory response in the tumor, decreases the size of the tumor, decreases the growth rate of the tumor, and/or increases overall survival of the subject as compared to a non-intratumoral injection of a saRNA virus comprising a first and second nucleic acid encoding a TAA and IL-12, or as compared to an intratumoral or non-intratumoral injection of saRNA alone.
- TAA tumor- associated antigen
- the present invention includes a method for enhancing the immune response, reducing tumor size, and/or increasing survival in a subject having a cancerous tumor, the method comprising intratumorally and/or intravenously administering to the subject a recombinant modified saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA), a second nucleic acid encoding IL-12 wherein the administration of the saRNA enhances an inflammatory response in the cancerous tumor, increases tumor reduction, and/or increases overall survival of the subject as compared to an injection of saRNA alone or injection of a saRNA comprising a first and second nucleic acid encoding a TAA, and IL-12 antigen administered by a different route of injection (i.e., non-intratumoral or non-intravenous injection).
- a different route of injection i.e., non-intratumoral or non-intravenous injection.
- the invention includes a method for enhancing the immune response, reducing tumor size, and/or increasing survival in a subject having a tumor, the method comprising intratumorally administering to the subject a recombinant modified saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12, wherein the administration of the saRNAs enhances T cell responses specific to the TAA as compared to intratumoral injection of saRNA alone or as compared to a non-intratumoral injection of a saRNA virus comprising a first and second nucleic acid encoding a TAA and IL-12.
- TAA tumor-associated antigen
- the TAA is encoded by a second saRNA that also encodes IL-12.
- the invention includes a method for reducing tumor size, and/or increasing survival in a subject having more than one tumor, the method comprising intratumorally administering to a particular tumor in the subject a recombinant modified saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA), a second nucleic acid encoding IL-12, whereby the administration of the saRNA to said tumor decreases the growth rate and/or size of another tumor in the subject that was not injected intratumorally with said saRNA(s).
- TAA tumor-associated antigen
- the invention provides a method of stimulating an immune response against a tumor and/or decreasing the size or growth rate of a tumor comprising intratumoral injection of a different tumor.
- the present invention includes a method of inducing an enhanced inflammatory response in a cancerous tumor of a subject and/or systemically in the subject, the method comprising intratumorally administering to the subject a recombinant modified saRNA comprising a nucleic acid encoding a first heterologous tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12, wherein the intratumoral administration of the saRNA generates an enhanced inflammatory response in the tumor as compared to an inflammatory response generated by or expected to be generated by a non-intratumoral injection of a saRNA virus comprising a first and second nucleic acid encoding a heterologous tumor-associated antigen and IL-12.
- TAA tumor-associated antigen
- the present invention includes a method of inducing an increased and/or enhanced inflammatory response in a cancerous tumor of a subject, the method comprising intratumorally administering to the subject a recombinant modified saRNA comprising a nucleic acid encoding a heterologous tumor-associated antigen (TAA) and IL-12, wherein the administration of the saRNA generates an enhanced inflammatory response in the tumor as compared to an inflammatory response generated by an intratumoral or non-intratumoral injection of saRNA alone or a non- intratumoral injection of a saRNA comprising a nucleic acid encoding a heterologous tumor-associated antigen and IL-12.
- TAA tumor-associated antigen
- the present invention provides a recombinant modified saRNA for use in preparing a medicament to treat cancer or to enhance the immune response in a subject to a cancerous tumor, the saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA) and IL-12.
- TAA tumor-associated antigen
- the saRNA further comprises a third nucleic acid encoding CD40L.
- the saRNA is provided in combination with a second saRNA comprising a nucleic acid encoding CD40L and optionally a TAA that is the same or is different from the TAA encoded by the first nucleic acid.
- the present invention includes a recombinant modified saRNA for use in enhancing the immune response of a subject to a tumor, the saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12.
- the present invention includes a recombinant modified saRNA for use in preparing a medicament to treat cancer or enhance the immune response in a subject having cancer, the saRNA comprising: a first nucleic acid encoding a tumor-associated antigen (TAA); a second nucleic acid encoding IL-12.
- the saRNAs can be administered at the same time or at different times so long as they are present in the subject together for some period of time.
- the saRNAs can be administered by the same route(s) and/or location of administration or by a different location and/or route or routes of administration. That is, in some embodiments, a first saRNA is administered intratumorally to a particular tumor in the subject and a second or subsequent saRNA is administered intratumorally to a different tumor in the subject, or is administered intravenously, subcutaneously, intraperitoneally, or by some other route of administration.
- a first saRNA is administered intraperitoneally to a subject and a second or subsequent saRNA is administered by a different route of administration, e.g., is administered intravenously, subcutaneously, intratumorally, or by some other route of administration.
- the TAA encoded by at least one saRNA is selected from the group consisting of: carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine related protein 1 (TRP1), tyrosine related protein 2 (TRP2), Brachyury, Preferentially Expressed Antigen in Melanoma (PRAME), Folate receptor 1 (FOLR1), Human endogenous retrovirus-K envelope (HERV-K-env), Human endogenous retrovirus-K-gag (HERV-K-gag), and combinations thereof.
- CEA carcinoembryonic antigen
- MUC-1 mucin 1 cell surface associated
- PAP prostatic acid phosphatase
- PSA prostate specific antigen
- HER-2 human epidermal growth factor receptor 2
- PRAME Preferentially Expressed Antigen in Mel
- the TAA encoded by a saRNA is expressed by at least one tumor in the subject to be treated or is likely or is suspected to be expressed by at least one tumor in the subject to be treated.
- the compositions and methods of the present invention enhance multiple aspects of a subject’s immune response.
- the invention provides improved treatment of a subject having at least one tumor, including for example a cancer patient. More particularly, the inventors demonstrated that various embodiments of the present invention injected intratumorally caused increased inflammatory responses in the tumor and that may also be detectable in the blood serum of the subject.
- indicia of systemic inflammation can include increased production of IL-12 p70, M-CSF, and IL- 33; increased antigen-specific CD8+ T cells, increased percentages of CD8+ T cells expressing IFN-gamma and TNF-alpha, decrease in tumor size and/or growth rate, improved survival of treated subjects, and the like, and can be detected by assays known in the art by evaluating the tumor and/or the peripheral blood serum, assessing survival at regular intervals, and the like. Definitions Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary.
- a first option refers to the applicability of the first element without the second.
- a second option refers to the applicability of the second element without the first.
- a third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein.
- any of the aforementioned terms (comprising, containing, including, having), whenever used herein in the context of an aspect or embodiment of the present invention may be substituted with the term “consisting of”, though less preferred.
- Consisting of excludes any element, step, or ingredient not specified in the claim element.
- consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.
- “Mutated” or “modified” protein or antigen as described herein is as defined herein any modification to a nucleic acid or amino acid, such as deletions, additions, insertions, and/or substitutions.
- the term “antigen” includes all related epitopes of a particular compound, composition or substance.
- epitopes refers to a site on an antigen to which B- and/or T-cells respond, either alone or in conjunction with another protein such as, for example, a major histocompatibility complex (“MHC”) protein or a T-cell receptor.
- MHC major histocompatibility complex
- Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by secondary and/or tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
- An epitope typically includes at least 5, 6, 7, 8, 9, 10 or more amino acids but generally less than 20 amino acids in a unique spatial conformation.
- Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., “Epitope Mapping Protocols” in Methods in Molecular Biology, Vol.66, Glenn E. Morris, Ed (1996).
- An antigen can be a tissue-specific (or tissue-associated) antigen or a disease-specific (or disease-associated) antigen. Those terms are not mutually exclusive, because a tissue-specific antigen can also be a disease-specific antigen. A tissue-specific antigen is expressed in a limited number of tissues.
- Tissue-specific antigens include, for example, prostate-specific antigen (“PSA”).
- PSA prostate-specific antigen
- a disease-specific antigen is expressed coincidentally with a disease process, where antigen expression correlates with or is predictive of development of a particular disease.
- Disease-specific antigens include, for example, HER-2, which is associated with certain types of breast cancer, or PSA, which is associated with prostate cancer.
- a disease-specific antigen can be an antigen recognized by T-cells or B-cells.
- a malignant growth arising from a particular body tissue that has undergone characteristic loss of structural differentiation, generally accompanied by increased capacity for cell division, invasion of surrounding tissue, and the capacity for metastasis. Tumors may be benign or malignant.
- prostate cancer is a malignant neoplasm that arises in or from prostate tissue
- ovarian cancer is a malignant neoplasm that arises in or from ovarian tissue
- colon cancer is a malignant neoplasm that arises in or from colon tissue
- lung cancer is a malignant neoplasm that arises in or from lung tissue.
- Residual cancer is cancer that remains in a subject after treatment given to the subject to reduce or eradicate the cancer.
- Metastatic cancer is a cancer at one or more sites in the body other than the site of origin of the original (primary) cancer from which the metastatic cancer is derived.
- a “conservative” variant is a variant protein or polypeptide having one or more amino acid substitutions that do not substantially affect or decrease an activity or antigenicity of the protein or an antigenic epitope thereof.
- conservative substitutions are those in which a particular amino acid is substituted with another amino acid having the same or similar chemical characteristics. For example, replacing a basic amino acid such as lysine with another basic amino acid such as arginine or glutamine is a conservative substitution.
- conservative variant also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide, and/or that the substituted polypeptide retains the function of the unstubstituted polypeptide.
- Non-conservative substitutions are those that replace a particular amino acid with one having different chemical characteristics, and typically reduce an activity or antigenicity of the protein or an antigenic epitope thereof.
- conservative substitutions include the following examples: Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn ; Gln Ile Leu, Val Leu Ile ; Val Lys Arg ; Gln ; Glu Met Leu ; Ile Phe Met ; Leu ; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp ; Phe Val Ile ; Leu “A disease-associated antigen” is expressed coincidentally with a particular disease process, where antigen expression correlates with or predicts development of that disease.
- Disease-associated antigens include, for example, HER-2, which is associated with certain types of breast cancer, or prostate-specific antigen (“PSA”), which is associated with prostate cancer.
- a disease-associated antigen can be an antigen recognized by T-cells or B-cells. Some disease-associated antigens may also be tissue- specific. A tissue-specific antigen is expressed in a limited number of tissues. Tissue- specific antigens include, for example, prostate-specific antigen PSA.
- the term “tumor antigen” and “tumor-associated antigen” refers to antigens present expressed exclusively on, associated with, or over-expressed in tumor tissue.
- Exemplary tumor antigens include, but are not limited to, 5- ⁇ -reductase, ⁇ -fetoprotein (“AFP”), AM- 1, APC, April, B melanoma antigen gene (“BAGE”), ⁇ -catenin, Bcl12, bcr-abl, Brachyury, CA-125, caspase-8 (“CASP-8”, also known as “FLICE”), Cathepsins, CD19, CD20, CD21/complement receptor 2 (“CR2”), CD22/BL-CAM, CD23/Fc ⁇ RII, CD33, CD35/complement receptor 1 (“CR1”), CD44/PGP-1, CD45/leucocyte common antigen (“LCA”), CD46/membrane cofactor protein (“MCP”), CD52/CAMPATH-1, CD55/decay accelerating factor (“DAF”), CD59/protectin, CDC27, CDK4, carcinoembryonic antigen (“CEA”), c-myc, cyclooxygena
- An “adjuvant” means a vehicle to enhance antigenicity.
- An adjuvant can include: (1) suspensions of minerals (alum, aluminum hydroxide, and/or phosphate) on which antigen is adsorbed; (2) water-in-oil emulsions in which an antigen solution is emulsified in mineral oil (Freund’s incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund’s complete adjuvant) to further enhance antigenicity by inhibiting degradation of antigen and/or causing an influx of macrophages; (3) immunostimulatory substances including but not limited oligonucleotides such as, for example, those including a CpG motif can also be used as adjuvants (for example see U.S.
- affecting an immune response includes the development, in a subject, of a humoral and/or a cellular immune response to a protein and/or polypeptide produced by the saRNA or VRP and/or compositions and/or vaccines comprising the saRNA and VRP of the invention.
- a “humoral” immune response refers to an immune response comprising antibodies
- the “cellular” immune response refers to an immune response comprising T-lymphocytes and other white blood cells, especially the immunogen-specific response by HLA-restricted cytolytic T-cells, i.e., “CTLs.”
- a cellular immune response occurs when the processed immunogens, i.e., peptide fragments, are displayed in conjunction with the major histocompatibility complex.
- alphavirus has its conventional meaning in the art, and includes the various species of Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), and eastern equine encephalitis virus (EEEV).
- VEEV Venezuelan equine encephalitis virus
- WEEV western equine encephalitis virus
- EEEV eastern equine encephalitis virus
- Equine encephalitis virus (EEV) includes VEEV, WEEV and EEEV and its strains and isolates.
- the virion-like structural complex includes one or more alphavirus structural proteins embedded in a lipid envelope enclosing a nucleocapsid comprised of capsid and replicon RNA.
- the lipid envelope is typically derived from the plasma membrane of the cell in which the particles are produced.
- the alphavirus replicon RNA is surrounded by a nucleocapsid structure comprised of the alphavirus capsid protein, and the alphavirus glycoproteins are embedded in the cell-derived lipid envelope.
- replicon particles are propagation- defective (or synonymously “replication defective”), which means that the particles produced in a given host cell cannot produce progeny particles in the host cell, due to the absence of the helper function, i.e. the alphavirus structural proteins required for packaging the replicon nucleic acid.
- the replicon nucleic acid is capable of replicating itself and being expressed within the host cell into which it has been introduced.
- Replicon particles of this invention may be referred to as VEETC83 replicon particles, and this refers to particles comprising either a TC83 replicon RNA or TC83 structural proteins, or both a TC83 replicon RNA and TC83 structural proteins.
- the terms “expressed”, “express”, “expression” and the like which can be used interchangeable denote the transcription alone as well as both the transcription and translation of a sequence of interest.
- the product resulting from this expression may be either RNA (resulting from transcription alone of the sequence to be expressed) or a polypeptide sequence (resulting from both transcription and translation of the sequence to be expressed).
- expression thus also includes the possibility that both RNA and polypeptide product result from said expression and remain together in the same shared milieu. For example, this is the case when the mRNA persists following its translation into polypeptide product.
- an expression cassette is defined as a part of a vector or recombinant virus typically used for cloning and/or transformation.
- An expression cassette is typically comprised of a) one or more coding sequences (e.g., open reading frame (ORF), genes, nucleic acids encoding a protein and/or antigen), and b) sequences controlling expression of one or more coding sequences (e.g., a promoter).
- an expression cassette may comprise a 3’ untranslated region (e.g., a transcriptional terminator such as a vaccinia transcriptional terminator). “Expression cassette” can be used interchangeable with the term “transcriptional unit”.
- Formulation refers to a composition containing an active pharmaceutical or biological ingredient e.g., a saRNA of the present invention, along with one or more additional components.
- the term “formulation” is used interchangeably with the terms “pharmaceutical composition,” “vaccine composition,” and “vaccine formulation” herein.
- the formulations can be liquid or solid (e.g., lyophilized).
- the vaccine may be monovalent or polyvalent and more than one cancer protective nucleic acid molecule may be provided in the vaccine; either more than one of the selected nucleic acid molecule, or different nucleic acid molecules.
- the prime and boost vaccine will include at least one nucleic acid molecule that is the same in both the prime and the boost.
- genes are used broadly to refer to any segment of polynucleotide associated with a biological function.
- genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs or viral RNA and/or the regulatory sequences required for their expression.
- gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.
- nucleic acid “nucleotide sequence”, “nucleic acid sequence” and “polynucleotide” can be used interchangeably and refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof.
- RNA/DNA hybrids encompasses RNA/DNA hybrids.
- polynucleotides a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
- a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches.
- sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component.
- modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support.
- the polynucleotides can be obtained by chemical synthesis or derived from a microorganism.
- “operably linked” means that the components described are in relationship permitting them to function in their intended manner e.g., a promoter to transcribe the nucleic acid to be expressed.
- a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
- a promoter is operably linked to a coding sequence if the promoter is placed in a position where it can direct transcription of the coding sequence.
- operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
- Percent (%) sequence homology or identity with respect to nucleic acid sequences described herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the reference sequence (i.e., the nucleic acid sequence from which it is derived), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity or homology can be achieved in various ways that are within the skill in the art, for example, using publically available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software.
- nucleic acid sequences are provided by the local homology algorithm of Smith and Waterman, (1981), Advances in Applied Mathematics 2:482- 489. This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov (1986), Nucl. Acids Res. 14(6):6745-6763.
- “pharmaceutical”, “pharmaceutical composition” and “medicament” are used interchangeably herein referring to a substance and/or a combination of substances being used for the prevention or treatment of a disease.
- “Pharmaceutically acceptable” means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effect(s) in the subject(s) to which they are administered.
- “Pharmaceutically acceptable carriers” are for example described in Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975); Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company (1990); Pharmaceutical Formulation Development of Peptides and Proteins, S.
- parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like, as a vehicle.
- non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
- Pharmaceutical compositions can also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, pH-buffering agents and the like such as, for example, sodium acetate or sorbitan monolaurate.
- prevent”, “preventing”, “prevention”, or “prophylaxis” of a disease or infection means preventing that such disease occurs in subject (e.g., human or animal).
- Prime-boost vaccination refers to a vaccination strategy using a first, priming injection of a vaccine targeting a specific antigen followed at intervals by one or more boosting injections of the same vaccine.
- Prime-boost vaccination may be homologous or heterologous.
- a homologous prime-boost vaccination uses a vaccine comprising the same immunogen and vector for both the priming injection and the one or more boosting injections.
- a heterologous prime-boost vaccination uses a vaccine comprising the same immunogen for both the priming injection and the one or more boosting injections but different vectors for the priming injection and the one or more boosting injections.
- a homologous prime-boost vaccination may use a saRNA vector comprising the same nucleic acids expressing alphavirus antigens for both the priming injection and the one or more boosting injections.
- a heterologous prime-boost vaccination may use a saRNA vector comprising nucleic acids expressing one alphavirus protein for the priming injection and another saRNA vector expressing a second alphavirus protein not contained in the priming injection or vice versa.
- Heterologous prime-boost vaccination also encompasses various combinations such as, for example, use of a plasmid encoding an immunogen in the priming injection and use of a saRNA encoding the same immunogen in the one or more boosting injections, or use of a recombinant protein immunogen in the priming injection and use of a saRNA vector encoding the same protein immunogen in the one or more boosting injections.
- promoter denotes a regulatory region of nucleic acid, usually DNA, located upstream of the sequence of a nucleic acid to be expressed, which contains specific DNA sequence elements, that are recognized and bound e.g., by protein transcription factors and polymerases responsible for synthesizing the RNA from the coding region of the gene being promoted.
- promoters are typically immediately adjacent to the gene in question, positions in the promoter are designated relative to the transcriptional start site, where transcription of DNA begins for a particular gene (i.e., positions upstream are negative numbers counting back from -1, for example -100 is a position 100 base pairs upstream).
- the promoter sequence may comprise nucleotides until position -1.
- SEQ ID NOs: 7 or 8 are polynucleotides comprising promoters of the invention.
- the term “26S promoter” is well known to the skilled person and refers to a subgenomic promoter of a 26S RNA of an alphavirus which is usually contained in a single open reading frame (e.g., of capsid-E3-E2-6K-E1 of VEEV).
- the mRNA encoding the structural proteins of EEVs e.g., VEEV is usually transcribed from a replication intermediate and a 26S subgenomic RNA promoter.
- the nucleic acid encoding the alphavirus structural proteins i.e., the capsid, E1 glycoprotein and E2 glycoprotein, contains at least one attenuating mutation.
- helper nucleic acid(s) include at least one attenuating mutation.
- protein protein
- peptide polypeptide
- polypeptide fragment are used interchangeably herein to refer to polymers of amino acid residues of any length.
- the polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids.
- the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
- a “recombinant” when applied to a nucleic acid, vector refers to a nucleic acid, vector, or made by an artificial combination of two or more otherwise heterologous segments of nucleic acid sequence, or to a nucleic acid, vector or comprising such an artificial combination of two or more otherwise heterologous segments of nucleic acid sequence.
- the artificial combination is most commonly accomplished by artificial manipulation of isolated segments of nucleic acids, using well-established genetic engineering techniques.
- a “recombinant” saRNA as described herein refers to saRNAs that are produced by standard genetic engineering methods, i.e., saRNAs of the present invention are thus genetically engineered or genetically modified saRNAs.
- saRNA thus includes saRNAs (e.g., VRPs) which have stably integrated recombinant nucleic acid, preferably in the form of a transcriptional unit, in their genome.
- a transcriptional unit may include a promoter, enhancer, terminator and/or silencer.
- saRNAs of the present invention may express heterologous antigenic determinants, polypeptides or proteins (antigens) upon induction of the regulatory elements.
- reference sample refers to a sample which is analyzed in a substantially identical manner as the sample of interest and whose information is compared to that of the sample of interest. A reference sample thereby provides a standard allowing for the evaluation of the information obtained from the sample of interest.
- a reference sample may be identical to the sample of interest except for one component which may be exchanged, missing or added.
- the term "structural protein" of an EEV refers to a structural protein/polyprotein encoded by the RNA of an EEV (e.g., any of the WEEVs, VEEVs or EEEVs as described herein).
- the structural protein is usually produced by the virus as a structural polyprotein of five proteins i.e., C, E3, E2, 6k and E1 and is represented generally in the literature as C-E3- E2-6k-E1.
- E3 and 6k are also described as membrane translocation/transport signals for the two glycoproteins, E2 and E1.
- Nucleotide sequences encoding “structural proteins” as used herein means a nucleotide sequence encoding proteins which are required for encapsidation (e.g., packaging) of the viral genome, and include the capsid protein, E1 glycoprotein, and E2 glycoprotein.
- Structural polyprotein of EEV refers to the polyprotein C-E3-E2-6k-E1 of an EEV.
- transcription level or “protein level” related to a specific promoter as used herein refers to the amount of gene/nucleic acid product present in the body or a sample at a certain point of time.
- the transcription or protein level (e.g., transcription of nucleic acid as mRNA or protein amount translated form the mRNA) can for example be determined, measured or quantified by means of the mRNA or protein expressed from the gene/polynucleotide e.g., as encoded by the saRNA of the present invention.
- Gene expression can result in production of the protein, by transcription of the gene by RNA polymerase to produce a messenger RNA (mRNA) that contains the same protein- encoding information and translation of the mRNA by ribosomes to produce the protein.
- mRNA messenger RNA
- transcription refers to the process of copying a DNA sequence of the gene by RNA polymerase into the mRNA, using the DNA as a template.
- transcription or protein level refers to the process by which the information contained in the mRNA is used as a blueprint to synthesize the protein.
- the transcription or protein level can for example be quantified by normalizing the amount mRNA or of protein of interest present in a sample with the total amount of gene product of the same category (mRNA or total protein) in the same sample or a reference sample (e.g., taken at the same time from the same sample).
- the transcription can be measured or detected by means of any method as known in the art, e.g., methods for the indirect detection and measurement of the gene product of interest that usually work via binding of the gene product of interest with one or more different molecules or detection means (e.g., primer(s), probes, antibodies, protein scaffolds) specific for the gene product of interest. Such methods include for example RT-PCR and/or quantitative PCR.
- detection means e.g., primer(s), probes, antibodies, protein scaffolds
- RT-PCR e.g., primer(s), probes, antibodies, protein scaffolds
- the determination of the level of protein can be measured or detected by means of any known method as known to the artisan, e.g., western blot, ELISA, or mass spectrometry.
- treat means the prevention, reduction, amelioration, partial or complete alleviation, or cure of a disease e.g., an EEV- caused disease. It can be one or more of reducing the severity of the disease, limiting or preventing development of symptoms characteristic of the disease being treated, inhibiting worsening of symptoms characteristic of the disease being treated, limiting or preventing recurrence of the disease in a subject who has previously had the disease, and limiting or preventing recurrence of symptoms in subjects.
- trivalent in combination with vaccine or saRNA means that the vaccine or saRNA has a valence against three different viruses and generates a protective immune response against antigens (e.g., structural proteins or structural polyproteins) of those different viruses.
- antigens e.g., structural proteins or structural polyproteins
- trivalent means a valence against three different viruses of which antigens are encoded by the saRNA vaccine or vaccine comprising a saRNA expressing the nucleic acids encoding for the antigens e.g., structural proteins or structural polyproteins of VEEV, WEEV and EEEV.
- the three different viruses are different virus strains e.g., two WEEV strains such as for example 71V-1658 and Fleming in addition to a VEEV or EEEV strain.
- the saRNA of the present invention for example comprises a nucleotide sequence encoding for the proteins (e.g., structural protein, structural polyprotein, envelope protein) of WEEV 71V-1658, WEEV Fleming and of an EEEV strain e.g., EEEV V105-00210.
- “monovalent” means that the vaccine or saRNA has a valence against only one virus of a particular species, such as only VEEV, only WEEV or only EEEV and generates a protective immune response against only one structural protein or structural polyprotein of one virus. It does not exclude however the generation of protective immune responses against several closely related virus subtypes. “Divalent” thus means that the vaccine or saRNA has a valence against two viruses.
- a “vector” refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to be delivered to a target cell, either in vitro or in vivo.
- the heterologous polynucleotide may comprise a sequence of interest for purposes of prevention or therapy and may optionally be in the form of an expression cassette.
- a vector needs not be capable of replication in the ultimate target cell or subject.
- the term includes cloning vectors and viral vectors.
- viral replicon as used in the context of the present invention is used to refer to RNA or DNA comprising portions of the 49S viral genomic RNA that are essential for transcription and for cytoplasmic amplification of the transported RNA and for subgenomic RNA expression of a heterologous nucleic acid sequence.
- the replicon encodes and expresses viral non-structural proteins necessary for cytoplasmic amplification of the virus RNA.
- virus refers to an infectious or non-infectious virus comprising a viral genome.
- nucleic acids, promoters, recombinant proteins, and/or expression cassettes as mentioned herein are part of the viral genome of the respective recombinant virus.
- the recombinant viral genome is packaged and the obtained recombinant viruses can be used for the infection of cells and cell lines, in particular for the infection of living animals including humans.
- TCID50 is the abbreviation of “tissue culture infectious dose”, that amount of a pathogenic agent that will produce pathological change in 50% of cell cultures inoculated, expressed as TCID50/ml.
- a method for determining TCID50 is well known to the person skilled in the art. It is for example described in e.g., Example 2 of WO 03/053463.
- the term “subject” as used herein is a living multi-cellular vertebrate organisms, including, for example, humans, non-human mammals and (non-human) primates. The term “subject” may be used interchangeably with the term “animal” herein.
- an increase in survival rate can be characterized as an increase in survival of a subject (e.g., a human cancer patient), but can also be characterized in terms of clinical trial endpoints understood in the art.
- the present invention comprises a saRNA comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12, that when administered intratumorally induces both an inflammatory response and an enhanced T cell response as compared to an inflammatory response and a T cell response induced by a non-intratumoral administration of saRNA alone or non-intratumoral administration of a saRNA comprising a first nucleic acid encoding a TAA and a second nucleic acid encoding IL-12.
- OS Overall Survival rate
- PFS Progression Free Survival
- a saRNA of the invention induces an increased or enhanced inflammatory response, as compared to an administration of saRNA alone.
- This increased or enhanced inflammatory response can be detected, for example, by measuring cytokine levels in the subject’s blood and/or plasma, or may be detected at or near the site of administration, such as, for example, in a tumor that was injected intratumorally.
- an intratumoral administration of a saRNA of the invention induces an increased or enhanced inflammatory response in a tumor, as compared to an administration of saRNA alone.
- a saRNA encoding a tumor-associated antigen (TAA) and encoding IL-12 is injected intraperitoneally to treat a subject and induces an increased or enhanced inflammatory response in at least one peritoneal tumor and/or in the omentum.
- the subject being treated with the methods of the invention has at least one tumor that is peritoneal carcinomatosis or has malignant ascites or a metastatic tumor of the omentum, preferably derived from an abdominal malignancy, more preferably derived from ovarian or colorectal cancer.
- the subject is being treated for a tumor that is an abdominal malignancy, preferably metastasizing into the peritoneal cavity and/or the omentum.
- the subject has a tumor that is a tumor of ovarian or colorectal cancer.
- treatment of a subject with a method of the invention increases the likelihood of survival of the subject.
- treatment of a subject with a method of the invention induces an antigen-specific immune or T cell response, or IFN- ⁇ production in the peritoneal cavity of a subject, and/or in the omentum.
- intraperitoneal administration is carried out in a prime-boost regimen.
- the invention provides a pharmaceutical preparation or composition comprising the saRNA of the invention which pharmaceutical preparation or composition is adapted to intraperitoneal administration.
- the invention provides the saRNA of the invention for use in increasing the overall survival of a subject, preferably a human, preferably suffering from peritoneal carcinomatosis or malignant ascites or a metastatic tumor of the omentum, preferably derived from an abdominal malignancy, more preferably derived from ovarian or colorectal cancer, wherein the saRNA is administered intraperitoneally.
- the invention provides the saRNA of the invention for use in reducing signs and symptoms of peritoneal carcinomatosis or malignant ascites or a metastatic tumor of the omentum in a subject, preferably a human; in some embodiments, the tumor is derived from an abdominal malignancy, such as, for example, ovarian or colorectal cancer, wherein the saRNA is administered intraperitoneally.
- the invention provides the saRNA of the invention for use in inducing an antigen-specific immune or T cell response, or IFN- ⁇ production in the peritoneal cavity of a subject suffering from peritoneal carcinomatosis or malignant ascites or a metastatic tumor of the omentum, for example, derived from an abdominal malignancy, such as ovarian or colorectal cancer, wherein the saRNA is administered intraperitoneally.
- an “increased inflammatory response” or “enhanced inflammation response” is characterized by one or more of the following: increased production of IL-12 p70, M-CSF, and/or IL-33; increased antigen- specific CD8+ T cells, increased percentages of CD8+ T cells expressing IFN-gamma and TNF-alpha, decrease in tumor size and/or growth rate, improved survival of treated subjects, and the like, which can be detected by assays known in the art.
- “increased inflammatory response” generally refers to an increase in production of a particular cytokine or cell type associated with inflammation, in comparison to baseline levels prior to treatment according to methods of the invention and/or treatment with compositions of the invention.
- the amount of a cytokine or cell type is increased by at least 10%, 20%, 30%, 50%, 70%, or 100% or more in comparison to baseline levels prior to treatment according to methods of the invention and/or treatment with compositions of the invention.
- “enhanced inflammatory response” generally refers to an inflammatory response in which a new cytokine or new cell population is produced that was not detectable or was only detectable at trace amounts prior to treatment according to methods of the invention and/or treatment with compositions of the invention.
- the compositions and methods of the present invention enhance multiple aspects of a subject’s immune response. In this manner, the invention provides improved treatment of a subject having at least one tumor, including for example a cancer patient.
- saRNAs and combinations thereof of the present invention when injected intratumorally caused increased inflammatory responses in the tumor that may be detectable in the tumor and may also be detectable in the blood serum of the subject.
- indicia of systemic inflammation can include increased production of IL-12 p70, M-CSF, and IL-33; increased antigen-specific CD8+ T cells, increased percentages of CD8+ T cells expressing IFN-gamma and TNF-alpha, decrease in tumor size and/or growth rate, improved survival of treated subjects, and the like, and can be detected by assays known in the art by evaluating the tumor and/or the peripheral blood serum, assessing survival at regular intervals, and the like.
- an inflammatory response is enhanced or increased in a tumor and/or tumor cells in accordance with present disclosure can be determined by measuring to determine whether there is an increase in expression of one or more molecules which are indicative of an increased inflammatory response, including the secretion of chemokines and cytokines as is known in the art.
- Exemplary inflammatory response markers include one or more of IL-12 p70, M-CSF, IL-33, IFN-gamma, and TNF-alpha. These molecules and the measurement thereof are validated assays that are understood in the art and can be carried out according to known techniques. See, e.g., Borrego et al. ((1999) Immunology 7(1): 159-165).
- the increased or enhanced inflammatory response provided by the compositions and methods of the invention can also produce decreases in the volume and/or mean diameter of at least one tumor in the treated subject.
- the invention provides methods of decreasing the volume, size, and/or growth rate of at least one tumor in a subject.
- treatment with the compositions and/or methods of the invention produces a decrease in the volume, size, and/or growth rate of at least one tumor of at least 10%, 20%, 30%, 50%, or more in comparison to the volume, size, and/or growth rate of said tumor prior to treatment.
- an “enhanced T cell response” is characterized by one or more of the following: (1) an increase in frequency of CD8 + T cells; (2) an increase in CD8 + T cell activation; and (3) an increase in CD8 + T cell proliferation.
- whether a T cell response is enhanced in accordance with the present application can be determined by measuring the expression of one or more molecules which are indicative of: (1) an increase in CD8 + T cell frequency; (2) an increase in CD8 + T cell activation; and/or (3) an increase CD8 + T cell proliferation.
- Exemplary markers that are useful in measuring CD8 + T cell frequency, activation, and proliferation include IFN- ⁇ , TNF- ⁇ , and/or CD44, as is known in the art.
- Measuring antigen specific T cell frequency can also be measured by MHC multimers such as pentamers or dextramers; such measurements and assays as well as others suitable for use in evaluating methods and compositions of the invention are validated and understood in the art.
- an increase in CD8 + T cell frequency is characterized by an increase of at least 2-fold, 3-fold, 5-fold, or 10-fold or more in IFN- ⁇ and/or dextramer+ CD8 + T cells compared to the pre-treatment/baseline.
- An increase in CD8 + T cell activation is characterized, for example, as at least a 2-fold increase in the number of CD8+ T cells and/or at least a 2-fold increase in CD69 and/or CD44 expression compared to pre- treatment/baseline expression.
- An increase in CD8 + T cell proliferation is characterized, for example, as at least a 2-fold increase in Ki67 expression compared to pre- treatment/baseline expression.
- an increased or enhanced T cell response is characterized by an increase in CD8 + T cell expression of effector cytokines and/or an increase of cytotoxic effector functions.
- An increase in expression of effector cytokines can be measured, for example, by expression of one or more of IFN- ⁇ , TNF- ⁇ , and/or IL-2 compared to pre-treatment/baseline.
- An increase in cytotoxic effector functions for example, can be measured by expression of one or more of CD107a, granzyme B, and/or perforin and/or antigen-specific killing of target cells.
- the combinations and methods described herein are for use in treating a human cancer patient.
- the cancer patient is suffering from and/or is diagnosed with a cancer selected from the group consisting of: breast cancer, lung cancer, head and neck cancer, thyroid cancer, melanoma, gastric cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, ovarian cancer, urothelial cancer, cervical cancer, or colorectal cancer.
- the combinations and methods described herein are for use in treating a human cancer patient suffering from and/or diagnosed with a breast cancer, colorectal cancer, or melanoma.
- Tumor-Associated Antigens for use in the compositions and methods of the invention.
- an immune response is produced in a subject against a cell- associated polypeptide antigen.
- a cell-associated polypeptide antigen is a tumor-associated antigen (TAA).
- the TAA is HER2, PSA, PAP, CEA, MUC-1, survivin, TRP1, TRP2, Brachyury, Preferentially Expressed Antigen in Melanoma (PRAME), Folate receptor 1 (FOLR1), Human endogenous retrovirus-K envelope (HERV-K-env), or Human endogenous retrovirus-K- gag (HERV-K-gag), alone or in any combination thereof.
- PRAME Preferentially Expressed Antigen in Melanoma
- FOLR1 Folate receptor 1
- HERV-K-env Human endogenous retrovirus-K envelope
- HERV-K-gag Human endogenous retrovirus-K- gag
- the TAA may include, but is not limited to, 5 alpha reductase, alpha-fetoprotein, AM-1, APC, April, BAGE, beta-catenin, Bcl12, bcr-abl, CA-125, CASP- 8/FLICE, Cathepsins, CD19, CD20, CD21, CD23, CD22, CD33 CD35, CD44, CD45, CD46, CD5, CD52, CD55, CD59, CDC27, CDK4, CEA, c-myc, Cox-2, DCC, DcR3, E6/E7, CGFR, EMBP, Dna78, farnesyl transferase, FGF8b, FGF8a, FLK-1/KDR, folic acid receptor, G250, GAGE-family, gastrin 17, gastrin-releasing hormone, GD2/GD3/GM2, GnRH, GnTV, GP1, gp100/Pmel17, gp-100-in
- the TAA is an Endogenous Retroviral Protein (ERV), or derivative thereof.
- ERV Endogenous Retroviral Protein
- Such an ERV can be an ERV from the Human HERV-K protein family and, for example, can be a HERV-K envelope (env) protein, a HERV-K group specific antigen (gag) protein, and a HERV-K “marker of melanoma risk” (mel) protein (see, e.g., Cegolon et al. (2013) BMC Cancer 13:4).
- Any TAA may be used so long as it accomplishes at least one objective or desired end of the invention, such as, for example, stimulating an immune response following administration of the saRNA containing it.
- the TAA encoded by the one or more saRNAs is known to be expressed by at least one tumor in the subject, for example, based on previous testing of a sample of the tumor.
- Exemplary sequences of TAAs including TAAs mentioned herein, are known in the art and are suitable for use in the compositions and methods of the invention.
- Sequences of TAAs for use in the compositions and methods of the invention may be identical to sequences known in the art or disclosed herein, or they may share less than 100% identity, such as at least 90%, 91%, 92%, 95%, 97%, 98%, or 99% or more sequence identity to either a nucleotide or amino acid sequence known in the art or disclosed herein.
- a sequence of a TAA for use in a composition or method of the invention may differ from a reference sequence known in the art and/or disclosed herein by less than 20, or less than 19, 18, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides or amino acids, so long as it accomplishes at least one objective or desired end of the invention (for example, to help stimulate an immune response when administered to a subject as a component of a saRNA).
- One of skill in the art is familiar with techniques and assays for evaluating TAAs to ensure their suitability for use in a saRNA or method of the invention.
- modifications to one or more of the TAAs are made such that, after administration to a subject, polyclonal antibodies are elicited that predominantly react with the one or more of the TAAs described herein.
- polyclonal antibodies could attack and eliminate tumor cells as well as prevent metastatic cells from developing into metastases.
- the effector mechanism of this anti-tumor effect would be mediated via complement and Antibody-Dependent Cellular Cytotoxicity (“ADCC”).
- ADCC Antibody-Dependent Cellular Cytotoxicity
- a modified TAA polypeptide antigen comprises a CTL epitope of the cell-associated polypeptide antigen and a variation, wherein the variation comprises at least one CTL epitope or a foreign TH epitope.
- modified TAAs can include (in one non-limiting example) one or more HER2 polypeptide antigens comprising at least one CTL epitope and a variation comprising at least one CTL epitope of a foreign TH epitope; these HER2 antigens and methods of producing the same are described in U.S. Patent No.7,005,498 and U.S. Patent Pub. Nos.2004/0141958 and 2006/0008465, herein incorporated by reference.
- the heterodimer form is also referred to as IL- 12-p70 or IL-12-p35/p40.
- IL-12 has many effects that promote an immune response, but some clinical studies with IL-12 had unacceptable levels of adverse events (see Lasek et al. (2014) Cancer Immunol. Immunother.63: 419-35). IL-12 has been demonstrated to induce production of IFN-gamma, to induce T H 1 cell differentiation ,and also to increase activation and cytotoxic function of T and NK cells (see Nguyen et al. (2020) Front. Immunol.11: 575597).
- IL-12 A variety of modified forms of IL-12 are known in the art and are useful in embodiments of the invention so long as they retain IL-12 function, such as, for example, increasing secretion of IFN-gamma (“IFN- ⁇ ”), etc.
- IFN- ⁇ IFN-gamma
- a modified form of IL-12 known in the art is “single chain Interleukin-12,” also referred to as “IL-12 sc” or “sc IL-12.”
- This IL-12 sc provides the advantage of automatically having the correct stoichiometry of the p35 and p40 subunits, so that there is not excess p40 subunit produced that might exert an inhibitory effect on the full length IL-12 (see, e.g., Anderson et al. (1997) Hum.
- IL-12 is encoded by a saRNA along with a tumor- associated antigen (“TAA”).
- TAA tumor- associated antigen
- a saRNA encodes IL-12 and, optionally, also encodes a TAA.
- the IL-12 sequence is a murine IL-12 sequence.
- the IL-12 has an amino acid sequence with at least 90%, 95%, 97% 98%, or 99% identity to SEQ ID NO: 6, or has an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 6 by less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids, or is identical to the sequence set forth in SEQ ID NO: 6.
- a nucleic acid encoding IL-12 comprises a nucleic acid sequence having at least 90%, 95%, 97% 98%, or 99% identity to SEQ ID NO: 5, i.e., differing from the nucleic acid sequence set forth in SEQ ID NO: 5 by less than 20, 10, 5, 4, 3, 2, or 1 nucleic acid in the sequence, or is identical to the sequence set forth in SEQ ID NO: 5.
- IL-12 is well studied, so it is expected that one of skill in the art would be able to introduce sequence modifications in more variable or less conserved regions to avoid affecting gene function.
- any IL-12 sequence is suitable for use in embodiments of the invention so long as it provides at least one function of IL-12 in an assay, such as any of the assays for IL-12 used in the working examples or otherwise known in the art.
- Self-amplifying RNA There are currently two different types of synthetic RNA vaccines: Conventional mRNA and self-amplifying RNA (saRNA). Use of conventional mRNA strategies (also referred to as nonreplicating or non-amplifying mRNA) against infectious diseases and cancers has been investigated in several preclinical and clinical trials.
- RNA vaccines which are genetically engineered replicons derived from self-replicating single-stranded RNA viruses address this limitation.
- VRPs viral replicon particles
- saRNA packaged into the viral particle
- envelope proteins are provided in trans as defective helper constructs during production. Resulting VRPs therefore lack the ability to form infectious viral particles following a first infection, and only the RNA is capable of further amplification.
- VRPs may be derived from both positive-sense and negative-sense RNA viruses, however the latter are more complex and require reverse genetics to rescue the VRPs.
- gene therapy there are several issues associated with the use of viral vectors for vaccine development.
- saRNA vaccines can be produced and delivered in a similar manner to conventional mRNA vaccines.
- Positive-sense alphavirus genomes that have been commonly used for saRNA vaccine design include the Venezuelan equine encephalitis virus (VEE), Sindbis virus (SINV), and Semliki forest virus (SFV).
- the alphavirus replicase genes encode an RNA-dependent RNA polymerase (RdRP) complex which amplifies synthetic transcripts in situ.
- the antigenic or therapeutic sequence is expressed at high levels as a separate entity and further proteolytic processing of the immunogen is not required.
- saRNAs can be delivered at lower concentrations than conventional mRNA vaccines to achieve comparable antigen expression.
- the saRNA constructs have historically been delivered from alphaviruses, such as the Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV) or Sindbis virus.
- saRNA constructs contain the four non-structural proteins, a subgenomic promoter, and the gene of interest (replacing the viral structural proteins). By deleting the viral structural proteins, the RNA is incapable of producing an infectious virus. After delivery to the cytoplasm, the non-structural proteins form an RNA- dependent RNA polymerase (RDRP) that replicates both the genomic RNA (entire RNA strand) and subgenomic RNA (gene of interest). Each of the four non-structural proteins plays a role in the formation of the RDRP, which is a complex and multistage process. This RNA replication is what leads to higher antigen expression than non-replicating mRNA.
- RDRP RNA- dependent RNA polymerase
- EEV viruses, proteins and nucleotide sequences EEV are alphavirus belonging to the family of Togaviridae.
- EEV are small, enveloped positive-strand RNA viruses well known in the art.
- the viral nucleocapsid is surrounded by host derived lipid membranes in which a trimer of envelope proteins of E1 and E2 heterodimers are embedded.
- the nucleocapsid consists of a capsid protein (C) surrounding the single-strand RNA genome.
- C capsid protein
- the RNA genome (49S RNA) of EEV viruses is approximately 11-12 kb in length and contains a 5 ⁇ cap and 3 ⁇ polyadenylation tail and is immediately translated upon entry into the cell.
- the 5 ⁇ region of the genome encodes for four non-structural proteins (NSP1, NSP2, NSP3, and NSP4).
- the 3 ⁇ region of the genome encodes for five structural proteins (C, E3, E2, 6k, E1) which are expressed as a structural polyprotein from 26S subgenomic RNA.
- the mRNA encoding for the structural proteins is transcribed from a replication intermediate and a 26S subgenomic promoter. Protease cleavage of the polyprotein produces the mature structural proteins C, E3, E2, 6k, E1.
- the nucleocapsid (C) protein possesses auto- proteolytic activity which cleaves the C protein from the precursor protein soon after the ribosome transits the junction between the C and E3 protein coding sequence.
- envelope glycoproteins E2 and E1 are derived by proteolytic cleavage and form heterodimers.
- E2 initially appears in the infected cell as a precursor, pE2, which consists of E3 and E2.
- E3 is cleaved from E2 by furin-like protease activity at a cleavage site.
- Live-attenuated vaccines have been used in the US military and laboratory workers and formalin-inactivated vaccines are available for use in horses.
- One such live-attenuated vaccine is TC-83, originally developed by the US Army for vaccine use (Pittman et al., 1996).
- TC-83 was created by serially passaging the Trinidad Donkey VEEV strain in guinea pig heart cells (Alevizatos et al., 1967). Point mutations in E2 and the 5′ untranslated region are responsible for the attenuated phenotype of TC- 83 (Kinney et al., 1993). TC-83 has been noted to be effective in preventing disease in humans, but 15–37.5% of vaccine recipients develop febrile symptoms (Berge et al., 1961; McKinney et al., 1963; Alevizatos et al., 1967; Pittman et al., 1996) and only 82% of vaccinees seroconvert upon vaccination.
- any combination of any WEEV, EEEV and/or VEEV as mentioned above is also encompassed with any of the embodiments as described herein.
- adjuvantation herein is intended that a particular encoded protein or component of a saRNA increases the immune response produced by the other encoded protein(s) or component(s) of the saRNA.
- the compositions, methods, and combinations of the invention increase overall survival of a treated subject.
- crease overall survival as used herein is intended that there is a statistically significant improvement in the survival rate of treated subjects as compared to untreated subjects.
- the one or more nucleic acids described herein are embodied in in one or more expression cassettes in which the one or more nucleic acids are operably linked to expression control sequences.
- “Operatively linked” or “operably linked” means that the components described are in relationship permitting them to function in their intended manner e.g., a promoter to transcribe the nucleic acid to be expressed.
- An expression control sequence operatively linked to a coding sequence is joined such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences.
- the expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon at the beginning of a protein-encoding open reading frame, splicing signals for introns, and in-frame stop codons.
- Suitable promoters include, but are not limited to, the SV40 early promoter, an RSV promoter, the retrovirus LTR, the adenovirus major late promoter, the human CMV immediate early I promoter, and various poxvirus promoters, including but not limited to the following vaccinia virus or MVA-derived and FPV-derived promoters: the 30K promoter, the I3 promoter, the PrS promoter, the PrS5E promoter, the Pr7.5K, the PrHyb promoter, the Pr13.5 long promoter, the 40K promoter, the MVA-40K promoter, the FPV 40K promoter, 30k promoter, the PrSynIIm promoter, the PrLE1 promoter, and the PR1238 promoter.
- MVA-derived and FPV-derived promoters including but not limited to the following vaccinia virus or MVA-derived and FPV-derived promoters: the 30K promoter, the I3 promoter, the PrS promoter, the PrS
- Additional promoters are further described in WO 2010/060632, WO 2010/102822, WO 2013/189611, WO 2014/063832, and WO 2017/021776, which are incorporated fully by reference herein.
- Additional expression control sequences include, but are not limited to, leader sequences, termination codons, polyadenylation signals, and any other sequences necessary for the appropriate transcription and subsequent translation of the nucleic acid sequence encoding the desired heterologous protein (e.g., a TAA, and/or IL-12) in the desired host system.
- the vector may also contain additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the desired host system.
- the combinations of the present invention can be administered as part of a homologous and/or heterologous prime-boost regimen. As shown by the working examples, a homologous prime boost regimen increases a subject’s specific T cell responses.
- a combination and/or method for stimulating the immune response, reducing tumor size and/or increasing survival in a subject comprising administering to the subject a combination of the instant invention, wherein the combination is administered as part of a homologous or heterologous prime- boost regimen.
- Methods for production of saRNA Conventional and synthetic saRNA vaccines are essentially produced in the same manner. Briefly, an mRNA expression plasmid (pDNA) encoding a DNA-dependent RNA polymerase promoter (typically derived from the T7, T3, or SP6 bacteriophages) and the RNA vaccine candidate is designed as a template for in vitro transcription.
- pDNA mRNA expression plasmid
- a DNA-dependent RNA polymerase promoter typically derived from the T7, T3, or SP6 bacteriophages
- the flexibility of gene synthesis platforms is a key advantage.
- RNA vaccines For conventional mRNA vaccines the antigenic or immunomodulatory sequence is flanked by 5′ and 3′ untranslated regions (UTRs).
- a poly(A) tail can either be incorporated from the 3′ end of the pDNA template, or added enzymatically after in vitro transcription.
- saRNA vaccine pDNA templates contain additional alphavirus replicon genes and conserved sequence elements.
- the nonstructural proteins 1, 2, 3, and 4 (nsP1-4) are essential for replicon activity as they form the RdRP complex.
- In vitro transcription is performed on typically on a linearized pDNA template or a linear DNA fragment, typically with a T7 DNA-dependent RNA polymerase, resulting in multiple copies of the RNA transcript. The 5’ end is capped for an efficient translation.
- RNA product is then undergoing purification which can include steps to remove a by-product of the in vitro transcription in form of double-stranded dsRNA. These can be removed, e.g. by a double-strand specific enzymatic RNase or by chromatography employing material with specific dsRNA affinity.
- the saRNA of the present disclosure can be formulated as part of a vaccine or used to prepare a medicament that is a vaccine.
- the saRNA can be converted into a physiologically acceptable form. An exemplary preparation follows.
- Purified virus is stored at -80°C with a titer of 5 x 10 8 TCID50/ml formulated in 10 mM Tris, 140 mM NaCl, pH 7.4.
- a titer of 5 x 10 8 TCID50/ml formulated in 10 mM Tris, 140 mM NaCl, pH 7.4.
- 1 x10 8 -1 x 10 9 particles of the virus can be lyophilized in phosphate- buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule, preferably a glass ampoule.
- the vaccine doses or shots can be prepared by stepwise freeze-drying of the virus in a formulation.
- the formulation contains additional additives such as, for example, mannitol, dextran, sugar, glycine, lactose, polyvinylpyrrolidone, and optionally other additives, such as antioxidants or inert gas, stabilizers, or recombinant proteins (e.g. human serum albumin) suitable for in vivo administration.
- additional additives such as, for example, mannitol, dextran, sugar, glycine, lactose, polyvinylpyrrolidone, and optionally other additives, such as antioxidants or inert gas, stabilizers, or recombinant proteins (e.g. human serum albumin) suitable for in vivo administration.
- the ampoule is then sealed and can be stored at a suitable temperature, for example, between 4°C and room temperature for several months. However, for long-term storage, the ampoule is stored preferably at temperatures below -20°C, most preferably at about -80°C.
- the lyophilisate is dissolved in 0.1 to 0.5 ml of an aqueous solution, preferably physiological saline or Tris buffer such as 10mM Tris, 140mM NaCl pH 7.7. It is contemplated that the saRNA vaccine or pharmaceutical composition of the present disclosure can be formulated in solution in a concentration range of 10 4 to 10 10 TCID 50 /ml, 10 5 to 5 ⁇ 10 9 TCID 50 /ml, 10 6 to 5x10 9 TCID 50 /ml, or 10 7 to 5x10 9 TCID 50 /ml.
- a preferred dose for humans comprises between 10 6 to 10 10 TCID 50 , including a dose of 10 6 TCID 50 , 10 7 TCID 50 , 10 8 TCID 50 , 5 ⁇ 10 8 TCID 50 , 10 9 TCID 50 , 5 ⁇ 10 9 TCID 50 , or 10 10 TCID 50 .
- Dosages in humans and animals can range from about 1 ⁇ 10 4 to about 1 ⁇ 10 10 , advantageously at a dose of about 1 ⁇ 10 6 to about 1 ⁇ 10 8 per dose.
- the present inventors contemplate weekly, biweekly or monthly doses for a period of about 1 to about 12 months, or longer.
- the saRNA is administered to a cancer patient intratumorally. In other embodiments, the saRNA is administered to a cancer patient intraperitoneally. In other embodiments, the saRNA is administered to a cancer patient either intratumorally, intravenously, subcutaneously, and/or intraperitoneally at the same time or at different times. Kits, and Methods of Use. In various embodiments, the invention encompasses kits, pharmaceutical combinations, pharmaceutical compositions, and/or immunogenic combinations comprising one or more saRNAs that include the nucleic acids described herein.
- kits and/or composition of the invention can comprise one or multiple containers or vials of one or more recombinant poxviruses of the present disclosure together with instructions for the administration of the saRNAs. It is contemplated that in a more particular embodiment, the kit can include instructions for administering the saRNA(s) in a first priming administration and then administering one or more subsequent boosting administrations of the saRNA(s) in a homologous or heterologous prime-boost regimen, as appropriate.
- the kits and/or compositions provided herein may generally include one or more pharmaceutically acceptable and/or approved carriers, additives, antibiotics, preservatives, diluents and/or stabilizers.
- auxiliary substances can include water, saline solution, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, and the like.
- Suitable carriers are typically large, slowly-metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and the like.
- Embodiment 1 is a saRNA for use in stimulating an immune response to a Tumor Associated Antigen (TAA) in a subject, comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); and (b) a second nucleic acid encoding IL-12; wherein the intratumoral administration of saRNA increases and/or enhances an inflammatory response in a tumor, reduces the growth rate and/or size of the tumor, and/or increases overall survival of the subject as compared to a non-intratumoral injection of said saRNA or an injection of a saRNA that does not comprise a nucleic acid encoding IL-12, wherein the saRNA is administered intratumorally.
- TAA Tumor Associated Antigen
- Embodiment 2 is a saRNA for use according to embodiment 1, further comprising (c) a third nucleic acid encoding 4-1BBL.
- Embodiment 3 is a saRNA for use according to embodiment 1, wherein said TAA is an endogenous retroviral (ERV) protein.
- ERP retroviral
- Embodiment 4 is a saRNA for use according to embodiment 1, wherein said TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine related protein 1 (TRP1), tyrosine related protein 1 (TRP2), Brachyury, FOLR1, PRAME, HERV- K-env, HERV-K-gag, and combinations thereof.
- CEA carcinoembryonic antigen
- MUC-1 mucin 1 cell surface associated
- PAP prostatic acid phosphatase
- PSA prostate specific antigen
- HER-2 human epidermal growth factor receptor 2
- survivin tyrosine related protein 1
- TRP1 tyrosine related protein 1
- TRP2 tyrosine related protein 1
- FOLR1 HER
- Embodiment 5 is a saRNA for use in the treatment of tumors, comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); and (b) a second nucleic acid encoding IL-12; wherein the intratumoral administration of the saRNA enhances an inflammatory response in a tumor, reduces the growth rate and/or size of the tumor, and/or increases overall survival of the subject as compared to a non-intratumoral injection of said saRNA or an injection of a saRNA that does not comprise a nucleic acid encoding IL-12; and wherein the saRNA is administered intratumorally.
- TAA tumor-associated antigen
- Embodiment 6 is a saRNA for use according to embodiment 5, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine related protein 1 (TRP1), tyrosine related protein 2 (TRP2), Brachyury, FOLR1, PRAME, HERV- K-env, HERV-K-gag, and combinations thereof.
- CEA carcinoembryonic antigen
- MUC-1 mucin 1 cell surface associated
- PAP prostatic acid phosphatase
- PSA prostate specific antigen
- HER-2 human epidermal growth factor receptor 2
- survivin tyrosine related protein 1
- TRP1 tyrosine related protein 1
- TRP2 tyrosine related protein 2
- FOLR1 HER
- Embodiment 7 is a pharmaceutical combination comprising: (i) a saRNA, comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); and (b) a second nucleic acid encoding IL-12; wherein the intratumoral administration of the saRNA enhances an inflammatory response in a tumor, reduces the growth rate and/or size of the tumor, and/or increases overall survival of the subject as compared to a non- intratumoral injection of said saRNA or an injection of a saRNA that does not comprise a nucleic acid encoding IL-12; and (ii) a pharmaceutically acceptable carrier.
- a saRNA comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); and (b) a second nucleic acid encoding IL-12; wherein the intratumoral administration of the saRNA enhances an inflammatory response in a tumor, reduces the growth rate and
- Embodiment 8 is a method for reducing tumor growth and/or increasing survival in a subject having a cancerous tumor, the method comprising intratumorally administering to the subject a saRNA comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12 and optionally a third nucleic acid encoding 4-1BBL, wherein the intratumoral administration of the saRNA enhances an inflammatory response in the tumor, decreases tumor growth and/or size, and/or increases overall survival of the subject as compared to injection of a saRNA that does not comprise a nucleic acid encoding IL-12 or saRNA alone.
- TAA tumor-associated antigen
- Embodiment 9 is a method according to embodiment 8, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine related protein 1 (TRP1), tyrosine related protein 1 (TRP2), Brachyury, PRAME, FOLR1, HERV-K-env, HERV-K-gag, and combinations thereof.
- Embodiment 10 is a method according to embodiment 8, wherein the subject is a human cancer patient.
- Embodiment 11 is a method for reducing tumor size or growth and/or increasing survival in a subject having an tumor, the method comprising intraperitoneally administering to the subject a saRNA comprising a first nucleic acid encoding IL-12 and optionally a second nucleic acid encoding a tumor-associated antigen (TAA), wherein the administration of the saRNA enhances Natural Killer (NK) cell response and enhances CD8 T cell responses specific to the TAA as compared to baseline levels prior to treatment or as compared to the expected result of injection with saRNA alone.
- TAA tumor-associated antigen
- Embodiment 12 is the method is according to embodiment 11, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine related protein 1 (TRP1), tyrosine related protein 1 (TRP2), Brachyury, PRAME, FOLR1, HERV- K-env, HERV-K-gag, and combinations thereof.
- Embodiment 13 is a method according to embodiment 11, wherein the subject is human and the tumor is intraperitoneal.
- Embodiment 14 is a method of inducing an enhanced inflammatory response in a peritoneal tumor of a subject, the method comprising intraperitoneally administering to the subject a saRNA comprising a first nucleic acid encoding IL-12 or IL-12sc and optionally a second nucleic acid encoding a heterologous tumor-associated antigen (TAA), wherein the intraperitoneal administration of the saRNA generates an enhanced inflammatory response in the tumor as compared to an inflammatory response that would be generated by a non-intraperitoneal injection of a saRNA alone.
- Embodiment 15 is a method according to embodiment 14, further comprising intraperitoneally administering to the subject a boosting dose of the same saRNA.
- Embodiment 16 is a vaccine comprising any of embodiments 1-6 and a pharmaceutically acceptable carrier.
- Embodiment 17 is a saRNA according to any one of embodiments 1-6, a vaccine according to embodiment 16, or a pharmaceutical combination according to embodiment 7, for use in reducing tumor size and/or increasing survival in a subject having a cancerous tumor.
- Embodiment 18 is a saRNA according to any one of embodiments 1-6, a vaccine according to embodiment 16, or a pharmaceutical combination according to embodiment 7, for use in a method for reducing tumor size and/or increasing survival in a subject having a cancerous tumor, the method comprising intratumorally or intraperitoneally administering to the subject said saRNA, vaccine, or pharmaceutical combination, wherein the intratumoral or intraperitoneal administration enhances an inflammatory response in the cancerous tumor, decreases tumor growth rate, increases tumor reduction, and/or increases overall survival of the subject as compared to injection of saRNA alone.
- Embodiment 19 is a saRNA according to any one of embodiments 1-6, a vaccine according to embodiment 16, or a pharmaceutical combination according to embodiment 7, for use in a method for stimulating an immune response in a subject, the method comprising intratumorally or intraperitoneally administering to the subject said saRNA, vaccine, or pharmaceutical combination, wherein the intratumoral or intraperitoneal administration enhances an inflammatory response in the cancerous tumor that is detectable by analysis of the tumor or by analysis of blood or sera of the subject as compared to administration of saRNA alone or as compared to a non-intratumoral or non-intraperitoneal administration of said saRNA, or as compared to an intratumoral or intraperitoneal administration of a saRNA lacking one or more of the components encoded by said saRNA.
- Embodiment 20 is a saRNA according to any one embodiments 1-6, a vaccine according to embodiment 16, or a pharmaceutical combination according to embodiment 7 for use in a method for treating cancer in subject.
- Embodiment 21 is a saRNA according to any one of embodiments 1-6, a vaccine according to embodiment 16, or a pharmaceutical combination according to embodiment 7 for use in a method for treating cancer, wherein the cancer is selected from the group consisting of: breast cancer, lung cancer, head and neck cancer, thyroid, melanoma, gastric cancer, bladder cancer, kidney cancer, liver cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer, urothelial, cervical, or colorectal cancer.
- Embodiment 22 is a saRNA according to any one of embodiments 1-6, wherein the enhanced inflammatory response is localized to the tumor.
- Embodiment 23 is a method of inducing an enhanced inflammatory response in a peritoneal tumor of a subject, the method comprising intratumorally administering to the subject a saRNA comprising a first nucleic acid encoding a first heterologous tumor- associated antigen (TAA) and a second nucleic acid encoding IL-12 or IL-12sc, wherein the intratumoral administration of the saRNA generates an enhanced inflammatory response in the tumor as compared to an inflammatory response that would be generated by or would be expected to result from intratumoral injection of saRNA virus alone.
- TAA tumor- associated antigen
- Embodiment 24 is a method according to embodiment 23, wherein the saRNA further comprises a nucleic acid encoding 4-1BBL.
- Embodiment 25 is a saRNA, comprising: (a) a first nucleic acid encoding a tumor- associated antigen (TAA); and (b) a second nucleic acid encoding IL-12; wherein the intratumoral administration of the saRNA enhances an inflammatory response in a tumor, reduces the growth rate and/or size of the tumor, and/or increases overall survival of the subject as compared to a non-intratumoral injection of said saRNA or an injection of a saRNA that does not comprise a nucleic acid encoding IL-12.
- TAA tumor- associated antigen
- Embodiment 26 is the saRNA of embodiment 25, further comprising (c) a third nucleic acid encoding 4-1BBL.
- Embodiment 27 is the saRNA of embodiment 25, wherein said TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine related protein 1 (TRP1), tyrosine related protein 1 (TRP2), Brachyury, FOLR1, PRAME, HERV-K-env, HERV-K- gag, p15, and combinations thereof.
- CEA carcinoembryonic antigen
- MUC-1 mucin 1 cell surface associated
- PAP prostatic acid phosphatase
- PSA prostate specific antigen
- HER-2 human epidermal growth factor receptor 2
- survivin tyrosine related
- Embodiment 28 is a pharmaceutical combination comprising the saRNA according to embodiment 25 and a pharmaceutically acceptable carrier.
- Embodiment 29 is a method of stimulating an immune response in a subject having a plurality of tumors, comprising a step of locally (intratumorally) administering to fewer than all of the tumors in said subject a saRNA comprising at least one first nucleic acid encoding a TAA and a second nucleic acid encoding IL-12, wherein an immune response to the TAA is stimulated in the subject.
- Embodiment 30 is a method of treating a subject having at least one inaccessible tumor and at least one accessible tumor, comprising locally (intratumorally) administering to at least one accessible tumor in the subject a saRNA comprising at least one first nucleic acid encoding a TAA and a second nucleic acid encoding 4-1-BBL, whereby the growth of the inaccessible tumor is decreased or stopped.
- Embodiment 31 is a method of preventing or decreasing the extent of tumor recurrence or metastasis in a subject having at least one tumor, comprising intratumorally or intraperitoneally administering to at least one tumor in the subject a saRNA comprising at least one first nucleic acid encoding IL-12 and optionally a second nucleic acid encoding a TAA, whereby the growth of the inaccessible tumor is decreased or stopped.
- Embodiment 32 is the method of embodiment 29, 31, or 31, wherein said saRNA further comprises a nucleic acid encoding 4-1BBL.
- Embodiment 33 is a saRNA for use in stimulating an immune response to a Tumor Associated Antigen (TAA) in a subject, comprising: (a) a first nucleic acid encoding IL- 12, for example, scIL-12; and (b) a second nucleic acid encoding a TAA; wherein the intraperitoneal administration of the saRNA enhances or increases an inflammatory response in a tumor, reduces the growth rate and/or size of the tumor, and/or increases overall survival of the subject as compared to a non- intraperitoneal injection of said saRNA or an injection of a saRNA that does not comprise a nucleic acid encoding IL-12, wherein the saRNA is administered intraperitoneally.
- TAA Tumor Associated Antigen
- Embodiment 34 is a saRNA for use according to embodiment 33, further comprising (c) a third nucleic acid encoding 4-1BBL.
- Embodiment 35 is a saRNA for use according to embodiment 33, wherein said TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine related protein 1 (TRP1), tyrosine related protein 1 (TRP2), Brachyury, FOLR1, PRAME, HERV- K-env, HERV-K-gag, and combinations thereof.
- CEA carcinoembryonic antigen
- MUC-1 mucin 1 cell surface associated
- PAP prostatic acid phosphatase
- PSA prostate specific antigen
- HER-2 human epidermal growth factor receptor 2
- Embodiment 36 is a saRNA for use in the treatment of tumors, comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); and (b) a second nucleic acid encoding IL-12; wherein the intratumoral administration of the saRNA enhances and/or increases an inflammatory response in a tumor, reduces the growth rate and/or size of the tumor, and/or increases overall survival of the subject as compared to a non- intratumoral injection of said saRNAor an injection of a saRNA that does not comprise a nucleic acid encoding IL-12; and wherein the saRNA is administered intratumorally.
- TAA tumor-associated antigen
- Embodiment 37 is a saRNA for use according to embodiment 36, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine related protein 1 (TRP1), tyrosine related protein 2 (TRP2), Brachyury, FOLR1, PRAME, HERV- K-env, HERV-K-gag, and combinations thereof.
- CEA carcinoembryonic antigen
- MUC-1 mucin 1 cell surface associated
- PAP prostatic acid phosphatase
- PSA prostate specific antigen
- HER-2 human epidermal growth factor receptor 2
- survivin tyrosine related protein 1
- TRP1 tyrosine related protein 1
- TRP2 tyrosine related protein 2
- FOLR1
- Embodiment 38 is a pharmaceutical combination comprising: (i) a saRNA, comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); and (b) a second nucleic acid encoding IL-12; wherein the intratumoral administration of the saRNA enhances and/or increases an inflammatory response in a tumor, reduces the growth rate and/or size of the tumor, and/or increases overall survival of the subject as compared to a non-intratumoral injection of said saRNA or an injection of a saRNA that does not comprise a nucleic acid encoding IL-12; and (ii) a pharmaceutically acceptable carrier.
- a saRNA comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); and (b) a second nucleic acid encoding IL-12; wherein the intratumoral administration of the saRNA enhances and/or increases an inflammatory response in
- Embodiment 39 is a saRNA for use in stimulating an immune response to a Tumor Associated Antigen (TAA) in a subject, comprising a nucleic acid encoding IL-12; wherein the intraperitoneal administration of the saRNA increases an inflammatory response in a tumor, optionally a peritoneal tumor, and/or in the omentum, reduces the growth rate and/or size of the tumor, and/or increases overall survival of the subject as compared to a non-intraperitoneal injection of said saRNA or an injection of a saRNA that does not comprise a nucleic acid encoding IL-12, wherein the saRNA is administered intraperitoneally.
- TAA Tumor Associated Antigen
- Example 1 Material and Methods Construction of saRNAVRP (Alphavirus replicon particles) The following sections describe construction of saRNAVRPs comprising one or more heterologous nucleic acids expressing an antigenic determinant. All other constructs described herein are made using similar methods.
- the recombinant alphavirus replicon particles VRP-BN001, -BN005 and -BN006 are all derived from the VEEV TC83 attenuated strain genome.
- the open reading frame (ORF) downstream of the subgenomic promoter encoding the polyprotein C-E3-E2-6k-E1 in the VEEV TC83 genome was completely deleted and a single A3G nucleotide substitution in the genomic 5’UTR was introduced to increase expression levels.
- the resulting replicon sequence (containing the 5’UTR, the ORF coding for the nonstructural proteins 1-4 (nsp1-4), the subgenomic promoter and the 3’UTR of the VEEV TC83 genome) was placed under the transcriptional control of a CMV promoter.
- a poly-A stretch and the HDV antigenomic ribozyme sequence were added downstream of the replicon’s 3’UTR to generate a polyadenylated replicon with an exact 3’ end.
- Different antigens of interest were cloned and placed under the transcriptional control of the subgenomic promoter in this TC83- based replicon.
- VRPs containing the VEEV TC83 based replicon construct expressing different transgenes were generated: 1) VRP-BN001 encodes a fusion protein composed of the Tag blue fluorescent protein (BFP) ORF followed by the self-cleaving P2A peptide and the ovalbumin (OVA) ORF, herein termed BFP-OVA.
- BFP Tag blue fluorescent protein
- OVA ovalbumin
- VRP-BN005 encodes an N-terminal FLAG-tagged (DYKDDDDK) version of the murine endogenous retroviral protein (MuLV) envelope glycoprotein 70 (gp70) containing two amino acid mutations (E552R und A558F) in the conserved immunosuppressive domain (ISD). In addition, the original signal sequence (aa1- 31) was replaced with the Ig-kappa (IgK) leader sequence for more efficient transport to the cellular membrane. 3) VRP-BN006 encodes a single chain murine IL-12 protein in which the p40 and p35 subunits are linked with a 3x (GGGGS) linker sequence.
- GGGGS 3x
- the secretion signal of the p35 subunit (aa1-22) was deleted so that the protein encodes only for one secretion signal (from the p40 subunit).
- the coding sequences for the transgene described above were codon optimized and synthetized (including also sequences necessary for cloning) as DNA strings and cloned into the replicon vector. Alternatively, the sequences were PCR amplified from pre-existing constructs and cloned into the replicon. Cloning of replicon plasmids launching SFV based saRNAs
- the recombinant alphavirus replicon particle VRP-BN010 is derived from the Semliki forest virus (SFV) genome.
- the open reading frame (ORF) downstream of the subgenomic promoter encoding most of the polyprotein C-E3-E2-6k-E1 was deleted, except for the first 102 nucleotides (or aa1-34) of the ORF encoding the capsid protein.
- This sequence from the SFV capsid contains a known translational enhancer element sequence and can increase transgene expression levels.
- a self-cleaving P2A peptide was introduced downstream of the truncated capsid.
- the resulting replicon sequence (containing the 5’UTR, the nsp1-4 ORF, the subgenomic promoter, capsid translational enhancer element [C(enh)], P2A sequence and the 3’UTR of the SFV genome) was placed under the transcriptional control of a CMV promoter.
- a poly-A stretch and the HDV antigenomic ribozyme sequence were added downstream of the replicon’s 3’UTR to generate a polyadenylated replicon with an exact 3’ end.
- the BFP-OVA transgene was cloned and placed under the transcriptional control of the SFV subgenomic promoter and in frame with the upstream capsid translational enhancer and P2A sequence resulting in the generation of a polycistronic mRNA encoding for C(enh)-P2A-BFP-P2A- OVA).
- the BFP-OVA ORF is identical to the encoded in VRP-BN001.
- the two P2A sequences in this mRNA have different nucleotide sequences to avoid recombination but encode for the same protein sequence and allow for the expression of BFP and OVA as independent proteins and separated from the C(enh) sequence.
- VRP-BN stocks suspension HEK293T cells were transiently transfected with two packaging plasmids and the replicon plasmid encoding the transgene of interest.
- the CMV-driven replicon launches the first saRNA encoding the nonstructural proteins (nsp1-4) and the antigen of interest which gets self-amplified in the transfected cells by means of the nsp1-4 alphavirus replication machinery.
- the two packaging plasmids separately encoding the capsid and envelope proteins provide all the necessary alphavirus structural proteins for packaging of the saRNA and VRP formation.
- VRPs bud out of the cell into the supernatants.
- the supernatants from transfected cells are then harvested, concentrated, and pelleted down by centrifugation through a 40% sucrose cushion.
- the resulting VRP pellet is resuspended in an appropriate buffer and titered.
- the purified VRP stocks are titrated in Vero cells by infection with serially diluted stocks.
- the transduced cells are detected by staining for double stranded RNA (dsRNA) replication intermediates using a dsRNA specific mouse monoclonal antibody (J2, Jena Bioscience) and analysed by flow cytometry.
- dsRNA double stranded RNA
- the two packaging plasmids used for production of VEEV-VRPs express: 1) A mutant VEEV TC83 capsid under the control of the CMV promoter.
- This mutant capsid (Cm) has amino acid substitutions that inactivate the nuclear localization signal (NLS) of the protein (DAPPEGPSAKKPKK sequence was mutated to GGGGEGPSAAAPKK) 2)
- the VEEV TC83 envelope proteins (E3-E2-6k-E1 polyprotein) which gets proteolytically processed in the cells resulting in the expression of mature VEEV TC83 envelope proteins under the control of a CMV promoter.
- the two packaging plasmids used for production of SFV-VRPs encode a CMV-driven SINV/SFV “mini genome” composed of the first 302 bp of the SINV genome [i.e., 5’UTR and known nsp1 replication enhancer element, nsp1(enh)], the 3’UTR of SFV followed by A poly-A stretch plus the HDV antigenomic ribozyme sequence and one of the following SFV structural proteins: 1) A mutant SFV capsid containing an S219A mutation that abolishes the self- cleaving activity of the protein.
- the mutant SFV capsid was cloned in frame with the partial SINV nsp1(enh) and separated by a F2A self-cleaving peptide [SINVnsp1(enh) – F2A- Capsid].
- the F2A peptide allowed for expression of a SFV capsid separated from SINV nsp1 sequences.
- the SFV envelope proteins E3-E2-6k-E1 polyprotein
- VRP-BN001 VRP-BN010 using different cell lines in vitro.
- VRP-BN001 VEEV-VRP-OVA-BFP
- VRP-BN010 SFV-VRP-OVA-BFP
- BFP blue fluorescent protein
- OVA ovalbumin
- VEEV-VRP- or SFV-VRP-induced OVA secretion in two different murine cancer cell lines; 4T1 mammary carcinoma cell line and CT26 colon carcinoma cells.
- cells were infected with 30 transduction unit/cell (TU/cell) VRPs. After one hour, VRP containing cell culture media was removed and the cells were cultured for 5 days. Cell culture media was replaced with fresh media in every 24h after infection for 5 days and frozen at -20°C. At the end of the experiment, OVA protein amount in all supernatant samples was measured by ELISA to determine the kinetics of VRP-induced antigen expression after infections.
- VEEV-VRP-OVA-BFP VRP-BN001
- SFV-VRP-OVA-BFP VRP-BN010
- VEEV-VRP-induced OVA expression was stronger than SFV-VRP, and at 120h time point we could still detect very low amounts of OVA in the supernatant of VEEV-VRP infected cells (Figure 1B).
- VEEV-VRP induced OVA secretion by CT26 cells were much higher than 4T1 cells ( Figure 1A, 1B).
- This data shows that level of antigen expression upon VRPs might alter depending on the cell line.
- antigen expression is strongest during first 48h after infection and starts to decrease after this time point for both VRPs ( Figure 1A, 1B). Fibroblasts are important components of tumor microenvironment and might play an essential role in the outcome of immunotherapies.
- VRP-infection leads to decreased cell viability in CT26, 4T1 and A31 cells in vitro.
- 4T1 and CT26 cell lines we have observed reduced cell confluency and abnormal morphology of the cells after VRP-BN001 and VRP-BN010 infections.
- Example 4 Detection of VEEV-VRP biodistribution by qPCR 6 hours after intratumoral administration
- Our VRP platform is a promising vaccine candidate based on our results showing the efficient activation of antigen-specific T cells both in tumor and infectious disease models as well as strong antibody responses seen in our EBV-NHP study.
- our knowledge about how VRPs induce immune responses is limited. Regarding their smaller size and possibly different cell tropism, we assume that biodistribution of the VRPs as well as their mechanism of action to prime T and B cells could differ from our vaccine platform MVA.
- VRPs end up in distant organs upon intratumoral (IT) administration.
- Mice bearing 4T1 tumors received either TNE buffer as a control or 1x10 8 Transfection Unit (TU) VEEV-VRP-OVA-BFP (VRP-BN001), which express Ovalbumin (OVA).
- TNE buffer as a control
- VEEV-VRP-OVA-BFP VRP-BN001
- Ovalbumin Ovalbumin
- VRPs could be detected not only in the tumor but also in distant organs after local injection.
- the highest OVA mRNA amount was detected 6h after the IT injection and the signal was significantly decreased at 24h time point.
- the strong signal we see in TdLN could be due to the smaller particle size of VRPs.
- VRPs could be more efficient in infecting target cells and expressing antigens.
- Example 5 Detection of VEEV-VRP biodistribution by immunofluorescence (IF) staining 6 hours after intratumoral administration As next step, we decided to employ a second method to check the biodistribution of VRP.
- IF immunofluorescence
- mice bearing B16.F10 tumors received either TNE buffer as a control or 1x10 8 TU VEEV-VRP-OVA-BFP (VRP-BN001), which express blue fluorescence protein (BFP) as an antigen.
- TNE buffer 1x10 8 TU VEEV-VRP-OVA-BFP
- BFP blue fluorescence protein
- BFP signal itself was very weak to be visualized, we used an anti- tagRFP antibody, which binds to the VRP-encoded BFP protein and a AF647-labelled secondary antibody (since anti-tag RFP antibody was not fluorescently labelled).
- DAPI was used to stain nuclei.
- BFP signal was seen both in the tumor and TdLN 6h after IT injection ( Figure 4A, 4B).
- both FITC-positive and negative cells were BFP + , suggesting that VRP might infect tumor cells, tumor infiltrating CD45 + leukocytes and other components of the tumor microenvironment (TME), like fibroblasts.
- Example 6 VEEV-VRP expressing tumor associated antigen (TAA) Gp70 (VRP- BN005) induces a strong anti-tumorigenic immune response in CT26 colon carcinoma model.
- TAA tumor associated antigen
- Vaccine Development department generated VEEV-VRP expressing the endogenous retroviral element Gp70 (VEEV-VRP-Gp70; VRP-BN005; will be mentioned as VRP-Gp70 from here on) as a tumor associated antigen (TAA).
- CT26 murine colorectal carcinoma model Treatment of subcutaneously implanted CT26.WT tumor bearers started when tumor size reached approximately 5.5 x 5.5 mm and mice received 1.00E+08 TU VRP-Gp70 via intratumoral (IT) injection. This time point was defined as day 0 and boost immunizations has been performed on day 4 and day 7, as indicated on table 1. Table 1.
- Study Design 1st injection; day 0 2nd injection; day 4 3rd injection; day 7 Group Analysis Test Article Dose Test Article Dose Test Article Dose A PBS n/a PBS n/a PBS n/a Blood- VRP-Gp70 VRP-Gp70 VRP-Gp70 restimulation 1.00E+08 1.00E+08 1.00E+08 B (VRP- TU (VRP- TU (VRP- TU day 9 BN005) BN005) BN005) IT treatment with PBS resulted in a slight delay in tumor growth, reflecting that CT26.WT tumors are immunogenic.
- VRP-BN005 VRP-Gp70 induces an anti-tumorigenic immune response in B16.F10 melanoma model.
- VRP-Gp70 induces an anti-tumorigenic immune response in B16.F10 melanoma model.
- B16.F10 melanoma model Treatment of subcutaneously implanted B16.F10 tumor-bearing mice started when tumors reached a size of approximately 4.5 x 4.5 mm.
- VRP-BN005 VRP-Gp70
- VRP-Gp70 VRP-Gp70
- CT26 colon carcinoma model has been classified as a hot tumor, which is rich for lymphocyte infiltration and shown to be responsive to checkpoint inhibitor treatments (Mosely et al., Cancer Imm Res.2016).
- VRP-Gp70 repetitive IT injections resulted in eradication of CT26.WT tumors in 5 out of 8 mice and in some animals, VRP-Gp70-induced antigen-specific CD8 T cell response correlated with the anti-tumor effect, as shown by the tumor growth graphs with dashed lines ( Figure 5A).
- Figure 5A dashed lines
- TCM central memory
- TEM effector memory
- T RM tissue-resident memory T cells
- T CM tissue-resident memory T cells
- T EM tissue-resident memory T cells
- the main role of T RM cells is to protect epithelial tissues against inflammation or infectious diseases.
- the main cell surface markers used to define these cells are CD103, CD49a and CD69, and these markers might vary according to the tissue.
- CD103 is expressed by most CD8 + T RM cells and suggested to be involved in T cell homing into epithelia.
- C- type lectin CD69 has a role in the retention of cells in the tissues.
- CD49a is used to differentiate two T RM cell subsets. It has been shown that CD49a expressing cells produce perforin and IFN ⁇ (Mami-Chouaib at al., J Immunother Cancer.2018). While the invention is not bound or limited by any particular scientific principle or mechanism of operation, since no tumor growth was observed after tumor rechallenge in VRP-Gp70 cured mice, we explored the presence of T RM cells, since they might be involved during the prevention of growth and/or elimination of tumor cells at the injection area.
- CD69 and CD103 were checked in CD8 T cell T EM subset. As shown in Figure 8D and 8E, both CD69 + CD103 + and CD69 + CD103- TRM cells were detected in the skin samples of cured mice. We observed much higher percentage of CD69 + CD103- T RM cells in the skin than CD69 + CD103 + cells, suggesting that these two populations might differ in their function ( Figure 8D, 8E).
- CD4 T cells in the TdLN, non-dLN, spleen and skin samples on day thirty-three after tumor rechallenge. Since we do not have any tools to detect antigen specific CD4 T cells by flow cytometry, we could not detect the percentage of these cells.
- CD4 TCM and TEM subsets using the same gating strategy to CD8 T cells as explained before.
- Increased accumulation of CD4 TCM subset was detected in the TdLN tissues of cured mice in comparison to control mice, whereas in skin we could not really detect these cells (Figure 9A).
- the elevated accumulation of CD4 TEM subset was more prominent in the tissues of cured mice compared to controls and the highest accumulation was detected in the skin ( Figure 9B).
- frequency of CD69 + CD103 + and CD69 + CD103- TRM in all organs of cured mice showed similarity.
- the highest amount of AH-1 + specific CD8 T cells were detected in the skin of cured mice, and they were comprised of mostly T EM cells.
- CD69 + CD103- T RM cells created the largest part of this population.
- CD69 + CD103 + T RM cells could detect some CD69 + CD103 + T RM cells as well, suggesting that T EM and T RM cells are having important roles during the elimination and /or growth inhibition of subcutaneously injected tumor cells.
- CD4 T cells we do not have working tetramers or pentamers to check antigen specific CD4 T cell responses as we do with the CD8 counterparts. Still, we have observed an increase of CD4 T EM cells in the organ of all cured mice and the increase was the most pronounced in the skin, like what we observed in CD8 T cells. Indeed, half of the skin-infiltrating CD4 T cells expressed TRM – associated markers such as CD103 and/ or CD69. This result suggests that, as antigen specific CD8 + TRM, CD4 + TRM do play a role in the control of tumor regrowth upon rechallenge.
- Example 9 VRP-BN005 (VRP-Gp70) IT administration modulates tumor microenvironment (TME), which results in elevated infiltration of T lymphocytes and NK cells.
- TME tumor microenvironment
- One strategy of cancer immunotherapy is to induce reprogramming of the immunosuppressive tumor microenvironment (TME) into an inflammatory one, where innate and adaptive anti-tumor immune responses can be developed.
- IT injection of rMVA induces inflammation in the TME and adjuvating of MVA-OVA with the co-stimulatory receptor 4-1BBL resulted in increased levels of inflammatory cytokines in the tumor, thereby resulting in a profoundly enhanced therapeutic efficacy (Hinterberger et al, JITC 2021).
- VRP-Gp70 IT injected mice showed higher infiltration of CD45 + leukocytes into the tumor at day one compared to the control and as expected, this increase was more pronounced on day seven ( Figure 10B).
- Both CD4 + and CD8 + T cells were increased in the TME one week after the immunization and as cell numbers showed, CD8 + T cells largely contributed to the increase in CD45 + cells in the tumor by day seven.
- NK cell number in the TME was higher in VRP-Gp70 treated mice compared to saline on day one and day seven (Figure 10B). To sum up, our data indicate that single IT immunization of VRP-Gp70 leads to significantly smaller B16.F10 tumors in weight one week after the treatment.
- IL-12 is an important cytokine, which has strong effects on CD8 + cytotoxic T cells and type 1 CD4 + T helper cells as well as NK cells. It has been shown that intratumoral (i.t.) injection of a lipid nanoparticle (LNP)-formulated IL-12 mRNA or IL-12-engineered tumor-specific CD8 + T cells have been proven successful in several preclinical models (Etxeberria et al., Cancer Cell 2019; Hewitt et al., Trans Cancer Mec and Ther.2020). Furthermore, i.t.
- LNP lipid nanoparticle
- IL-12-encoding plasmid DNA was leading to a significant clinical response in metastatic melanoma patients and the treatment was well-tolerated (Heinzerling et al., Hum Gene Ther. 2005). Based on its crucial role in modulating immune system and efficacy in inducing a potent anti-tumorigenic response against various cancer types, we decided to produce a VEEV-VRP expressing vector expressing a single chain IL-12 p40p35 fusion protein (IL-12sc) and test its effect in vitro and in vivo.
- IL-12sc single chain IL-12 p40p35 fusion protein
- VRP-IL12 IL-12 expressing VEEV-VRP
- VRP-BN006 recombinant IL- 12p70
- VRP-BN015 recombinant IL-12p70
- VRP-BN015 recombinant IL-12p70
- VRP-IL12 infection resulted in increased activation and cytotoxicity of both CD8 + T cells and NK cells in vitro, compared to VRP infection ( Figure 11A, 11B).
- analysis of cell supernatants after 18h showed enhanced secretion of IL- 12 and IL-12 induced IFN ⁇ by VRP-IL12 infected cells compared to VRP infected cells ( Figure 11C, 11D).
- VRP-IL12 IT administration improves tumor growth control and survival in poorly immunogenic B16.F10 tumor model without causing any toxicity.
- VRP-BN006 VRP-BN006
- VRP-BN005 VRP-BN005
- VRP-BN005 VRP-BN005
- IL-12 adjuvating VRP with IL-12 could strengthen the therapeutic outcome of VRP.
- treatment of subcutaneously implanted B16.F10 tumor bearers started when tumor size reached approximately 5 x 5 mm and mice received 1.00E+08 TU VRP or increasing titers of VRP-IL12 via intratumoral (IT) injection. This time point was defined as day 0 and boost immunizations have been done on day 5 and day 8, as indicated on table 3.
- VRP-IL12 For the VRP titration, 1x10 6 , 1x10 7 or 1x10 8 TU VRP-IL12 was intratumorally administered. Interestingly, we did not observe big differences in the anti-tumorigenic effect of VRP-IL12 between different doses and the lowest dose was already strong enough to promote an anti-tumor response in mice ( Figure 12C-F). Furthermore, compared to VRP treated control mice, it is clearly seen that adjuvating of IL-12 improved the anti-tumorigenic efficacy of VRP and increased the survival of mice. Very importantly, even after the administration of the highest dose of VRP-IL12, no IL-12 related toxic effects have been observed.
- Example 12 Combination of VRP-IL12 with a TAA expressing VRP elicits a more potent anti-tumorigenic response in poorly immunogenic B16.F10 tumor model. Since VRP-IL12 did not express a tumor-associated antigen (TAA), we hypothesized that combining VRPs expressing the TAA Gp70 and IL-12 might strengthen the anti- tumorigenic immune response via induction of tumor-specific CD8 + T cell responses in B16.F10 melanoma model.
- TAA tumor-associated antigen
- B16.F10 tumor-bearing mice received three consecutive IT injections of TNE buffer, a combination of VRP-BN015 (VRP) plus VRP- BN005 (VRP-Gp70) or a combination of VRP-BN005 (VRP-Gp70) plus VRP-BN006 (VRP-IL12).
- VRP VRP-BN015
- VRP-BN005 VRP-BN005
- VRP-IL12 VRP-IL12
- Example 13 Comparison of different IT immunization schedules for VRP-IL12. After showing enhanced anti-tumorigenic effect of VRP upon adjuvating with IL-12, we questioned whether using different immunization schedules could differ the outcome of this therapy or not. To test this, B16.F10 tumor bearing mice have been vaccinated intratumorally (IT) with 1x10 8 TU VRP (VRP-BN0015) or VRP-IL12 (VRP-BN006) and the first immunization day was accepted as day 0. Boost immunizations were repeated either on day 5 and 8 or on day 7 and 14 as shown on Table 4.
- IT intratumorally
- mice were IT injected with TNE buffer on day 0, 5 and 8.
- Table 4 Study Design G roup Day 0 Day 5 Day 7 Day 8 Day 14 A TNE buffer TNE b uffer TNE buffer B VRP (VRP- VRP (VRP- VRP (VRP- B N015) BN015) BN015) 1.00E+08 TU 1.00E+08 TU 1.00E+08 TU C VRP-IL12 (VRP- VRP-IL12 (VRP- VRP-IL12 B N006) BN006) (VRP-BN006) 1.00E+08 TU 1.00E+08 TU 1.00E+08 TU D VRP (VRP- VRP (VRP- VRP (VRP- B N015) BN015) BN015) 1.00E+08 TU 1.00E+08 TU E VRP-IL12 (VRP- VRP-IL12 VRP-IL12 B N006) (VRP-BN006) (VRP-BN006) (VRP-BN006) (V
- VRP-IL12 IT administration on day 0-5-8 schedule improved the tumor growth control and survival compared to VRP treated mice ( Figure 14C).
- VRP and VRP-IL12 IT immunizations were done on weekly schedules, we 10 have observed that although both treatments caused tumor growth control similarly to the mice immunized on day 0-5-8 schedule ( Figure 14D, E), weekly treatments resulted in shorter median survival.
- median survival was detected as 29 vs 21 days, and for VRP-IL12 groups it was 56 days vs 46 days after day 0-5-8 immunization and day 0-7-14 immunization, respectively.
- VRP encoding IL-12 causes enhanced tumor growth control and longer survival even if it is administered to the mice weekly, compared to VRP-treated mice.
- repetition of VRP or VRP-IL12 IT treatment with shorter intervals might be more effective in prolonging the survival of mice than weekly administration.
- this experiment should be repeated with bigger group sizes.
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Abstract
La présente invention concerne un ARN auto-amplifiant (saRNA) destiné à être utilisé dans le traitement de tumeurs. Le traitement est fourni en utilisant le saRNA, en particulier un VRP comprenant un acide nucléique codant pour un antigène associé à une tumeur (TAA) ainsi que l'IL-12. Dans certains modes de réalisation de l'invention, les procédés comprennent l'injection de ces saRNA par voie intratumorale. Dans certains modes de réalisation, les saRNA sont injectés par voie intrapéritonéale pour stimuler une réponse immunitaire à des tumeurs péritonéales.
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