Patent Application
20220370581
The sequence listing submitted via EFS, in compliance with 37 CFR § 1.52(e)(5), is incorporated herein by reference. The sequence listing text file submitted via EFS contains the file “CP-5231-US_SEQ_LIST”, created on May 16, 2022, which is 12,586 bytes in size.
The present disclosure relates to a vaccine and a method for treating cancer. More particularly, the present disclosure relates to a vaccine specific for tumor antigen and a method for treating cancer used thereof.
The main therapeutic strategies for cancer therapy are radiotherapy, chemotherapy, targeted therapy and surgery. Even the advance in drug development and surgery techniques, the 5-year survival rate of late-stage cancer patients is still poor, suggesting that developing novel therapeutic strategies is urgent such as cancer immunotherapy. Immunotherapy depends on the reactivation of immune system to eliminate tumors such as immune checkpoint blockade, cell therapy and cancer vaccine. Although the clinical response of immune checkpoint blockade is promising in several malignancies, the application is limited by the status of DNA mismatch repair deficiency (10-15% cancer patients) and density of immune cell infiltration, indicating majority of cancer patients is not suitable for immune checkpoint blockade. Therefore, developing novel immunotherapy strategies such as neoantigen-based immunotherapy is critical.
Neoantigens are derived from the somatic mutations during cancer progression, which elicit tumor-specific immune response. With the breakthrough of next-generation sequencing technology, identifying personalized neoantigens to develop cancer vaccines to activate tumor-specific immune responses is feasible, while radiotherapy (RT) and chemotherapy (CT) cannot only increase tumor antigen release but also change tumor microenvironment to a more permissible one. It is anticipated that neoantigen-based cancer vaccine when combined with RT and immunogenic CT can potentially achieve sustainable disease control in those who refractory to the conventional treatments.
Moreover, monotherapy with neoantigens peptide vaccine alone was not effective enough in eliminating tumor and the majority of mutations differ from patient to patient, making neoantigens more personalized with high cost and long time. Therefore, targeting the frequently shared neoantigens and optimizing the delivery of neoantigen-based immunotherapy are good way to solve this problem.
According to one aspect of the present disclosure is to provide a vaccine including a vector and a transgene. The transgene encodes a plurality of peptides and is packaged in the vector, wherein the peptides in order include a secretion signal peptide, at least one tumor antigen, at least one co-inhibitory peptide and a toll-like receptor 9 (TLR9) antagonist. The at least one tumor antigen is a subtraction of tumor and normal cell antigens. The at least one co-inhibitory peptide includes programmed death-ligand 1 (PD-L1) antagonist, programmed cell death protein-1 (PD-1) antagonist or a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antagonist.
According to another aspect of the present disclosure is to provide a method for treating cancer. The method includes administering the vaccine according to the foregoing aspect to a subject in need for a treatment of cancer to induce an anti-tumor immune response in the subject.
According to still another aspect of the present disclosure is to provide a method for treating cancer by a cancer vaccine cocktail therapy including following steps. The vaccine according to the foregoing aspect is administered to a subject in need for a treatment of cancer to induce an immune priming against the at least one tumor antigen in the subject. An enhancer is administered to the subject to enhance local tumor control in the subject. A booster is administered to the subject to prevent local recurrence and metastasis in the subject.
The present disclosure can be more fully understood by reading the following detailed description of the embodiment, with reference made to the accompanying drawings as follows:
Please refer to
The vector 110 is for enhancing tumor antigens expression with diverse tropism, and can be a vaccinia viral vector, an adeno-associated virus (AAV) vector or a nanoparticle. Preferably, the AAV vector can be an adeno-associated virus 2 (AAV2) vector or an adeno-associated virus 6 (AAV6) vector. The nanoparticle can include but not limit to a liposome-derived delivery system [such as dicetyl phosphate-tetraethylenepentamine-based polycation liposome (TEPA-PCL), lipoplex (like DOTMA:cholesterol:TPGS lipoplex or DDAB:cholesterol:TPGS lipoplex), cationic liposome-hyaluronic acid (LPH) nanoparticle], a lipid nanoparticle (LNP), a polyethyleneimine (PEI) or PEI-conjugate, a dendrimer nanoparticle, a poly(amidoamine) (PAMAM) nanoparticle, poly(lactide-co-glycolide) (PLGA) nanoparticle, an atelocollagen nanoparticle and a silica nanoparticle.
The transgene 120 encodes a plurality of peptides, wherein the peptides in order include a secretion signal peptide 121, at least one tumor antigen 122, a co-inhibitory peptide 123 and a toll-like receptor 9 (TLR9) antagonist 124.
The secretion signal peptide 121 is for assisting tumor antigen secretion. Preferably, the secretion signal peptide 121 can be an interleukin 2 signal peptide (IL2 sp) or an interleukin 12 signal peptide (IL12 sp).
The at least one tumor antigen 122 is for increasing an anti-tumor immune response in a subject in need for a treatment of cancer, wherein is the at least one tumor antigen 122 is a subtraction of tumor and normal cell antigens. Preferably, the at least one tumor antigen 122 can be selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an oncogenic mutation, an aberrantly expressed tumor-specific antigen (aeTSA) and a shared neoantigen (neoAg). In addition, the at least one tumor antigen 122 can be selected by comparing whole exome sequencing of matched tumor and normal cell DNA from the subject to identify tumor-specific somatic mutations (neoantigens), and then selecting polynucleotides encoding the neoantigens from a pre-existing library of neoantigen-encoding polynucleotides. The TAA was highly expressed on tumor cells with lower expression on normal cells. For example, but are not limited to, the TAA in breast cancer includes mammaglobin-A overexpressed in breast cancer, prostate specific antigen (PSA), melanoma antigen recognized by T cells (MART 1), melanocyte protein PMEL, Bcr/Abl tyrosin-kinase, HPVE6, E7, MZ2-E, MAGE-1 and MUC-1. The TSAs were found on cancer cells only, not on healthy cells. For example, but are not limited to, the TSA includes driver genes KRAS-G12/13 codon hotspot mutations, TP53 hotspot mutations, PIK3CA hotspot mutations, BRAF mutations and frameshift mutations. The aeTSA derives from aberrant expression of unmutated transcripts that are not expressed in any normal somatic cell, including medullary thymic epithelial cells (mTEC), which orchestrate central immune tolerance.
The co-inhibitory peptide 123 is for blocking the co-inhibitory signals in dendritic cell (DC) and increase antigen presentation ability of DC, wherein the co-inhibitory peptide 123 includes programmed death-ligand 1 (PD-L1) antagonist, programmed cell death protein-1 (PD-1) antagonist or a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antagonist. Preferably, the PD-L1 antagonist can include a PL-L1 trap and a PD-1 peptide, the PD-1 antagonist can include a PD-1 trap and a PD-L1/PD-L2 peptide, and the CTLA4 antagonist can include a CTLA4 trap and an antagonistic antibody against CTLA4.
The TLR9 antagonist 124 is an anti-viral clearance sequence for attenuating the innate immunity of viral clearance and maintain high antigen load. Preferably, the TLR9 antagonist 124 can be selected from a CpG oligonucleotide TLR9 binding domain, a TLR decoy peptide or a CpG binding sequence.
In addition, the vaccine 100 of the present disclosure can includes a co-stimulatory peptide for increasing the recruitment and activation of DC, wherein the co-stimulatory peptide is selected from a granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 12 (IL12) and interferon (IFNs).
According to one embodiment, the method for treating cancer includes administering the vaccine according to the foregoing aspect to a subject in need for a treatment of cancer to induce an anti-tumor immune response in the subject. Preferably, the method for treating cancer in this embodiment can further include administering radiotherapy to the subject. In addition, the vaccine of the present disclosure can be under conditions wherein the peptides are expressed and synergistically promote a tumor-specific immune response in the subject, and synergistically prolong subject survival.
“Cancer” refers to a physiological condition in a mammal characterized by a disorder of cell growth. A “tumor” includes one or more cancer cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include breast cancer, colon cancer, rectal cancer, colorectal cancer, lung cancer including small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenoma, and lung squamous cell carcinoma, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, and head and neck cancer.
An “effective amount” refers to an amount of the vaccine of the present disclosure effective to “treat” a disease or disorder in a subject. The effective amount is to some extent related to the biological or medical response of the tissue, system, animal or human to whom it is administered, for example, when administered, it is sufficient to prevent the development of one or more diseases or conditions or to alleviate the symptoms of one or more conditions or conditions being treated to a certain extent. A therapeutically effective amount will vary depending on the disease and its severity, as well as the age and weight of the mammal to be treated.
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Reference will now be made in detail to the present embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings. However, following examples are for illustration only, but the present disclosure is not limited thereto. For example, the vector used in following examples is the AAV vector, but the vector is used to deliver the transgene to the target cell, so it is expected that other vectors, such as the vaccinia viral vector or the nanoparticle, can be used to achieve the same effect.
First, we uncovered that the shared neoantigens (hereafter “neoAgs”) profiles in the residual tumors after chemotherapy (CT) and radiotherapy (RT), and established the neoAgs profiles within refractory and relapse tumors. These neoAgs can utilize to develop in vitro diagnosis (IVD) testing and antibody-based immunotherapy drugs. Moreover, these neoAgs can be the key ingredients for improving the tumor-specific of DC vaccine and DC-DIK cell therapy, and develop a neoAgs peptide-based cancer vaccine immunotherapy to improve the therapeutic efficacy of RT, CT and cell therapy. Please refer to Table 1, which is a list of neoAgs in mouse colon carcinoma CT26 cell line (hereinafter referred to as “CT26 cell”).
Further, we developed a neoAg peptide-based cancer vaccine including the aforementioned neoAgs to confirm the therapeutic effect on cancer treatment.
Please refer to 3A, which is a schematic view showing a construction of Example 1 of a neoAg peptide-based cancer vaccine. In
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To demonstrate the therapeutic efficacy of the Example 1 of the neoAg peptide-based cancer vaccine, a colorectal cancer mouse model is established first. Six-week-old female BALB/c mice were inoculated subcutaneously 2×105 CT26 cells with 20% matrigel (Corning, Union City, Calif., USA) into the lower right leg. After 8 days, the colorectal cancer mice were randomly assigned into different groups, Example 1 and Comparison 1 (1×108 vg) were administered via intramuscular injection every 6 days for 3 times and boost the 4th times on Day 25. For radiotherapy, colorectal cancer mice after complete anesthesia were placed the right leg in the irradiation field, we give one treatment, the local tumors were received 5 Gy fractionated radiotherapy on Day 11. At the same time, the tumor volume was measured and calculated by the formula: V=(L×W2)/2 every 3 days until Day 28 sacrificed. The tumor tissues were collected for following immune analysis.
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To demonstrate the effect of the Example 1 of the neoAg peptide-based cancer vaccine on infiltration of immune cells for anti-tumor immunity, isolation of tumor-infiltrating lymphocytes was performed. Isolated fresh tumors from colorectal cancer mice of Example 1, Example 2, Comparison 1 and Comparison 2, place the tumor in a 6 cm dish containing 5 ml of RPMI 1640 media at room temperature, then mince the tumor into 1-2 mm small pieces using sterile blade. Prepare a 50 ml conical tube, place a 70 μm cell strainer in the top, and transfer all the tumor tissue to the strainer by sterile dropper. If there are pieces of tissue left on, use the rubber of 5 mL syringe and add another RPMI 1640 media to mesh the tissue through the strainer. Carefully transfer all the cell solution into the 15 mL conical tube containing Ficoll-Paque at the bottom. Centrifuge at 1025× g for 20 mins at 20° C. with slow acceleration and brakes turned off. Carefully transfer the layer of mononuclear cells to a new 50 ml conical tube using a sterile pipet, add 10 mL RPMI 1640 media then centrifuge at 650× g for 10 mins at 20° C. Remove the supernatant, and gently resuspend cells in 10 ml of complete RPMI 1640 media, centrifuge at 650× g for 10 mins at 20° C. again. Remove the supernatant and add 1 mL RPMI 1640 media resuspend, these are the extracted tumor-infiltrating lymphocytes (TILs).
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To demonstrate the effect of the Example 1 of the neoAg peptide-based cancer vaccine in tumor microenvironment (TME), isolation of tumor-infiltrating lymphocytes was performed. Colorectal cancer mice were randomly assigned into different groups, Example 1 and Comparison 1 (1×108 vg) and PBS were administered via intramuscular injection 4 times on Day 8, Day 14, Day 21 and Day 25, respectively. For radiotherapy, colorectal cancer mice after complete anesthesia were placed the right leg in the irradiation field, the local tumors were received 5 Gy fractionated radiotherapy twice on Day 11 and Day 18. The colorectal cancer mice were sacrificed on Day 28, and the tumor tissues were collected for immune analysis. The treatment strategy of Examples 1, 3, Comparisons 1, 3 and Controls 1, 3 are shown in Table 3.
Please refer to
Further, we developed AAV-based cancer vaccines including the TLR9 antagonist and different tumor antigens to confirm the therapeutic effect on cancer treatment. Please refer to
In
To demonstrate the therapeutic efficacy of the Examples 4, 6 and 8 of the AAV-based cancer vaccines, colorectal cancer mice were randomly assigned into different groups. Examples 4, 6 and 8 of the AAV-based cancer vaccines and Comparison 1 (1×108 vg) were administered via intramuscular injection 4 times on Day 8, Day 14, Day 21 and Day 25, respectively. For radiotherapy, colorectal cancer mice after complete anesthesia were placed the right leg in the irradiation field, the local tumors were received 5 Gy fractionated radiotherapy on Day 11. The tumor volume was measured and calculated by the formula: V=(L×W2)/2 every 3 days until Day 30 sacrificed, and the tumor tissues were collected for immune analysis. The treatment strategy of Examples 4-9 and Comparisons 1-2 are shown in Table 5.
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Further, we developed a vaccine of the present disclosure to confirm the therapeutic effect on cancer treatment. Please refer to
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To demonstrate the therapeutic efficacy of the Example 10 of the vaccine of the present disclosure, colorectal cancer mice were randomly assigned into different groups. Example 10 of the vaccine of the present disclosure and Comparison 4 (1×108 vg) were administered via intramuscular injection 4 times on Day 8, Day 14, Day 21 and Day 25, respectively. For radiotherapy, colorectal cancer mice after complete anesthesia were placed the right leg in the irradiation field, the local tumors were received 5 Gy fractionated radiotherapy once on Day 11 or twice on Day 11 and Day 17. At the same time, the tumor volume was measured and calculated by the formula: V=(L×W2)/2 every 3 days until Day 28 sacrificed.
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In
Further, we developed another vaccine of the present disclosure to confirm the therapeutic effect on cancer treatment. Please refer to
In
To demonstrate the therapeutic efficacy of the Example 13 of the vaccine of the present disclosure, colorectal cancer mice were randomly assigned into different groups. Example 13 of the vaccine of the present disclosure, Comparison 7 (1×108 vg) and PBS were administered via intramuscular injection 4 times on Day 8, Day 14, Day 21 and Day 25, respectively. For radiotherapy, colorectal cancer mice after complete anesthesia were placed the right leg in the irradiation field, the local tumors were received 5 Gy fractionated radiotherapy once on Day 11 or twice on Day 11 and Day 18. In addition, colorectal cancer mice were inoculated subcutaneously 3×105 CT26 cells with 20% matrigel for tumor rechallenge on Day 56. At the same time, the tumor volume was measured and calculated by the formula: V=(L×W2)/2 every 3 days, and flow cytometry was performed on Day 30. In addition, after intramuscular injection immunization with Example 13 of the vaccine of the present disclosure and Comparison 7, the levels of Glud1+ CD8 cells were measured in the blood of colorectal cancer mice by using a Glud1/MHC-I-specific tetramer assay.
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In
To further demonstrate that the vaccine of the present disclosure triggers high neoantigen immunogenicity to boost the therapeutic efficacy of radiotherapy, we generated still another vaccine carrying eight mutated TSAs, then vaccinated BALB/c mice bearing 4T1 tumors. Please refer to
In
To demonstrate the therapeutic efficacy of the Example 16 of the vaccine of the present disclosure, a mammary cancer mouse model is established first. Six-week-old female BALB/c mice were inoculated subcutaneously 3×105 4 T1 cells with 20% matrigel (Corning, Union City, Calif., USA) to obtained BALB/c mice bearing 4T1 tumors, which are poorly immunogenic mammary cancer cells. After 8 days, the mammary cancer mice were randomly assigned into different groups, Example 16 of the vaccine of the present disclosure, Comparison 10 (1×108 vg) and PBS were administered via intramuscular injection 4 times on Day 8, Day 14, Day 21 and Day 25, respectively. For radiotherapy, mammary cancer mice after complete anesthesia were placed in the irradiation field, the local tumors were received 5 Gy fractionated radiotherapy twice on Day 11 and Day 18. At the same time, the tumor volume was measured and calculated by the formula: V=(L×W2)/2 every 3 days, and flow cytometry was performed on Day 31.
Please refer to
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According to another embodiment, the method for treating cancer by a cancer vaccine cocktail therapy includes following steps. The vaccine according to the foregoing aspect is administered to a subject in need for a treatment of cancer to induce an immune priming against the at least one tumor antigen in the subject. An enhancer is administered to the subject to enhance local tumor control in the subject. A booster is administered to the subject to prevent local recurrence and metastasis in the subject.
According to the present disclosure, the at least one tumor antigen can be selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an oncogenic mutation, an aberrantly expressed tumor-specific antigen (aeTSA) and a shared neoantigen (neoAg). The enhancer can be a radiation, a chemotherapeutic agent, an immunomodulating agent, a targeted therapy drug, an antibody drug, or a combination thereof. The booster can be a cancer vaccine including the at least one tumor antigen or a therapeutic cell including the at least one tumor antigen. Preferably, the cancer vaccine including the at least one tumor antigen can be a DC-based cancer vaccine or a virus-based cancer vaccine, and the therapeutic cell including the at least one tumor antigen can be a CIK (cytokine-induced killer cell), a DC-CIK or a neoAg-pulsed DC-CIK. The booster also can be a therapeutic cell including an immune checkpoint protein, an immunosuppressive factor and/or an immunostimulatory factor. Preferably, the therapeutic cell including the immune checkpoint protein can be a CAR-T cell, a CAR-NK cell or an adoptive T cell.
Please further refer to
To demonstrate the therapeutic efficacy of the cancer vaccine cocktail therapy of the vaccine of the present disclosure, colorectal cancer mice were randomly assigned into different groups. Example 13 of the vaccine of the present disclosure and Comparison 7 (1×108 vg) were administered via intramuscular injection twice on Day 8 and Day 14, respectively. For radiotherapy as the enhancer, colorectal cancer mice after complete anesthesia were placed the right leg in the irradiation field, the local tumors were received 5 Gy fractionated radiotherapy 3 times on Day 11, Day 18 and Day 25. A neoAg-DC-CIK was used as the booster, and the neoAg-DC-CIK was administered via intramuscular injection twice on Day 21 and Day 31. In addition, αPD-1 as the immune checkpoint blockade (ICB) was administered twice on Day 16 and Day 23. At the same time, the tumor volume was measured and calculated by the formula: V=(L×W2)/2 every 3 days until Day 40 sacrificed.
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In
In summary, the vaccine of the present disclosure coexpresses the at least one co-inhibitory peptide and TLR9 antagonist to increase the at least one tumor antigen expression to activate tumor antigen specific T-cell responses. Therefore, the vaccine of the present disclosure holds great promise and advantages due to its clinical safety and low immunological clearance prior to sufficient transgene expression. In addition, the vaccine of the present disclosure can increase the therapeutic efficacy of radiotherapy against cancer. Thus, radiotherapy synergistically increases the therapeutic efficacy of the vaccine of the present disclosure including the co-inhibitory peptide-armed tumor antigen, providing a novel, safe and efficient tumor antigen-based immunotherapy. Furthermore, the cancer vaccine cocktail therapy of the present disclosure including administering the vaccine of the present disclosure, the enhancer and the booster can effectively inhibit tumor growth and inhibit tumor recurrence.
Although the present disclosure has been described in considerable detail with reference to certain embodiments thereof, other embodiments are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the embodiments contained herein.
It will be apparent to those skilled in the art that various modifications and variations can be made to the structure of the present disclosure without departing from the scope or spirit of the disclosure. In view of the foregoing, it is intended that the present disclosure cover modifications and variations of this disclosure provided they fall within the scope of the following claims.
This application claims the benefits of priority of U.S. Provisional Application No. 63/189,861, filed on May 18, 2021 and U.S. Provisional Application No. 63/308,568, filed on Feb. 10, 2022, the content of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63189861 | May 2021 | US | |
| 63308568 | Feb 2022 | US |
