Superantigen vaccine conjugates for treatment of cancer

Abstract

The present disclosure provides a composition comprising a vaccine conjugate having an SMEZ-2 vector. Also provided are methods for treating cancer comprising administering the vaccine conjugates provided herein.

Description

[0001] Priority Claim

[0002] This application claims the priority benefit of U.S. Provisional Application Serial Nos. 63 / 413,457 and 63 / 413,468, both filed on October 5, 2022, the entire contents of which are hereby incorporated by reference. Background of the Invention 1. Field of the Technology

[0003] The present disclosure generally relates to the field of molecular biology. More particularly, it relates to compositions comprising vaccine conjugates for the treatment of cancer.

[0004] 2. Related Art

[0005] Pancreatic ductal adenocarcinoma (PDAC) resulted in 56,700 new cancer cases and 45,700 deaths in 2019, and the lowest 5-year survival rate among all cancers, only 9% (Siegeletal., 2019). Current treatment options are limited and have remained essentially the same, with surgery, chemotherapy, gemcitabine, or FOLFIRINOX as the main treatment options (Oettle et al., 2013; Oettle et al., 2007; Conroy et al., 2011; Conroy et al., 2018). Vaccine therapies have previously been used in clinical trials for PDAC patients, but their success has been very limited. Phase I / II trials with synthetic KRAS-derived peptides showed detectable immune responses when administered with GM-CSF and are currently in clinical trials with checkpoint inhibition (NCT04117087) (Gjertsen et al., 1995; Gjertsen et al., 2001). The telomerase 16-aa peptide vaccine (GV1001), designed to bind to multiple MHC molecules, showed significant efficacy in Phase I / II, but the Phase III PrimoVax and TeloVac trials showed no survival benefit (Bernhardt et al., 2006; Gunturu et al., 2013). In addition, clinical trials of the gastrin-based vaccine (G17DT) and the poxvirus-based vaccine encoding carcinoembryonic antigen and mucin 1 (PANVAC-V) both showed no improvement in survival (Gilliam et al., 2012; Arlen et al., 2007). It is an allogeneic, irradiated pancreatic cancer cell line vaccine expressing GM-CSF and is currently undergoing clinical trials (with checkpoint inhibition) together with the attenuated, mesothelin-expressing Listeria-based CRS-207, which are given in a prime / boost format (NCT03190265) (Tsujikawa et al., 2020). However, the phase IIb ECLIPSE study using this vaccine combination alone showed no survival advantage over chemotherapy (Le et al., 2019). In addition to the overall failure of these vaccine therapies for PDAC, objective responses to other immunotherapies are also largely absent. Therefore, the lack of response of PDAC to existing immunotherapy options necessitates new treatment options to overcome these barriers.

[0006] Treating patients with multiple malignancies using cancer vaccines by targeting immune stimulation is attractive, but objective clinical responses are mostly limited (Rosenberg et al., 2004). Although the lack of response is a complex issue, it is mainly attributed to the tolerance of the thymus and T cells to self-antigens upregulated in multiple cancers (Melief et al., 2015). Neoantigens and tumor-associated antigens (TAAs) have recently been focused on for generating cancer vaccines because their expression is usually restricted or absent in non-malignant cells but is abnormal or overexpressed in cancer cells (Romero et al., 2016). Computational methods are commonly used to predict peptide epitopes of TAAs or neoantigens, both of which are immunogenic and bind effectively to MHC molecules for recognition by T cells (Nat Biotechnol., 2017). These methods are still mainly theoretical and have the complexity of multiple factors (peptide processing prediction, epitopes binding to MHC molecules, mutation directions for T cells, different MHC alleles in patients, etc.), and there is a huge gap to cross for these software-based algorithms to be clinically useful.

[0007] Currently, sipuleucel-T is the only approved cancer vaccine and is used to treat metastatic castration-resistant prostate cancer. Sipuleucel-T is an autologous dendritic cell vaccine and is prepared by extracting peripheral blood mononuclear leukocytes from a patient by leukapheresis, sending them to a manufacturing facility, and pulsing them with several rounds of a recombinant fusion protein (prostatic acid phosphatase (PAP) and granulocyte-macrophage colony stimulating factor (GM-CSF)) to generate mature antigen presenting cells (APCs). These activated cells are then reinfused into the patient to generate an immune response. Overall, sipuleucel-T has shown a 4.1-month median survival improvement, but it is a complex and expensive process with significant manufacturing hurdles (Kantoff et al., 2010, Hammerstrom et al., 2011). To date, many other cancer vaccine strategies have been used for different cancer types but have generally been unsuccessful (Melero et al., 2014). Although recent phase I data from a combination of an RNA vaccine candidate and checkpoint inhibition in melanoma have shown promising results, invigorating the clinical utility of vaccination against TAAs (Sahin et al., 2020), the need for cancer vaccines for treating cancers such as PDAC remains unmet.

[0008] Although there have been considerable advancements in the treatment of pediatric acute lymphoblastic leukemia (pALL), the effective treatment of pediatric acute myeloid leukemia (pAML) has lagged behind. pAML is rarer than pALL, accounting for approximately 20% of pediatric leukemia cases (Morais et al., 2020). However, it is also more lethal, with a five-year survival rate of only 67% to 68% (Siegelet al., 2022). One of the challenges in treating pAML is the heterogeneity of the disease. The French-American-British classification of AML divides the disease into eight subtypes, M0 to M7. Each subtype is characterized by a specific hematopoietic lineage and stage of maturation. While adult AML (aAML) is thought to be caused by the accumulation of somatic mutations, these mutations are relatively rare in pAML. Cytogenetic abnormalities leading to de novo AML are much more common in pediatric patients (Bolouri et al., 2017). These features make it challenging to develop targeted therapies applicable to most patients. Additionally, the primary focus of AML treatment has been on aAML, with the hope that they will also be applicable to pAML. However, the differences between aAML and pAML suggest that a treatment may be effective for one but not the other.

[0009] Currently, a CD33-targeted therapy has been FDA-approved for pAML - the antibody-drug conjugate (ADC) gemtuzumab ozogamicin (GO). GO consists of an anti-CD33 monoclonal antibody conjugated to a cytotoxic calicheamicin payload. After endocytosis of the ADC, the linker is cleaved, and calicheamicin induces cell death through double-strand DNA breaks. However, GO has several drawbacks suggesting that it may not be the best approach for targeting CD33. A major drawback is that the binding epitope of GO is on the Immunoglobulin-like V (IgV) domain. 50% of CD33-positive AML patients express the single nucleotide polymorphism (SNP) rs12459419C>T, which results in a CD33 splice variant lacking the IgV domain (Lamba et al., 2017). This renders GO ineffective in half of the patients for whom it is indicated, as they express a CD33 protein lacking the gemtuzumab binding epitope.

[0010] In addition, premature cleavage of the linker between gemtuzumab and the calicheamicin payload can result in off-target effects. Another ADC, inotuzumab ozogamicin, is a CD22-targeting agent that also uses calicheamicin. It has been shown that the use of these drugs can increase the risk of venoocclusive disease (VOD) in patients undergoing hematopoietic stem cell transplant (HSCT) (Ladha et al., 2020). Another potential problem with GO is resistance to calicheamicin. Previous studies have shown that the expression of P-glycoprotein (Pgp) on AML blasts can lead to drug efflux and reduced potency of GO (Linenberger et al., 2001).

[0011] Finally, in clinical trials comparing pAML patients receiving standard chemotherapy with pAML patients receiving chemotherapy with added GO, GO showed only modest improvement. Relative to standard chemotherapy, event-free survival (EFS) was significantly improved in patients receiving GO, but there was no difference in overall survival (OS). When patients were divided into low-risk, intermediate-risk, and high-risk groups, no significant improvement in event-free survival was shown (Gamis et al., 2014). Thus, there remains a significant need for new pAML treatments, and CD33 has not yet reached its full potential as a target. SUMMARY OF THE INVENTION

[0012] In a first embodiment, the present disclosure provides a vaccine conjugate comprising a mutant Streptococcal Mitogenic Exotoxin Z-2 (SMEZ-2) conjugated to at least one target protein or a fragment thereof, wherein the at least one target protein is overexpressed in cancer.

[0013] In some aspects, the mutant SMEZ-2 comprises the mutations W75L and K182Q. In certain aspects, the mutant SMEZ-2 comprises the mutations W75L, K182Q, and / or D42C. In some specific aspects, the mutant SMEZ-2 comprises the mutations W75L, K182Q, and D42C.

[0014] In some aspects, the conjugate comprises a linker, such as a linker between SMEZ-2 and the at least one target protein. In certain aspects, the linker is a peptide linker, such as a glycine-serine linker. For example, the linker is AIA or GGGGS.

[0015] In some aspects, at least one target protein is a neoantigen or a tumor-associated antigen (TAA). In some specific aspects, the target protein is anterior gradient 2 (AGR2), such as human AGR2. In some aspects, at least one target protein is human epidermal growth factor receptor 2 (HER2), prostate-specific membrane antigen (PSMA), melanoma-associated antigen 3 (MAGE-A3), NY-ESO-1, IL-8, or GDF-15. In some aspects, at least one target protein is CD33 or a fragment thereof, mesothelin (MSLN), B-cell maturation antigen (BCMA), GPRC5D, CD123, CLL-1 (CD371), CD19, CD30, or CD20. In some aspects, CD33 or a fragment thereof contains one or more amino acid substitutions. In some aspects, full-length CD33 has an amino acid substitution at asparagine 98. In some aspects, CD33 contains a truncated form of CD33 consisting of the CD33-IgC domain, such as CD33-IgC containing an amino acid substitution at aspartic acid 231 or cysteine 154. In some aspects, one or more amino acid substitutions are located at D231, D246, C154, and / or C169. In some aspects, one or more amino acid substitutions are D231E, D246E, C154S, and / or C169S.

[0016] In some aspects, the vaccine conjugate contains at least a second target protein or a fragment thereof. In some aspects, at least one target protein is CD38.

[0017] In some aspects, at least one target protein is an immune checkpoint protein. In some specific aspects, the immune checkpoint protein is CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR.

[0018] Another embodiment provides a pharmaceutical composition comprising a vaccine conjugate of the present embodiment (e.g., a vaccine conjugate comprising a mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2) conjugated to at least one target protein or a fragment thereof, wherein at least one target protein is overexpressed in cancer) and an adjuvant. In some aspects, the adjuvant is incomplete Freund's adjuvant. In certain aspects, the conjugate and incomplete Freund's Adjuvant (IFA) are formulated as a 1:1 emulsion. In some aspects, the conjugate is formulated for intravenous infusion, subcutaneous injection, or intramuscular injection.

[0019] Another embodiment provides an expression vector comprising a sequence encoding mutant SMEZ-2 fused to a sequence encoding a target protein or a fragment thereof. In some aspects, the target protein is a neoantigen or a TAA. In certain aspects, the vector encodes a vaccine conjugate of the present embodiment (e.g., a vaccine conjugate comprising a mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2) conjugated to at least one target protein or a fragment thereof). Also provided herein are host cells comprising the expression vector of the present embodiment, such as Escherichia coli (E. coli), human embryonic kidney cells (HEK293), or Chinese hamster ovary (CHO) cells.

[0020] In another embodiment, a method for stimulating an immune response in a subject is provided, which comprises administering to the subject an effective amount of a vaccine conjugate of the present embodiment (e.g., a vaccine conjugate comprising a mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2) conjugated to at least one target protein or a fragment thereof, wherein at least one target protein is overexpressed in cancer) or a pharmaceutical composition of the present embodiment.

[0021] In some aspects, the immune response is an anti-cancer immune response. In certain aspects, the subject has cancer. In some particular aspects, the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, urogenital system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer, or hematopoietic system cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary cancer, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma tumor, multiple endocrine neoplasia type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In some specific aspects, the cancer is pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC). In certain aspects, the cancer is breast cancer. In some aspects, the cancer is multiple myeloma or lymphoma, such as T cell non-Hodgkin’s lymphoma.

[0022] In certain aspects, the immune response is an anti-AGR2 specific immune response. In some aspects, the anti-AGR2 specific immune response is detected by measuring an increase in the titer of AGR2-specific immunoglobulins in a blood sample of the subject. In other aspects, the immune response is an anti-CD38, anti-PD1, anti-CTLA4, or anti-PDL1 response, and is measured by measuring an increase in the titer of CD38-, PD1-, CTLA4-, or PDL1-specific immunoglobulins in a blood sample of the subject.

[0023] In some aspects, the conjugate is administered by injection, such as intravenous infusion.

[0024] In other aspects, the method further comprises administering a second anti-cancer treatment to the subject. In some aspects, the second anti-cancer treatment is immunotherapy, chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy. In some aspects, the second anti-cancer treatment is immunotherapy, such as immune checkpoint inhibitors. For example, immune checkpoint inhibitors are inhibitors selected from CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR. In some specific aspects, the immune checkpoint inhibitor comprises an anti-PD1 agent, such as an anti-PD1 antibody, an anti-PDL1 antibody or an anti-PDL2 antibody. In certain aspects, the anti-PD1 agent is nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA or AMP-224. In some aspects, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. For example, the anti-CTLA-4 antibody is tremelimumab, or ipilimumab. In some aspects, the additional anti-cancer treatment comprises a TLR9 agonist, such as CpG ODN1826, and / or a CD40 agonist, such as a CD40 agonist antibody. In certain aspects, an immune checkpoint inhibitor and a TLR9 agonist are administered to the subject.

[0025] Another embodiment provides a method of treating a subject having cancer, comprising administering to the subject the vaccine conjugate of this embodiment (e.g., a vaccine conjugate comprising a mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2) conjugated to at least one target protein or a fragment thereof, wherein at least one target protein is overexpressed in cancer) or the pharmaceutical composition of this embodiment.

[0026] In some aspects, the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, urogenital system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, cholangiocarcinoma, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma tumor, multiple endocrine neoplasia type I and type II tumors, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer. In some specific aspects, the cancer is pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC). In some specific aspects, the cancer is breast cancer. In some aspects, the cancer is multiple myeloma or lymphoma, such as T-cell non-Hodgkin lymphoma.

[0027] In some other aspects, the method further comprises administering to the subject at least one immune checkpoint inhibitor. In some aspects, administering to the subject at least one immune checkpoint inhibitor comprises administering at least one immune checkpoint inhibitor prior to the vaccine conjugate. In some other aspects, administering to the subject at least one immune checkpoint inhibitor comprises administering at least one immune checkpoint inhibitor after or concurrently with the vaccine conjugate. For example, the immune checkpoint inhibitor is selected from inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR. In some specific aspects, the immune checkpoint inhibitor comprises an anti-PD1 agent, such as an anti-PD1 antibody, an anti-PDL1 antibody or an anti-PDL2 antibody. In certain aspects, the anti-PD1 / PDL agent is nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA or AMP-224. In some aspects, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. For example, the anti-CTLA-4 antibody is tremelimumab, or ipilimumab. In some aspects, two immune checkpoint inhibitors are administered to the subject. In some specific aspects, the two immune checkpoint inhibitors are an anti-PD1 antibody and an anti-CTL4 antibody. In some aspects, the vaccine conjugate is administered two or more times. In some aspects, the additional anti-cancer treatment comprises a TLR9 agonist, such as CpG ODN1826, and / or a CD40 agonist, such as a CD40 agonistic antibody. In certain aspects, an immune checkpoint inhibitor and a TLR9 agonist are administered to the subject.

[0028] In other aspects, the method further includes administering an additional anti-cancer treatment to the subject. In some aspects, the additional anti-cancer treatment is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or cytokine therapy.

[0029] Another embodiment provides a kit that contains the vaccine conjugate of this embodiment (e.g., a vaccine conjugate containing mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2)) or the pharmaceutical composition of this embodiment. In some aspects, the kit further contains an immune checkpoint inhibitor. For example, the immune checkpoint inhibitor is an anti-PD1 antibody or a CTLA-4 antibody.

[0030] Another embodiment provides a composition for treating cancer, the composition containing the vaccine conjugate of this embodiment (e.g., a vaccine conjugate containing mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2)) or the pharmaceutical composition of this embodiment. In some aspects, the composition further contains an immune checkpoint inhibitor.

[0031] In another embodiment, the present disclosure provides a vaccine conjugate containing mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2) conjugated to CD33 or a fragment thereof.

[0032] In some aspects, the mutant SMEZ-2 contains the mutations W75L and K182Q. In certain aspects, the mutant SMEZ-2 contains the mutations W75L, K182Q, and / or D42C. In some specific aspects, the mutant SMEZ-2 contains the mutations W75L, K182Q, and D42C. In certain aspects, the vaccine conjugate contains at least a second target protein or a fragment thereof.

[0033] In some aspects, CD33 or a fragment thereof contains one or more amino acid substitutions. In some aspects, the full-length CD33 has an amino acid substitution at asparagine 98. In some aspects, CD33 contains a truncated form of CD33 consisting of the CD33-IgC domain, such as CD33-IgC that contains an amino acid substitution at aspartic acid 231 or cysteine 154. In some aspects, one or more amino acid substitutions are located at D231, D246, C154, and / or C169. In certain aspects, one or more amino acid substitutions are D231E, D246E, C154S, and / or C169S.

[0034] In certain aspects, the linker is a peptide linker, such as a glycine-serine linker, particularly a GGGGS linker.

[0035] Another embodiment provides a pharmaceutical composition comprising a vaccine conjugate of this embodiment (e.g., a vaccine conjugate comprising mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2) conjugated to CD33) and an adjuvant. In some aspects, the adjuvant is incomplete Freund's adjuvant. In certain aspects, the conjugate and incomplete Freund's adjuvant (IFA) are formulated as a 1:1 emulsion. In some aspects, the conjugate is formulated for intravenous infusion, subcutaneous injection, or intramuscular injection.

[0036] Another embodiment provides an expression vector comprising a sequence encoding mutant SMEZ-2 fused to a sequence encoding CD33 or a fragment thereof. Also provided herein are host cells comprising the expression vector of this embodiment, such as Escherichia coli, human embryonic kidney cells (HEK293), or Chinese hamster ovary (CHO) cells.

[0037] In another embodiment, a method for stimulating an immune response in a subject is provided, which comprises administering to the subject an effective amount of a vaccine conjugate of this embodiment (e.g., a vaccine conjugate comprising mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2) conjugated to CD33 or a fragment thereof) or a pharmaceutical composition of this embodiment.

[0038] In some aspects, the immune response is an anti-cancer immune response. In certain aspects, the subject has cancer. In some specific aspects, the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, urogenital system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer, or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, cholangiocarcinoma, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma tumor, multiple endocrine neoplasia type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In some specific aspects, the cancer is acute myeloid leukemia.

[0039] In certain aspects, the immune response is an anti-CD33 specific immune response. In some aspects, the anti-CD33 specific immune response is detected by measuring an increase in the titer of CD33-specific immunoglobulins in a blood sample of the subject.

[0040] In some aspects, the conjugate is administered by injection, such as intravenous infusion.

[0041] In other aspects, the method further comprises administering a second anti-cancer treatment to the subject. In some aspects, the second anti-cancer treatment is immunotherapy, chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy. In some aspects, the second anti-cancer treatment is immunotherapy, such as immune checkpoint inhibitors. For example, immune checkpoint inhibitors are inhibitors selected from CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR. In some specific aspects, the immune checkpoint inhibitor comprises an anti-PD1 agent, such as an anti-PD1 antibody, an anti-PDL1 antibody or an anti-PDL2 antibody. In certain aspects, the anti-PD1 agent is nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA or AMP-224. In some aspects, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. For example, the anti-CTLA-4 antibody is tremelimumab, or ipilimumab.

[0042] Another embodiment provides a method of treating a subject having cancer, which comprises administering to the subject a vaccine conjugate of this embodiment (e.g., a vaccine conjugate comprising mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2) conjugated to CD33 or a fragment thereof) or a pharmaceutical composition of this embodiment.

[0043] In some aspects, the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, urogenital system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, cholangiocarcinoma, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma tumor, multiple endocrine neoplasia type I and II tumors, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer. In some specific aspects, the cancer is acute myeloid leukemia.

[0044] In other aspects, the method further comprises administering to the subject at least one immune checkpoint inhibitor. In some aspects, administering to the subject at least one immune checkpoint inhibitor comprises administering at least one immune checkpoint inhibitor prior to the vaccine conjugate. In other aspects, administering to the subject at least one immune checkpoint inhibitor comprises administering at least one immune checkpoint inhibitor after or concurrently with the vaccine conjugate. For example, the immune checkpoint inhibitor is selected from inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In some specific aspects, the immune checkpoint inhibitor comprises an anti-PD1 / PDL agent, such as an anti-PD1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody. In certain aspects, the anti-PD1 agent is nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA, or AMP-224. In some aspects, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. For example, the anti-CTLA-4 antibody is tremelimumab, or ipilimumab. In some aspects, two immune checkpoint inhibitors are administered to the subject. In some specific aspects, the two immune checkpoint inhibitors are an anti-PD1 antibody and an anti-CTL4 antibody. In some aspects, the vaccine conjugate is administered two or more times.

[0045] In other aspects, the method further comprises administering to the subject additional anti-cancer therapy. In some aspects, the additional anti-cancer therapy is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or cytokine therapy.

[0046] Another embodiment provides a kit comprising the vaccine conjugate of this embodiment (e.g., a vaccine conjugate comprising mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2)) or the pharmaceutical composition of this embodiment. In some aspects, the kit further comprises an immune checkpoint inhibitor. For example, the immune checkpoint inhibitor is an anti-PD1 antibody or an anti-CTLA-4 antibody.

[0047] Another embodiment provides a composition for treating cancer, the composition comprising the vaccine conjugate of this embodiment (e.g., a vaccine conjugate comprising mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2)) or the pharmaceutical composition of this embodiment. In some aspects, the composition further comprises an immune checkpoint inhibitor.

[0048] It is expected that any method or composition described herein can be implemented relative to any other method or composition described herein. For example, a compound synthesized by one method can be used to prepare a final compound according to a different method.

[0049] When used in the claims and / or the specification in conjunction with the term "comprising", the use of a noun without a quantifier can mean "one", but it is also consistent with the meanings of "one or more", "at least one", and "more than one". The term "about" means plus or minus 5% of the specified number.

[0050] Other objects, features, and advantages of the present disclosure will be apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, although indicating some specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will be apparent to those skilled in the art from this detailed description. Brief Description of the Drawings

[0051] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present invention. The present invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments given herein.

[0052] Figure 1 : Mechanism of promoting antigen presentation by mutant SMEZ-2.

[0053] Figure 2 : SDS-PAGE of purified proteins. (1) Molecular weight marker (2) AGR2 (3) SMEZ-2 (W75L, K182Q, D42C) (4) AGR2-SMEZ-2 (W75L, K182Q, D42C).

[0054] Figure 3 : Engineered construct of AGR2-SMEZ-2 conjugate expressed in E. coli.

[0055] Figure 4 : ELISA data of anti-AGR2 IgG in the plasma of C57BL / 6 mice vaccinated with 100 μg of AGR2-SMEZ2 or an equimolar amount of the specified control. The protein was administered by intramuscular injection.

[0056] Figures 5A - 5B: C57BL / 6 mice were treated according to the schedule shown in Figure 5A. Mice were injected with 100 μg AGR2 - SMEZ or an equimolar amount of AGR2 (Figure 5A), 50 μg of ODN 1826 (CpG, C), and implanted with 100,000 AGR2 - expressing KPC cells (T). Proteins were administered subcutaneously in IFA. ODN1826 was given by intramuscular injection. (Figure 5B) Tumor volume was measured 16 days after implantation.

[0057] Figures 6A - 6B: Recombinant murine CD38, SMEZ2, and CD38 - SMEZ2 conjugates were expressed and purified from mammalian cells. (Figure 6A) shows SDS - PAGE analysis. (Figure 6B) Plasma was drawn from Balb / c mice 10 days after two injections of 50 μg mCD38 - SMEZ subQ in IFA (or an equimolar equivalent of control). Serum was diluted 1:10,000 and anti - CD38 IgG was measured by ELISA.

[0058] Figures 7A - 7C: SDS - PAGE of purified proteins. (Figure 7A) murine CTLA - 4 and CTLA - 4SMEZ. (Figure 7B) murine PD - 1 and murine PD1 - SMEZ (Figure 7C) murine PD - L1 and PDL1 - SMEZ.

[0059] Figures 8A - 8B: In vivo activity of SMEZ - PD1, SMEZ - PD - L1, and SMEZ - CTLA4. (Figure 8A) C57BL / 6 mice were given three injections of a mixture consisting of SMEZ conjugates of murine PD - 1, PDL1, and CTLA - 4. Blood was drawn and IgG specific for each antigen was quantified by ELISA. (Figure 8B) Spleen cells were harvested from the mice in Figure 8A and CD4+ and CD8+ T cells were analyzed by flow cytometry. Both T cell populations were significantly reduced by the SMEZ mixture.

[0060] Figures 9A - 9C: Pre - clinical efficacy of CD33 - SMEZ - 2 in an AML mouse model. (Figure 9A) The indicated proteins were expressed and purified from Expi293 HEK cells. Western blots of each protein expressing a 6×His tag for affinity purification are shown. (Figure 9B) C57BL / 6 mice were injected subcutaneously with 40 pmol of the indicated protein in IFA emulsion. After three administrations, blood was drawn and ELISA assays were performed to detect anti - CD33 - specific IgG in the plasma of inoculated mice. ELISA data are shown. (Figure 9C) Two weeks after treating C57BL / 6 mice with CD33 - SMEZ - 2, by intravenous injection with 1×10 6C1498 - hCD33 cells were used to attack mice. Survival data are shown.

[0061] Figures 10A - 10C: (Figure 10A) 15,000 KPC cells were orthotopically injected into C57BL / 6 mice. The KPC cells were derived from a syngeneic pancreatic tumor of LSL - Kras G12D ; LSL - Trp53 R172H ; Pdx1 - cre mice (which overexpress hAGR2). The KPC cells were injected into the head of the pancreas on day 0. Mice were treated twice with 50 μg SMEZ - AGR2 or SMEZ - MSLN (mesothelin) (subQ; emulsified in IFA) before orthotopic injection and twice after orthotopic injection (days - 24, - 10, 4, and 18). Starting from day 3, mice were treated 3 times / week for 3 weeks (days 3, 5, 7, 10, 12, 14, 17, 19, 21) with 50 μg ODN 1826 (IM) and 250 μg α - mPD - 1 (IM; BioXCell; clone RMP1 - 14). For all 3 treatments, control mice received the vehicle. Mice were sacrificed on day 25, and the tumors were excised, (Figure 10B) photographed, and (Figure 10C) weighed.

[0062] Figures 11A to 11C: (Figure 11A) shows the protein structures of various CD33-SMEZ constructs. The upper panel shows full-length CD33-SMEZ, which contains the two extracellular domains (IgV and IgC) of CD33 linked to a modified SMEZ-2 superantigen. The model in the lower left depicts CD33IgC-SMEZ, which contains only the IgC domain of CD33 conjugated to a modified SMEZ-2. Finally, CD33 IgC-SMEZ-3Mut is shown in the lower right, which is an optimized form of CD33 IgC-SMEZ with three amino acid substitutions, assuming that these three amino acid substitutions can stabilize the protein, reduce aggregation, and improve the developability of the protein. (Figure 11B) Western blot (left panel) shows the original and modified versions of CD33 IgC and CD33 IgC-SMEZ under non-reducing conditions. The proteins were visualized by blotting for a 6×His affinity tag engineered into all proteins. Both CD33 IgC and CD33 IgC-SMEZ showed protein aggregation, and protein aggregation was reduced in the 3Mut candidates after introduction of three amino acid mutations. (Right panel) Protein aggregation was quantified by Proteostat protein aggregation assay, and it was confirmed that aggregation was significantly reduced for both CD33 IgC and CD33 IgC-SMEZ (referred to here as M2T) compared to the 3Mut form of the protein. (Figure 11C) C57BL / 6 mice were treated intramuscularly with 10 μg or 2 μg of CD33 IgC-SMEZ, an equimolar dose of CD33 IgC alone, or a vehicle control (PBS). One week after the fourth dose, the α-CD33 antibody titers from mouse plasma were measured by enzyme-linked immunosorbent assay (ELISA). As expected, the SMEZ-conjugated protein induced a high level of anti-IgC IgG in mice compared to the IgC protein alone.

[0063] Description of exemplary embodiments

[0064] Anterior gradient protein 2 (AGR2) is a member of the protein disulfide isomerase (PDI) family and is involved in disulfide bond formation of newly synthesized proteins in the endoplasmic reticulum (ER). Like other proteins in the PDI family, AGR2 has an N-terminal ER leader sequence and a C-terminal ER retention signal (Pateletal., 2013). Although these sequence features localize and retain AGR2 to the ER, it has also been observed extracellularly interacting with the extracellular matrix and associated with increased invasion, proliferation, survival, and metastasis in several human cancers, including PDAC (Fessart etal., 2016; Moidu et al., 2020; DiMaro et al., 2014; Ma et al., 2017; Wang et al., 2008). Indeed, a significant increase in extracellular AGR2 was observed in both immunohistochemical (IHC) staining and tissue microarrays of PDAC, but not in normal pancreatic tissues from patient samples. Thus, the elevated expression level of AGR2 and its pro-cancer characteristics led to the hypothesis that AGR2 is an attractive TAA immunotherapeutic target in PDAC.

[0065] Accordingly, in certain embodiments, vaccine conjugates are provided herein, such as the detoxified AGR2-SMEZ-2 conjugate, which can stimulate a robust immune response against PDAC expressing high levels of AGR2. The conjugate utilizes the MHCII-binding ability of SMEZ-2 to enable efficient uptake and presentation of AGR2 peptides by APCs. The conjugate can result in a robust anti-PDAC immune response and tumor eradication.

[0066] This vaccine approach offers several advantages over monoclonal antibody (mAb) therapy, including adaptability to other tumor types and conjugation with different TAAs, lower manufacturing costs, and higher durability. Additionally, the polyclonal response elicited is less likely to result in treatment resistance, as a single AGR2 mutation is unlikely to escape the immune response. This technology overcomes the challenges currently presented by peptide-based cancer vaccines, where in silico modeling of peptide-MHC molecule binding is no longer required due to the high-affinity binding properties of SMEZ-2. Accordingly, other embodiments provide vaccine conjugates for other TAA- and neoantigen-based vaccine candidates.

[0067] In some specific aspects, bacterial superantigen conjugates are provided herein by cis-cloning a tumor-associated antigen or immune target (i.e., AGR2, CD38, PD-1, PD-L1, CTLA-4) in the form of a mutant of the streptococcal mitogenic exotoxin Z-2 (SMEZ-2) bacterial superantigen. The mutant superantigen has a high affinity for MHC class II molecules, thereby promoting and enhancing the presentation of antigens to immune effector cells and stimulating an immune response against target cells expressing the antigen linked to SMEZ2. Current data show that AGR2-SMEZ2, CD38-SMEZ, PD-1-SMEZ, PDL1-SMEZ, and CTLA4-SMEZ conjugates induced robust humoral responses, and high titers of specific immunoglobulins were detected in the plasma of treated mice.

[0068] Sialic acid-binding Ig-like lectin 3 (CD33) is an attractive therapeutic target for acute myeloid leukemia (AML). It is a member of the Siglec family, characterized by cell-cell interactions mediated by binding to sialylated glycans. CD33 consists of two extracellular domains (an immunoglobulin-like V (IgV) domain and an immunoglobulin-like C (IgC) domain), a transmembrane domain, an immunoreceptor tyrosine-based inhibitory motif (ITIM) domain, and an ITIM-like domain. Although the signaling functions and roles of CD33 are not fully understood, it is thought to inhibit cell activation and proliferation upon ligand binding. CD33 is normally expressed on myeloid cells and is present in approximately 85% to 90% of both cases of pAML and aAML. Notably, it is not expressed on lymphocytes or red blood cells.

[0069] Streptococcal mitogenic exotoxin Z-2 (SMEZ-2) is a streptococcal superantigen (SAg) that cross-links MHC class II and the T cell receptor (TCR), leading to the proliferation of mature T cells, overproduction of cytokines, and ultimately T cell anergy (Deacy et al., 2021). Radcliff et al. previously showed that a modified form called SMEZ-2M1 can prevent TCR binding and eliminate the subsequent cytokine storm. SMEZ-2M1 retains the ability to bind to MHC class II and can enhance the presentation of conjugated antigens to the immune system (Radcliff et al., 2012).

[0070] Accordingly, in certain embodiments, vaccine conjugates are provided herein, such as the detoxified CD33-SMEZ-2 conjugate. In the present study, the inventors developed CD33-targeted immunotherapy by cloning human CD33 linked to a mutant form of the SMEZ-2 bacterial superantigen. The mutant superantigen has a high affinity for MHC class II molecules, thereby promoting and enhancing the presentation of CD33 peptides to immune effector cells and stimulating an anti-CD33 specific immune response against AML. Data showed that the CD33-SMEZ-2 conjugate induced a robust anti-CD33 humoral response, and high titers of CD33-specific immunoglobulins were detected in the plasma of treated mice. Additional animal studies demonstrated that these effects were sufficient to reduce the AML burden in mice and significantly prolong survival.

[0071] Another embodiment provides the CD33 vaccine conjugate of this embodiment (e.g., a vaccine conjugate conjugated to mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2)), wherein strategic amino acid substitutions are made to the full-length or truncated CD33 protein. In some aspects, the full-length CD33 is modified at predicted deamidation sites (including asparagine at position 98) to improve stability and prevent protein aggregation, and overall improve the drug suitability of the candidate. In some aspects, the truncated form of CD33 consisting only of the CD33-IgC domain is further modified by mutations of amino acids predicted to be high-risk sites for isomerization (including aspartic acid at position 231) or free cysteines (including cysteine at position 154) to improve stability and prevent protein aggregation, and overall improve the drug suitability of the candidate.

[0072] I. Cancer Vaccine Conjugates

[0073] The compositions and methods of the present invention may include a cancer vaccine in the form of active immunotherapy, wherein an antigenic peptide, polypeptide, or protein, or an autologous or allogeneic tumor cell composition or "vaccine" is administered to a subject. The vaccine can be administered systemically, such as intravenously, intradermally, or by intramuscular injection. The vaccine can also be administered multiple times to enhance the immune response against the administered antigen.

[0074] The term "vaccine" is used according to its ordinary and common meaning in medicine and immunology and refers to a composition containing an antigenic component (such as an antigenic protein) for administration to a subject (such as a human), which elicits an immune response against the antigenic component (such as an antigenic protein). In some embodiments, the vaccine is therapeutic. In some embodiments, the vaccine is prophylactic. In some embodiments, the vaccine comprises one or more adjuvants (such as aluminum adjuvants). A liquid vaccine is a vaccine in liquid form, which can be, for example, a solution, suspension, emulsion or dispersion, or the antigenic component (such as an antigenic protein) of the vaccine, and may optionally contain other components. A dry vaccine is a vaccine containing 5% or less water.

[0075] A vaccine is a preparation for enhancing immunity against a specific disease. A vaccine contains an agent for inducing a response from the immune system of a subject. Various agents commonly used in vaccines include, but are not limited to: killed but previously virulent microorganisms; live attenuated microorganisms; inactivated toxic compounds produced by disease-causing microorganisms; protein subunits of microorganisms; and conjugates.

[0076] When applied to methods of administering vaccines, the term "prime-boost" or "prime boost" is used according to its ordinary and common meaning in virology and immunology and refers to a method of vaccine administration in which a first dose of a vaccine or vaccine component is administered to a subject or patient to initiate administration (prime), and a second vaccine is administered to the same patient or subject at a later time (such as hours, days, weeks, months later) (boost). The first and second vaccines can be the same or different, but both are intended to elicit an immune response that can be used to treat or prevent the same disease or condition. In some embodiments, the prime is one or more viral proteins or portions thereof, and the boost is one or more viral proteins or portions thereof.

[0077] As used herein, the term "associated" or "associated with" when used to describe a disease (such as a virus-related disease or a bacterium-related disease) means that the disease is caused by what is described as disease-associated or what is described as associated with the disease, or the symptoms of the disease are caused by what is described as disease-associated or what is described as associated with the disease. As used herein, what is described as associated with a disease, if it is a causative agent, can be a target for the treatment of the disease.

[0078] The vaccine antigens described herein can be chemically conjugated to a carrier or recombinantly expressed with an immunogenic carrier peptide or polypeptide (e.g., an antigen-carrier fusion peptide or polypeptide) to enhance the immune response. Methods for conjugating polypeptides or peptides to immunogenic carrier proteins are well known in the art and include, for example, glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine. In some specific embodiments, the carrier is mutant SMEZ-2.

[0079] A. SMEZ-2 Bacterial Superantigen

[0080] Streptococcal mitogenic exotoxin Z-2 (SMEZ-2) from Streptococcus pyogenes is a bacterial superantigen (SAg) that is the most immunogenic SAg discovered to date (Kamezawa et al., 1997; Proft et al., 2000). Wild-type SMEZ-2 binds to both MHC class II molecules and the T cell receptor (TCR) with high affinity, which activates T cells indiscriminately and can stimulate up to 20% of the body's T cell pool (Li et al., 1999). This results in the generation of a non-specific immune response that, in the case of microgram amounts of SAg, leads to the massive release of cytokines and toxic shock syndrome (Alouf et al., 2003). The T cell-binding and mitogenic (and toxic) effects of SMEZ-2 can be abrogated by mutations in key residues (W75L, K182Q, and D42C) on the TCR Vβ-binding surface, which produces a protein that retains high affinity for MHC class II molecules without the toxic effects associated with wild-type SMEZ-2 (Radcliff et al., 2012). Thus, MHC class II binding by mutant SMEZ-2 provides an effective carrier system for directly targeting antigens to antigen-presenting cells (APCs). This effectively "hijacks" the function of SMEZ-2 through detoxification and generates a protein "carrier" for the efficient presentation of conjugated antigens (Dickgreber et al., 2009).

[0081] B. Target Protein

[0082] The target proteins of the antigens targeted by the vaccine conjugates of the present invention include those expressed in the context of the disease, disorder, or cell type to be targeted by the vaccine conjugate. Diseases and disorders include proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphoma, leukemia, and / or myeloma, such as B, T, and myeloid leukemia, lymphoma, and multiple myeloma. In some embodiments, the antigen is selectively expressed or overexpressed on the cells of the disease or disorder (e.g., tumor or pathogenic cells) compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells. Any suitable antigen can be used in the methods of the present invention. Exemplary antigens include, but are not limited to, antigen molecules from infectious agents, auto / self-antigens, tumor / tumor-associated antigens, and tumor neoantigens.

[0083] The terms “tumor-associated antigen,” “tumor antigen,” and “cancer cell antigen” are used interchangeably herein. In each case, the term refers to a protein, glycoprotein, or carbohydrate that is specifically or preferentially expressed by cancer cells.

[0084] The tumor-associated antigen can be of any kind, as long as it is expressed on the cell surface of tumor cells. Tumor-associated antigens can be derived from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, mesothelioma cancer, ovarian cancer, sarcoma cancer, or melanoma cancer. Exemplary tumor-associated antigens or antigens of tumor cell origin include MAGE 1, 3, and MAGE 4 (or other MAGE antigens, such as those disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY-ESO-1); SAGE; and HAGE or GAGE. Non-limiting examples of these tumor antigens are expressed in a wide range of tumor types (e.g., melanoma, lung cancer, sarcoma, and bladder cancer). See, for example, U.S. Patent No. 6,544,518. Prostate cancer tumor-associated antigens include, for example, prostate specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphate, NKX3.1, and six-transmembrane epithelial antigen of the prostate (STEAP).

[0085] Some exemplary embodiments of tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, etc. In some specific aspects, the antigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and CEA. In some specific aspects, the antigens of two or more antigen receptors include, but are not limited to, CD19, EBNA, WT1, CD123, NY-ESO, EGFRvIII, MUC1, HER2, CA-125, WT1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and / or CEA. The sequences of these antigens are known in the art, such as CD19 (accession number NG_007275.1), EBNA (accession number NG_002392.2), WT1 (accession number NG_009272.1), CD123 (accession number NC_000023.11), NY-ESO (accession number NC_000023.11), EGFRvIII (accession number NG_007726.3), MUC1 (accession number NG_029383.1), HER2 (accession number NG_007503.1), CA-125 (accession number NG_055257.1), WT1 (accession number NG_009272.1), Mage-A3 (accession number NG_013244.1), Mage-A4 (accession number NG_013245.1), Mage-A10 (accession number NC_000023.11), TRAIL / DR4 (accession number NC_000003.12), and / or CEA (accession number NC_000019.10).

[0086] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. Additionally, tumor antigens can be auto-peptide hormones, such as full-length gonadotrophin hormone releasing hormone (GnRH), a 10-amino acid long short peptide, which can be used to treat many cancers.

[0087] Tumor antigens include tumor antigens derived from cancers characterized by the expression of tumor-associated antigens (e.g., HER-2 / neu expression). Target tumor-associated antigens include lineage-specific tumor antigens such as the melanocyte-melanoma lineage antigens MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase, and tyrosinase-related protein. Illustrative tumor-associated antigens include, but are not limited to, tumor antigens derived from or comprising any one or more of the following: p53, Ras, c-Myc, cytoplasmic serine / threonine kinases (such as A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MART-1, MC1R, Gp100, PSA, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms' tumor antigen (WT1), AFP, beta-catenin / m, caspase-8 / m, CEA, CDK-4 / m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signal transducer 1 (TACSTD1), TACSTD2, receptor tyrosine kinases (such as epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor receptor (Platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), cytoplasmic tyrosine kinases (such as the src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducer and activator of transcription STAT3, STAT5, and STAT6, hypoxia-inducible factors (such as HIF-1 and HIF-2), nuclear factor-kappa B (NF-κB), Notch receptors (such as Notch1-4), c-Met, mammalian targets of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK), and its regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-α, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAP, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoint, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GloboH, NY-BR-1, RGS5, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1, and idiotype.,

[0088] Antigens can include epitope regions or epitope peptides derived from genes mutated in tumor cells or genes transcribed at different levels in tumor cells compared to normal cells, such as telomerase, survivin, mesothelin, mutated ras, bcr / abl rearrangement, Her2 / neu, mutated or wild-type p53, cytochrome P450 1B1, and aberrantly expressed intronic sequences, such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myelomas and B-cell lymphomas; tumor antigens including epitope regions or epitope peptides derived from oncoviral processes, such as human papillomavirus proteins E6 and E7; Epstein bar virus protein LMP2; non-mutated carcinoembryonic proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0089] In other embodiments, the antigen is obtained from or derived from a pathogenic microorganism or opportunistic pathogenic microorganism (also referred to herein as an infectious disease microorganism), such as a virus, fungus, parasite, and bacterium. In certain embodiments, the antigen derived from such a microorganism includes a full-length protein.

[0090] C. Vaccine conjugate production

[0091] A variety of commercially available vectors and expression systems can be used to produce the vaccine conjugates of the present invention, including those designed for mammalian cells, insect (Spodoptera; baculovirus-delivering) cells, and bacterial cells. In some aspects, purification is performed using Escherichia coli, such as Escherichia coli BL21(DE3) cells, HEK cells, or CHO cells.

[0092] In certain embodiments, the vaccine conjugate is purified. As used herein, the term "purified" is intended to refer to a composition separable from other components, wherein the protein is purified to any degree relative to its naturally-occurring state. Thus, a purified protein also refers to a protein that is removed from its naturally-occurring environment. In the case of using the term "substantially purified", the indication will refer to a composition wherein the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99% or more of the protein in the composition.

[0093] In certain embodiments, plasmid vectors are contemplated for transformation of host cells. Generally, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used with these hosts. The vector typically carries a replication site, as well as a marker sequence that enables phenotypic selection in the transformed cell. In one non-limiting example, derivatives of the plasmid pBR322, which is derived from the Escherichia coli species, are typically used to transform E. coli. pBR322 contains ampicillin and tetracycline resistance genes, thus providing an easy means for identifying transformed cells. The pBR plasmid or other microbial plasmids or bacteriophages must also contain or be modified to contain, for example, a promoter that can be used by the microbial organism for expressing its own proteins. In some aspects, the vaccine conjugate is cloned into pET15TEV_NESG, which expresses a protein with an N-terminal 6× histidine tag that can be cleaved by Tobacco Etch Virus (TEV) protease. The vaccine conjugate is expressed in BL21(DE3) E. coli and purified using immobilized metal affinity chromatography (IMAC). To avoid non-specific immunogenicity or unexpected side effects, the 6× histidine tag is removed by overnight digestion with TEV, and the purified protein is obtained in the flow-through by another round of IMAC purification.

[0094] Alternatively, bacteriophage vectors containing replicon and control sequences compatible with the host microorganism can be used as transformation vectors associated with these hosts. For example, bacteriophage λGEM TM- 11 can be used to prepare recombinant bacteriophage vectors that can be used to transform host cells (such as E. coli LE392).

[0095] Other useful plasmid vectors include the pIN vectors (Inouye et al., 1985); and the pGEX vectors, which are used to produce glutathione S-transferase (GST) soluble fusion proteins for later purification and isolation or cleavage. Other suitable fusion proteins are those with β-galactosidase, ubiquitin, etc.

[0096] Bacterial host cells containing the expression vector (such as E. coli) are grown in any of a number of suitable media (such as LB). As understood by those skilled in the art, expression of the recombinant protein in certain vectors can be induced by contacting the host cells with a reagent specific for certain promoters, such as by adding IPTG to the medium or by shifting the incubation to a higher temperature. After culturing the bacteria for an additional period of time (generally 2 to 24 hours), the cells are collected by centrifugation and washed to remove the remaining medium.

[0097] Protein purification techniques are well known to those skilled in the art. These techniques at one level involve the gross fractionation of the cellular milieu into a polypeptide fraction and a non-polypeptide fraction. After separating the polypeptide from other proteins, chromatographic and electrophoretic techniques can be used to further purify the polypeptide of interest to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for preparing pure peptides are ion exchange chromatography, size exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, etc. or by heat denaturation followed by centrifugation; gel filtration, reverse phase, hydroxyapatite, and affinity chromatography; and combinations of such techniques with other techniques. When purifying a protein, it may be desirable to extract the protein using denaturing conditions. A polypeptide can be purified from other cellular components using an affinity column that binds to a labeled portion of the polypeptide. As is generally known in the art, it is contemplated that the order in which the various purification steps are carried out can be altered, or certain steps can be omitted, and still yield a suitable method for preparing a substantially purified protein or peptide.

[0098] In accordance with the present disclosure, those skilled in the art will be aware of a variety of methods for quantifying the degree of purification of a protein or peptide. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptide within a fraction by SDS / PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, compare it to the specific activity of the initial extract, and thereby calculate the purity. Of course, the actual units used to represent the amount of activity will depend on the particular assay technique chosen after purification, and whether the expressed protein or polypeptide exhibits detectable activity. It is known that the migration of a polypeptide can vary with different SDS / PAGE conditions, sometimes significantly (Capaldi et al., 1977). Thus, it should be understood that the apparent molecular weight of a purified or partially purified expressed product can vary under different electrophoretic conditions.

[0099] One of ordinary skill in the art will be skilled in constructing vectors by standard recombinant techniques (e.g., see Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference) to express the antigen receptors of the present disclosure. Vectors include, but are not limited to, plasmids, cosmids, viruses (phages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., derived from Moloney murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.); lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.); adenovirus (Ad) vectors, including their replication-competent, replication-defective, and gutless forms; adeno-associated viral (AAV) vectors; simian virus 40 (SV-40) vectors; bovine papillomavirus vectors; Epstein-Barr virus vectors; herpesvirus vectors; vaccinia virus vectors; Harvey murine sarcoma virus vectors; murine mammary tumor virus vectors; Rous sarcoma virus vectors; parvovirus vectors; poliovirus vectors; vesicular stomatitis virus vectors; maraba virus vectors, and group B adenovirus enadenotucirev vectors.

[0100] 1. Regulatory elements

[0101] Expression cassettes contained in vectors useful for the present disclosure particularly contain (in the 5' to 3' direction) a eukaryotic transcriptional promoter operably linked to a protein coding sequence, splicing signals containing intervening sequences, and a transcriptional termination / polyadenylation sequence. Promoters and enhancers that control the transcription of protein coding genes in eukaryotic cells are composed of a variety of genetic elements. The cellular machinery is able to collect and integrate the regulatory information transmitted by each element, allowing different genes to evolve different, often complex, transcriptional regulatory patterns. Promoters for use in the context of the present disclosure include constitutive, inducible, and tissue-specific promoters.

[0102] 2. Promoter / Enhancer

[0103] The expression constructs provided herein contain promoters that drive the expression of antigen receptors. A promoter typically contains sequences that function to localize the start site of RNA synthesis. The best-known example is the TATA box, but in some promoters lacking a TATA box (such as the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late gene), discrete elements covering the start site itself contribute to determining the start position. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30 - 110 bp upstream of the start site, although many promoters have been shown to also contain functional elements downstream of the start site. To have a coding sequence "under the control" of a promoter, the 5' end of the transcriptional reading frame's start site is positioned "downstream" (i.e., 3') of the selected promoter. "Upstream" promoters stimulate the transcription of DNA and promote the expression of the encoded RNA.

[0104] The spacing between promoter elements is generally flexible such that promoter function is maintained when the elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be increased up to 50 bp before activity begins to decline. Depending on the promoter, individual elements have been shown to function either cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers", which are cis-acting regulatory sequences that participate in the transcriptional activation of nucleic acid sequences.

[0105] A promoter may be associated naturally with a nucleic acid sequence, such as can be obtained by isolating the 5' non-coding sequence located upstream of the coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be associated naturally with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages can be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the nucleic acid sequence in its natural environment. Recombinant or heterologous enhancers also refer to enhancers that are not normally associated with the nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, and promoters or enhancers that are not "naturally occurring", i.e., contain different elements of transcriptional regulatory regions and / or mutations that alter expression. For example, the promoters most commonly used in recombinant DNA constructs include the beta-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to nucleic acid sequences that are synthetically produced for promoters and enhancers, recombinant cloning and / or nucleic acid amplification techniques (including PCR TM ) can be used in combination with the compositions disclosed herein to generate sequences. Additionally, it is contemplated that control sequences directing the transcription and / or expression of sequences in non-nuclear organelles (such as mitochondria, chloroplasts, etc.) may also be employed.

[0106] Of course, it is important to use promoters and / or enhancers that effectively direct the expression of DNA segments in the organelles, cell types, tissues, organs, or organisms selected for expression. Those skilled in the art of molecular biology are generally aware of the use of promoter, enhancer, and cell type combinations for protein expression (see, e.g., Sambrook et al. 1989, which is incorporated herein by reference). The promoters used can be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segments, such as for facilitating large-scale production of recombinant proteins and / or peptides. The promoters can be heterologous or endogenous.

[0107] In addition, any promoter / enhancer combination (according to, e.g., the eukaryotic promoter database EPDB) can be used to drive expression. Use of the T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if a suitable bacterial polymerase is provided as part of the delivery complex or as an additional genetic expression construct.

[0108] Some non-limiting examples of promoters include early or late viral promoters, e.g., SV40 early or late promoter, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter; eukaryotic cell promoters, e.g., such as β-actin promoter, GADPH promoter, metallothionein promoter; and concatenated response element promoters, e.g., cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA), and response element promoter near the minimal TATA box (tre). The human growth hormone promoter sequence (e.g., the human growth hormone minimal promoter described in Genbank accession number X05244, nucleotides 283 to 341) or the mouse mammary tumor promoter (available from ATCC, catalog number ATCC45007) can also be used. In certain embodiments, the promoter is the CMV IE, dectin-1, dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, β-actin, MHC class I, or MHC class II promoter, however, any other promoter that can be used to drive therapeutic gene expression can be applied to the practice of the present disclosure.

[0109] In some aspects, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that enhance the activity of a promoter and have the potential for cis - acting even at relatively long distances (up to several thousand bases away from the target promoter), regardless of their orientation. However, enhancer function is not necessarily limited to such long distances, as they can also act in close proximity to a given promoter.

[0110] 3. Initiation signals and linked expression

[0111] Specific initiation signals can also be used in the expression constructs provided in the present disclosure for efficient translation of the coding sequence. These signals include the ATG start codon or adjacent sequences. It may be necessary to provide exogenous translation control signals, including the ATG start codon. One of ordinary skill in the art can readily determine this and provide the necessary signals. It is well - known that the start codon must be "in - frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translation control signals and start codons can be natural or synthetic. Expression efficiency can be enhanced by including appropriate transcriptional enhancer elements.

[0112] In certain embodiments, an internal ribosome entry site (IRES) element is used to generate polygenic or polycistronic messages. The IRES element is capable of bypassing the ribosome scanning model of 5'-methylated cap - dependent translation and initiating translation at an internal site. IRES elements from two members of the picornavirus family (poliovirus and encephalomyocarditis virus) and IRESs from mammalian messages have been described. The IRES element can be linked to a heterologous open reading frame. Multiple open reading frames can be transcribed together, each separated by an IRES, thus generating a polycistronic message. By virtue of the IRES element, each open reading frame can utilize ribosomes for efficient translation. Transcribing a single message using a single promoter / enhancer can effectively express multiple genes.

[0113] In addition, certain 2A sequence elements can be used to generate linked or co - expression of genes in the constructs provided in the present disclosure. For example, cleavage sequences can be used to co - express genes by linking open reading frames to form a single cistron. Exemplary cleavage sequences are F2A (foot - and - mouth disease virus 2A) or "2A - like" sequences (e.g., Thosea asigna virus 2A; T2A).

[0114] 4. Initiation of replication

[0115] To amplify the vector in a host cell, it may contain one or more origins of replication (commonly referred to as "ori"), for example, the oriP corresponding to EBV as described above or a nucleic acid sequence of a genetically engineered oriP with similar or enhanced functions in programming, which is a specific nucleic acid sequence that initiates replication. Alternatively, the origin of replication of other episomal replication viruses or autonomously replicating sequences (ARS) as described above can be used.

[0116] 5. Selection and Screenable Markers

[0117] In some embodiments, cells containing the constructs of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker will confer an identifiable change to the cell, thereby allowing easy identification of cells containing the expression vector. Generally, a selection marker is a marker that confers a property that allows selection. A positive selection marker is a marker where the presence of the marker allows its selection, while a negative selection marker is a marker where the presence of the marker prevents its selection. An example of a positive selection marker is a drug resistance marker.

[0118] Generally, including a drug selection marker facilitates the cloning and identification of transformants. For example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, bleomycin, and histidinol are useful selection markers. In addition to markers that confer phenotypes that allow the differentiation of transformants based on conditional implementation, other types of markers are also considered, including screenable markers such as GFP, which is based on colorimetric analysis. Alternatively, a screenable enzyme can be used as a negative selection marker, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT). Those skilled in the art also know how to use immunological markers, which can be used in combination with FACS analysis. It is considered that the marker used is not important as long as it can be co-expressed with the nucleic acid encoding the gene product. Other examples of selection markers and screenable markers are well known to those skilled in the art.

[0119] D. Formulations and Administration

[0120] The present disclosure provides a pharmaceutical composition comprising a mutant SMEZ-2 conjugated to a target protein. Such a composition comprises a prophylactically or therapeutically effective amount of the target protein or a fragment or peptide immunogen thereof and a pharmaceutically acceptable carrier. In one specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals and more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, etc.

[0121] If desired, the composition may also contain minor amounts of wetting or emulsifying agents or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate, etc. Some examples of suitable pharmaceutical agents are described in "Remington’s Pharmaceutical Sciences." Such compositions will contain a prophylactically or therapeutically effective amount of an antibody or a fragment thereof (preferably in purified form) and a suitable amount of a carrier to provide the form for proper administration to a patient. The formulation should be suitable for the mode of administration, which can be oral, intravenous, intra-arterial, buccal, nasal, aerosolized, bronchial inhalation, or delivery by mechanical ventilation.

[0122] An active vaccine can be formulated for parenteral administration, for example, formulated for injection by the intradermal, intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Administration by the intradermal and intramuscular routes is contemplated. Alternatively, the vaccine can be administered directly to the mucosa by a surface route, for example, by nasal drops, inhalation, or by nebulizer. Pharmaceutically acceptable salts include acid salts and salts formed with: inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from: inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0123] Typically, the components of the compositions of the present disclosure are provided separately or mixed together in unit dosage forms, such as as a dry lyophilized powder or an anhydrous concentrate in an airtight sealed container (e.g., an ampoule or sachette) indicating the amount of the active agent. When the composition is to be administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0124] The compositions of the present disclosure can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.; and those formed with cations such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0125] Also as is well known in the art, the immunogenicity of a particular immunogenic composition can be enhanced by using a non-specific immune response stimulant known as an adjuvant. Adjuvants have been experimentally used to promote a general increase in immunity against poorly immunogenic antigens (e.g., U.S. Patent 4,877,611). Immunization regimens have used adjuvants to stimulate responses for many years and thus adjuvants are well known to those of ordinary skill in the art. Some adjuvants affect the way antigens are presented. For example, when a protein antigen is adsorbed to alum, the immune response is enhanced. Emulsification of the antigen also prolongs the duration of antigen presentation and elicits an innate immune response. Suitable molecular adjuvants include all acceptable immunostimulatory compounds such as cytokines, toxins, or synthetic compositions.

[0126] The term "adjuvant" is used in its ordinary and common meaning in immunology and refers to a substance that is commonly used as a component of an immunogenic composition. When administered to a subject as part of an immunogenic composition together with one or more specific antigens, an adjuvant can increase the antigen-specific immune response in the subject. In some embodiments, the adjuvant accelerates the immune response to the antigen. In some embodiments, the adjuvant prolongs the immune response to the antigen. In some embodiments, the adjuvant enhances the immune response to the antigen.

[0127] Those skilled in the art will be aware of the different types of adjuvants that can be conjugated to a vaccine according to the present disclosure, and which adjuvants are approved for human and experimental use. These include alkyl lysophospholipid (ALP); BCG; and biotin (including biotinylated derivatives), etc. Certain adjuvants that are particularly considered for use are from Gram-negative bacterial cells (Gram -Teichoic acids of (bacterial cell). These include lipoteichoic acid (LTA), ribitol teichoic acid (RTA), and glycerol teichoic acid (GTA). Active forms of their synthetic counterparts can also be used in combination with the compositions of the present disclosure (Takada et al., 1995).

[0128] In some aspects, the compositions described herein may also include adjuvants. Although alum is an approved adjuvant for humans, adjuvants in experimental animals include complete Freund's adjuvant (containing a non-specific immune response stimulant of killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants that can be used in animals and sometimes in humans include interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, interferons, Bacillus Calmette-Guérin (BCG), aluminum hydroxide, muramyl dipeptide (MDP) compounds such as thur-MDP and nor-MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine MDP), lipid A, and monophosphoryl lipid A (MPL). RIBI is also contemplated, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene / Tween 80 emulsion. Even MHC antigens can be used.

[0129] E. Methods of treatment

[0130] In particular, compositions are disclosed herein that can be used to treat cancer in an object (e.g., a human object). The above compositions are preferably administered to a mammal (e.g., a rodent, human, non-human primate, dog, cow, sheep, horse, cat, etc.) in an effective amount, i.e., an amount capable of producing a desired result (e.g., causing apoptosis of cancer cells or killing bacterial cells) in the treated object. The toxicity and therapeutic efficacy of the compositions used in the methods of the present disclosure can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, the dosage for any given animal depends on many factors, including the size of the object, body surface area, body weight, age, the particular composition to be administered, the time and route of administration, general health status, clinical signs of infection or cancer, and other drugs being administered concurrently. The compositions as described herein are typically administered in a dosage that inhibits the growth or proliferation of bacterial cells, inhibits the growth of biofilms, or induces the death of cancer cells (e.g., induces apoptosis of cancer cells), as determined by measuring the reduction in cancer cell growth or proliferation or hematological parameters (complete blood count (CBC)).

[0131] As used herein, the term "object" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include pre- and post-natal forms. In many embodiments, the object is a human. An object can be a patient, which refers to a person who presents to a healthcare provider for diagnosis or treatment of a disease. The term "object" may be used interchangeably herein with "individual" or "patient". An object may have or be susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0132] As used herein, the term "therapeutically effective amount" or "effective dose" refers to the dose or concentration of a drug that is effective in treating a disease or disorder. For example, with respect to using the monoclonal antibodies or antigen-binding fragments thereof disclosed herein to treat cancer, a therapeutically effective amount is the dose or concentration of the monoclonal antibody or antigen-binding fragment thereof that is capable of achieving any of the following: reducing tumor volume, eradicating all or part of the tumor, inhibiting or slowing tumor growth or infiltration of cancer cells into other organs, inhibiting the growth or proliferation of cells mediating a cancerous disorder, inhibiting or slowing tumor cell metastasis, improving any symptoms or markers associated with the tumor or cancerous disorder, preventing the tumor or cancerous disorder or delaying its occurrence, or some combination thereof.

[0133] As used herein, "treatment" of a disorder and variations thereof include preventing or alleviating the disorder, slowing the onset or rate of occurrence of the disorder, reducing the risk of developing the disorder, preventing symptoms associated with the disorder or delaying their occurrence, alleviating or terminating symptoms associated with the disorder, causing the disorder to completely or partially regress, curing the disorder, or some combination thereof.

[0134] The treatment methods of the present disclosure (which include prophylactic treatment) generally include administering to a subject in need thereof (including mammals, particularly humans) a therapeutically effective amount of the compositions described herein. Such treatment would be suitable for administration to a subject (particularly a human) suffering from, having, being susceptible to a disease, disorder or its symptoms, or at risk of developing a disease, disorder or its symptoms. Those subjects "at risk" can be determined by any objective or subjective measure, such as a diagnostic test or the opinion of the subject or a health care provider (e.g., genetic testing, enzyme or protein markers, markers as defined herein, family history, etc.).

[0135] In one embodiment, the present disclosure provides a method for monitoring the progress of treatment. The method comprises the steps of determining a hematological parameter and / or an altered level of analysis or diagnostic measurement (e.g., screening, assay) of cancer stem cells (CSCs) using a cell surface protein as a diagnostic marker (which may include, for example, but not limited to, CD34, CD38, CD90, and CD117) in a subject suffering from or susceptible to a condition or its symptoms associated with cancer (e.g., leukemia) in which the subject has been administered a therapeutically effective amount of the compositions described herein. The marker levels determined in the method can be compared with the known levels of the markers in healthy normal controls or in other diseased patients to establish the disease state of the subject. In some preferred embodiments, a second level of the marker in the subject is determined at a time point later than the time when the first level was determined, and the two levels are compared to monitor the disease progression or treatment efficacy. In certain preferred embodiments, a pre-treatment level of the marker in the subject is determined according to the methods described herein before the start of treatment; then the pre-treatment level of the marker can be compared with the level of the marker in the subject after the start of treatment to determine the treatment efficacy.

[0136] F. Combination Therapy

[0137] It is contemplated that the vaccine conjugates described herein can be used in combination therapy with additional anti-cancer agents or compounds that mitigate one or more side effects of the disease or treatment experienced by a patient. The following is a general discussion of treatments that can be used in conjunction with the treatments of the present disclosure. To treat cancer using the methods and compositions of the present disclosure, tumor cells or a subject are typically contacted with a composition of the present disclosure and at least one other treatment. These treatments will be provided in a combined amount effective to achieve a reduction in one or more disease parameters. The process can involve contacting the cell / subject simultaneously with both agents / treatments, such as using a single composition or pharmaceutical formulation that contains both agents, or contacting the cell / subject simultaneously with two different compositions or formulations, where one composition contains the compound and the other contains the other agent. Alternatively, the compositions of the present disclosure can be administered minutes to weeks before or after the other treatment. It is generally desirable to ensure that there is no significant interruption between the times of each delivery such that the treatments will still be able to have a beneficial combined effect on the cell / subject. In such cases, it is contemplated that the cell can be contacted with the two forms within about 12 to 24 hours of each other, within about 6 to 12 hours of each other, or with only about a 12-hour delay between them. However, in some cases, it may be desirable to significantly extend the treatment times, where the administrations are separated by days (2, 3, 4, 5, 6, or 7 days) to weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks).

[0138] It is also contemplated that more than one administration of the compound or other treatment may be desirable. A variety of combinations can be used, where the vaccine conjugate is “A” and the other treatment is “B”, as follows:

[0139]

[0140] The following are some examples of standard anti-cancer treatments that can be used in combination with the compositions and methods of the present application.

[0141] 1. Chemotherapy

[0142] The term “chemotherapy” refers to the use of drugs to treat cancer. “Chemotherapeutic agents” are used to denote compounds or compositions that are administered in the treatment of cancer. These agents or drugs are classified by their mode of activity within the cell (e.g., whether and at which stage they affect the cell cycle). Alternatively, agents can be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.

[0143] Some examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, prosulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (such as calicheamicin, especially calicheamicin γ1; I and calicheamicin ω1 I); dynemicin, including dynemicin A uncialamycin and its derivatives; bisphosphonates, such as clodronate; esperamicin; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyano-morpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxy-doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate;Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;Podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichloroethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids such as paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexe such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum compounds; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoid such as retinoic acid; capecitabine;Cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, actinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP-16), tamoxifen, raloxifene, estrogen receptor binders, taxol, paclitaxel, docetaxel, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.;

[0144] 2. Radiation Therapy

[0145] Radiation therapy, also known as radiation therapy, is the use of ionizing radiation to treat cancer and other diseases. Ionizing radiation deposits energy, which damages or destroys cells by damaging the genetic material of the cells in the area being treated such that these cells are unable to continue growing. Although radiation damages both cancer cells and normal cells, the latter are able to repair themselves and function properly.

[0146] Radiation therapy used in accordance with the present disclosure may include, but is not limited to, the use of -rays, X-rays, and / or radioisotopes delivered directionally to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwave and UV irradiation. Most likely, all of these agents induce extensive damage to DNA, DNA precursors, DNA replication and repair, and to the assembly and maintenance of chromosomes. The dose range of X-rays is from a daily dose of 12.9 to 51.6 mC / kg for an extended period of time (3 to 4 weeks) to a single dose of 0.516 to 1.55 C / kg. The dose range of radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake of the neoplastic cells.

[0147] Radiation therapy may include delivering a radiation dose directly to the cancer site using radiolabeled antibodies (radioimmunotherapy). Antibodies are highly specific proteins produced by the body in response to the presence of an antigen (a substance recognized as foreign by the immune system). Some tumor cells contain specific antigens that trigger the production of tumor-specific antibodies. Large quantities of these antibodies can be prepared in the laboratory and linked to a radioactive substance (a process called radiolabeling). Once injected into the body, the antibodies actively seek out cancer cells, which are destroyed by their radiation-induced cell-killing (cytotoxic) action. This approach minimizes the risk of radiation damage to normal cells. Conformal radiation therapy uses the same radiotherapy machine as conventional radiation therapy, a linear accelerator, but metal blocks are placed in the path of the x-ray beam to change its shape to match the shape of the cancer. This ensures that a higher radiation dose is delivered to the tumor. Normal surrounding cells and nearby structures receive a lower dose of radiation, thus reducing the likelihood of side effects. A device called a multileaf collimator has been developed and can be used as an alternative to the metal blocks. The multileaf collimator consists of multiple metal leaves fixed to the linear accelerator. The layers can be adjusted so that the radiotherapy beam can be shaped to the treatment area without the need for metal blocks. The precise positioning of the radiotherapy machine is very important for conformal radiation therapy, and the position of the internal organs can be checked using a special scanner at the start of each treatment.

[0148] High-resolution intensity-modulated radiation therapy also uses a multileaf collimator. During this treatment, the layers of the multileaf collimator are moved while the treatment is being delivered. This method is likely to achieve an even more precise shaping of the treatment beam and keep the radiotherapy dose constant across the treatment area.

[0149] Although studies have shown that conformal radiation therapy and intensity-modulated radiation therapy can reduce the side effects of radiation therapy, shaping the treatment area so precisely has the potential to prevent the destruction of tiny cancer cells just outside the treatment area. This means that the risk of future cancer recurrence may be higher when using these specialized radiation therapy techniques.

[0150] Scientists are also looking for ways to improve the effectiveness of radiation therapy. The effects of two types of investigational drugs on cells exposed to radiation are being studied. Radiosensitizers make tumor cells more likely to be destroyed, and radioprotectors protect normal tissues from the effects of radiation. The effectiveness of hyperthermia, the use of heat, in making tissues more sensitive to radiation is also being investigated.

[0151] 3. Immunotherapy

[0152] In the context of cancer treatment, immunotherapeutic agents generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Trastuzumab (Herceptin TM) is such an example. The immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody alone can act as an effector of the treatment, or it can recruit other cells to actually affect cell killing. The antibody can also be conjugated with a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and used only as a targeting agent. Alternatively, the effector can be a lymphocyte carrying surface molecules that directly or indirectly interact with the tumor cell target. A variety of effector cells include cytotoxic T cells and NK cells. The combination of treatment modalities (i.e., direct cytotoxic activity and inhibition or reduction of ErbB2) will provide a therapeutic benefit in treating ErbB2-overexpressing cancers.

[0153] In one aspect of immunotherapy, the tumor cells must carry some markers suitable for targeting (i.e., not present on most other cells). There are many tumor markers, and any of these tumor markers can be suitable for targeting in the context of the present disclosure. Common tumor markers include carcinoembryonic antigen, prostate-specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, SialylLewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine the anti-cancer effect with an immune-stimulating effect. There are also immune-stimulating molecules, which include: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, -IFN; chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand. Combining immune-stimulating molecules (either as proteins or using gene delivery in combination with tumor suppressors) has been shown to enhance the anti-tumor effect (Ju et al., 2000). In addition, antibodies against any of these compounds can be used to target the anti-cancer agents discussed herein.

[0154] Some examples of immunotherapies currently under investigation or in use are immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169); cytokine therapies such as interferon α, , and; IL-1, GM-CSF, and TNF; gene therapies such as TNF, IL-1, IL-2, p53 (U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies such as anti-ganglioside GM2, anti-HER-2, anti-p185 (U.S. Patent 5,824,311).

[0155] In active immunotherapy, antigenic peptides, polypeptides, or proteins, or autologous or allogeneic tumor cell compositions or “vaccines” are typically administered together with different bacterial adjuvants (Ravindranath and Morton, 1991; Morton et al., 1992; Mitchell et al., 1990; Mitchell et al., 1993).

[0156] In adoptive immunotherapy, a patient's circulating lymphocytes or tumor infiltrating lymphocytes are isolated in vitro, activated by lymphokines such as IL-2 or transduced with the tumor necrosis gene, and readministered.

[0157] Checkpoint inhibitors are an emerging class of immunotherapeutic agents. Checkpoint inhibitor therapy is a form of cancer immunotherapy currently under investigation. The therapy targets immune checkpoints (i.e., key regulators that stimulate or inhibit the action of the immune system), which tumors can utilize to protect themselves from attack by the immune system. Checkpoint therapy can block inhibitory checkpoints, restoring immune system function. The first anti-cancer drug to target an immune checkpoint was ipilimumab, a CTLA4 blocker approved in the United States in 2011.

[0158] Currently approved checkpoint inhibitors target the molecules CTLA4, PD-1, and PD-L1. PD-1 is the transmembrane programmed cell death 1 protein (also known as PDCD1 and CD279), which interacts with PD-L1 (PD-1 ligand 1, or CD274). PD-L1 on the cell surface binds to PD1 on the surface of immune cells, which inhibits the activity of immune cells. A key regulatory role in the function of PD-L1 includes exerting a key regulatory effect on T cell activity. Upregulation of PD-L1 on the cell surface (mediated by cancer) can inhibit T cells that might otherwise attack. Antibodies that bind to PD-1 or PD-L1 and thus block the interaction can allow T cells to attack tumors.

[0159] The first checkpoint antibody approved by the FDA was ipilimumab, which was approved in 2011 for the treatment of melanoma. It blocks the immune checkpoint molecule CTLA-4. Clinical trials have also shown some benefits of anti-CTLA-4 therapy for lung or pancreatic cancer, especially in combination with other drugs.

[0160] However, patients treated with checkpoint blockade (especially CTLA-4 blocking antibodies) or combinations of checkpoint blocking antibodies are at high risk of developing immune-related adverse events (such as cutaneous, gastrointestinal, endocrine, or hepatic autoimmune reactions). This is most likely due to the breadth of T cell activation induced when anti-CTLA-4 antibodies are administered by injection into the bloodstream.

[0161] Using a mouse model of bladder cancer, researchers found that local injection of low-dose anti-CTLA-4 in the tumor region had the same tumor-suppressive ability as when the antibody was delivered in the blood. At the same time, the circulating antibody levels were lower, indicating that local administration of anti-CTLA-4 therapy may result in fewer adverse events.

[0162] The initial clinical trial results of the IgG4 PD1 antibody nivolumab (under the trade name Opdivo and developed by Bristol-Myers Squibb) were published in 2010. It was approved in 2014. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, bladder cancer, head and neck cancer, and Hodgkin lymphoma.

[0163] Pembrolizumab (trade name Keytruda) is another PD1 inhibitor, which was approved by the FDA in 2014 and is the second checkpoint inhibitor approved in the United States. Keytruda is approved for the treatment of melanoma and lung cancer and is manufactured by Merck.

[0164] Spartalizumab (PDR001) is a PD-1 inhibitor currently being developed by Novartis for the treatment of solid tumors and lymphomas. In May 2016, the PD-L1 inhibitor atezolizumab was approved for the treatment of bladder cancer. Other modalities to enhance adoptive immunotherapy include targeting so-called innate checkpoint blockade, such as CISH.

[0165] Immune adverse effects can be caused by checkpoint inhibitors. Altering checkpoint inhibition can have different effects on most organ systems of the body. The exact mechanism is unknown but differs in some respects based on the molecule targeted.

[0166] 4. Surgery

[0167] Approximately 60% of people with cancer will undergo some type of surgery, which includes prophylactic, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that can be used in combination with other treatments, such as the treatments of the present disclosure, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0168] Curative surgery includes resections in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumorectomy refers to the physical removal of at least part of the tumor. In addition to tumorectomy, treatments performed surgically also include laser surgery, cryosurgery, electro-surgery, and microscopically controlled surgery (Mohs’ surgery). It is also contemplated that the present disclosure can be used in combination with the removal of superficial cancer, primary cancer, or an accompanying amount of normal tissue.

[0169] After removing some or all of the cancer cells, tissue, or tumor, a cavity may form in the body. Treatment can be accomplished by perfusion, direct injection, or local application of additional anti-cancer treatment in the area. Such treatments can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments can also have multiple doses.

[0170] In some specific embodiments, adjuvant treatment with the compounds of the present disclosure after tumor removal is considered to be particularly effective in reducing tumor recurrence. Additionally, the compounds of the present disclosure can also be used in neoadjuvant settings.

[0171] 5. Other agents

[0172] It is contemplated that other agents may be used in conjunction with the present disclosure. These additional agents include immunomodulators, agents that affect the upregulation of gap junctions and cell surface receptors, cytostatic and differentiating agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. Immunomodulators include tumor necrosis factor; interferons α, β, and γ; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1β, MCP-1, RANTES, and other chemokines. It is also contemplated that upregulation of cell surface receptors or their ligands (e.g., Fas / Fas ligand, DR4 or DR5 / TRAIL (Apo-2 ligand)) will enhance the apoptosis-inducing ability of the present disclosure by establishing autocrine or paracrine effects on hyperproliferative cells. Increasing intercellular signaling by increasing the number of gap junctions will enhance the antiproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiating agents may be used in combination with the present disclosure to improve the antiproliferative efficacy of the treatment. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present disclosure. Some examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is also contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis (e.g., the antibody c225) may be used in combination with the present disclosure to improve treatment efficacy.

[0173] With the introduction of cytotoxic chemotherapeutic agents, many advances have been made in cancer treatment. However, one of the consequences of chemotherapy is the development / acquisition of drug-resistant phenotypes and the development of multidrug resistance. The development of drug resistance remains a major obstacle in treating such tumors and, thus, there is a clear need for alternative methods, such as gene therapy. Another form of treatment that can be used in combination with chemotherapy, radiotherapy, or biotherapy includes hyperthermia, which is the process of exposing a patient's tissue to elevated temperatures (up to 106°F). External or internal heating devices may be involved in the application of local, regional, or whole-body hyperthermia. Local hyperthermia involves applying heat to a small area (e.g., a tumor). The heat can be generated externally by high-frequency waves targeted at the tumor from an extracorporeal device. Internal heating may involve sterile probes, including thin heated wires or hollow tubes filled with warm water, implanted microwave antennas, or radiofrequency electrodes.

[0174] The patient's organ or limb is heated for regional treatment, which is achieved using a device that generates high energy (e.g., a magnet). Alternatively, some of the patient's blood may be removed and heated before being perfused into the area to be internally heated. Whole-body heating may also be implemented in cases where the cancer has spread systemically. A warm water blanket, hot wax, induction coils, and a hyperthermia chamber can be used for this purpose.

[0175] Technicians are guided by "Remington’s Pharmaceutical Sciences", 15th Edition, Chapter 33, particularly pages 624 to 652. Depending on the condition of the subject being treated, it will be necessary to make some changes in the dosage. In any case, the person responsible for administration will determine the appropriate dosage for an individual subject. In addition, for human administration, the formulation should meet the standards of sterility, pyrogenicity, general safety and purity required by the FDA Office of Biological Standards.

[0176] It should also be noted that any of the foregoing treatments may prove itself useful in the treatment of cancer.

[0177] II. Kit

[0178] In some aspects of some embodiments, kits are envisioned that contain a therapeutic agent and / or other therapeutic and delivery agents. In some embodiments, the present disclosure contemplates kits for the preparation and / or administration of the vaccine compositions of the various embodiments. The kit may comprise one or more sealed vials containing any of the pharmaceutical compositions of the embodiments of the invention. The kit may contain, for example, a vaccine conjugate and reagents for preparing, formulating and / or administering the components of the various embodiments or performing one or more steps of the methods of the invention. In some embodiments, the kit may further comprise a suitable container that does not react with the components of the kit, such as an eppendorf tube, assay plate, syringe, bottle or tube. The container may be made of a sterilizable material (such as plastic or glass).

[0179] The kit may contain one or more reagents for biotechnological products or assays. The kit may also contain reagents for in vitro assays (such as western blot, flow cytometry, immunoprecipitation, ELISA or immunofluorescence).

[0180] The kit may also contain instructions that outline the procedural steps of the methods described herein and will follow substantially the same procedures as described herein or known to those of ordinary skill in the art. This instructional information may be in a computer-readable medium containing machine-readable instructions that, when executed by a computer, cause a real or virtual program for delivering a pharmaceutically effective amount of a therapeutic agent to be displayed.

[0181] III. Examples

[0182] The following examples are included to illustrate some preferred embodiments of the present disclosure. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that the inventors have found to function well in the practice of the present disclosure and can thus be considered to constitute a preferred mode for the practice of the present disclosure. However, in accordance with the present disclosure, those skilled in the art should understand that many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain the same or similar results.

[0183] Example 1 - Development of the SMEZ-2 Vaccine Conjugate

[0184] An inactivated AGR2-SMEZ-2 conjugate was developed that stimulates a robust immune response against PDAC with high extracellular AGR2. The conjugate utilizes the MHC-II binding ability of SMEZ-2 to enable efficient uptake and presentation of AGR2 peptides by APCs ( Figure 1 ). This technology overcomes the challenges currently presented by peptide-based cancer vaccines, where due to the high-affinity binding properties of SMEZ-2, in silico modeling of peptide binding to MHC molecules is no longer required. Thus, the method of the present invention provides AGR2-based PDAC vaccines, as well as other vaccine candidates based on TAAs and neoantigens.

[0185] The candidate vaccine conjugate was synthesized by: using the AsiSI restriction site to fuse the DNA sequence of human AGR2 (21 - 175) with the sequence of an inactivated and T cell receptor-binding-deficient mutant SMEZ-2 (W75L, K182Q, D42C). The fusion was cloned into pET15TEV_NESG, which expresses a protein with an N-terminal 6× histidine tag that can be cleaved by tobacco etch virus (TEV) protease ( Figure 3 ). The vaccine conjugate was expressed in BL21(DE3) Escherichia coli and purified using immobilized metal affinity chromatography IMAC. To avoid non-specific immunogenicity or unexpected side effects, the 6× histidine tag was excised by overnight digestion with TEV and purified by another round of IMAC, where the flow-through contained the purified protein. The purity of the conjugate was determined by SDS-PAGE (>95%) ( Figure 2 ). AGR2 and SMEZ-2 were cloned, expressed, and purified separately using the same method as for the conjugate. The proteins were highly soluble in Escherichia coli, and the yields were 33 mg / L of AGR2, 11 mg / L of SMEZ-2 (W75L, K182Q, D42C), and 4 mg / L of AGR2-SMEZ-2 (W75L, K182Q, D42C). As an alternative to expression in Escherichia coli, these proteins can be produced in other cell types (such as HEK and CHO cells).

[0186] The immune responses elicited by unconjugated AGR2, unconjugated SMEZ2, the unconjugated forms of AGR2 and SMEZ mixed together, the AGR2-SMEZ2 conjugate, and the TLR9 agonist CpG ODN1826 were tested in C57BL / 6 mice by intramuscular injection in PBS vehicle. CpG ODN1826 is a class B CpG oligonucleotide that contains a complete phosphorothioate backbone and one or more CpG dinucleotides. Mice (n = 10) were injected every two weeks with a total volume of 100 μL of 100 μg of AGR2-SMEZ-2 or equimolar amounts of the other proteins. Anti-AGR2 specific IgG from mouse plasma was analyzed by ELISA. Figure 4 The data in were taken from blood samples 10 days after the second injection. The results showed that 10 days after the second vaccination, the anti-AGR2 antibody titers in the AGR2-SMEZ-2 treated group were significantly higher than those in mice treated with AGR2 alone or AGR2 co-injected with SMEZ, indicating that the SMEZ2 vector can enhance the presentation of tumor-associated antigens such as AGR2 to the immune system.

[0187] Next, the anti-tumor potential of this treatment method was evaluated using a preclinical mouse model of pancreatic ductal adenocarcinoma (PDAC). Specifically, PDAC cells harvested from pancreatic tumors in LSL-KrasG12D; LSL-Trp53R172H; Pdx1-cre (KPC) mice were used. KPC32908 cells were engineered to express high levels of human AGR2, which has >95% sequence homology with mouse AGR2. Then, wild-type C57BL / 6 mice (syngeneic to KPC mice) were treated with the AGR2-SMEZ2 conjugate or appropriate controls with or without CpG ODN1826 according to the schedule shown in Figure 5A. After two cycles of treatment, the mice were challenged with KPC cells injected subcutaneously into the posterior flanks. This model rapidly developed aggressive KPC PDAC tumors that were resistant to standard-of-care chemotherapy and checkpoint inhibitor immunotherapy. However, the combination of AGR2-SMEZ2 and the TLR9 agonist CpG ODN1826 significantly reduced the tumor growth rate (Figure 5B), thus demonstrating tumor control of pancreatic tumors in a highly aggressive model system.

[0188] Next, the activity of the SMEZ2 conjugate system against other tumor-associated antigens and tumor types / models was evaluated. CD38 is an actionable therapeutic target in multiple myeloma (MM), as demonstrated by the clinical efficacy of the anti-CD38 monoclonal antibody daratumumab / DARZALEX in newly diagnosed and relapsed / refractory MM patients [45-47]. To further demonstrate the potential of the SMEZ2 conjugate platform to enhance the immune presentation of tumor-associated antigens and induce a native polyclonal humoral response, a murine CD38-SMEZ2 conjugate was constructed and expressed and purified in a mammalian expression system. The purified protein product is shown in Figure 6A. Wild-type BALB / C mice were then treated with two doses of 50 μg mCD38-SMEZ or an equimolar equivalent of control in IFA by subcutaneous administration. Plasma was obtained 10 days after the second injection, and CD38-specific IgG was quantified by ELISA. As shown in Figure 6B and consistent with the data observed using the AGR2-SMEZ2 protein, significantly higher levels of CD38-specific IgG were measured in the blood of mice receiving the CD38-SMEZ2 conjugate compared to groups receiving either CD38 alone or a mixture of free CD38 and SMEZ2. These results further demonstrate the ability of the SMEZ2 superantigen platform to enhance the presentation of multiple tumor-associated antigens to the immune system.

[0189] Next, the SMEZ2 platform was evaluated against additional targets, including immune cell localization antigens that regulate anti-tumor T cell responses. Specifically, constructs were created against two inhibitory immune checkpoint molecules that inhibit T cell function, Programmed Cell Death protein-1 (PD-1, also known as CD279) and Cytotoxic T Lymphocyte Associated protein-4 (CTLA-4, also known as CD152)

[35] . Antagonist antibodies or immune checkpoint inhibitors (ICIs) against these molecules have clinical activity in a range of tumor types, and the field has witnessed FDA approval of multiple ICIs, including pembrolizumab / KETRUDA (anti-PD-1), nivolumab / OPDIVO (anti-PD-1), and ipilimumab / YERVOY (anti-CTLA-4) [36-40]. Monoclonal antibodies against Programmed Death Ligand-1 (PD-L1) have also shown clinical utility, where PD-L1 engages with PD-1 to activate the PD-1 immune checkpoint and transduce inhibitory T cell signals. Atezolizumab / TECENTRIQ is the first FDA-approved anti-PD-L1 antibody

[41] . In addition to the role played by the immune checkpoint molecules CTLA-4 and PD-1 in suppressing anti-tumor T cell immunity, they are also highly expressed in malignant T cells, making them potential immunotherapeutic targets for lymphomas and leukemias arising from the T cell compartment. These include peripheral forms of T cell non-Hodgkin lymphoma, such as cutaneous T cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), and NK / T cell lymphoma, in which the expression of wild-type or gene fusions of PD-1 and CTLA-4 is at high levels compared to normal T cells [42-44]. Thus, immune checkpoints are actionable targets for driving anti-tumor immune responses in certain tumor types and can also be attractive therapeutic targets for T cell malignancies.

[0190] To explore the potential of using the SMEZ2 platform to target immune checkpoints, mouse PD1-SMEZ, CTLA4-SMEZ, and PDL1-SMEZ constructs were created and expressed and purified in a mammalian cell culture system (Figure 7). Next, the ability of the superantigen constructs to induce PD-1 / PD-L1 / CTLA-4-specific immune responses in vivo was evaluated. The results in Figure 8 show that a mixture containing all 3 conjugates was able to induce strong polyclonal humoral responses against mouse PD-1, PD-L1, and CTLA-4 (Figure 8A). Although it was hypothesized that the antibodies generated against these antigens might lead to blockade of immune checkpoints and increased T cell viability, proliferation, and activity, the results showed that the mixture actually depleted the T cell population in the spleen (Figure 8B). This indicates that the response is mainly an anti-T cell response rather than a T cell-supporting effect. These results further demonstrate the concept of the SMEZ platform and provide evidence for its versatility against multiple tumor and disease-specific antigens. They further suggest that SMEZ checkpoint constructs or mixtures can be used to deplete the T cell population in pathological conditions such as T cell non-Hodgkin lymphoma or autoimmune diseases.

[0191] Development of the Example 2 - CD33-SMEZ-2 Vaccine Conjugate

[0192] To evaluate the potential of SMEZ-2 superantigen to drive immune responses against CD33+ AML, the full-length human CD33-SMEZ-2 conjugate protein was cloned, produced, and purified in a mammalian cell culture system (Figure 9A). Then, the purified protein was tested in vivo using an immunocompetent syngeneic mouse model of AML in C57BL / 6 mice. The protein was injected subcutaneously in an emulsion of Freund's incomplete adjuvant (IFA).

[0193] At a dose of 40 pmol injected at 2-week intervals, the CD33-SMEZ-2 conjugate was able to induce significantly stronger humoral responses against CD33, as determined by ELISA measurement of anti-hCD33 IgG in the plasma of immunized mice (Figure 9B).

[0194] To evaluate the anti-AML effect of the CD33-SMEZ-2 therapeutic agent, a syngeneic AML cell model of C1498 AML cells that spontaneously arise in C57BL / 6 mice was used. Mice were treated with two doses of CD33-SMEZ-2 and then, two weeks later, intravenously injected with 1 × 10 6 C1498 cells transduced with the human CD33 gene. This treatment regimen significantly prolonged the median survival (p < 0.05, N = 5) and cured 60% of the treated mice, where "cured" was defined as survival beyond 100 days.

[0195] *********

[0196] According to the present disclosure, all of the methods disclosed and claimed herein can be made and carried out without undue experimentation. Although the compositions and methods of the present invention have been described in accordance with some preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the methods described herein and in the steps or the order of the steps of such methods without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain reagents, which are both chemically and physiologically relevant, can be substituted for the reagents described herein, while the same or similar results will be achieved. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0197] References

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Claims

1. A vaccine conjugate comprising a mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2) conjugated to at least one target protein or a fragment thereof, wherein the at least one target protein is overexpressed in cancer.

2. The conjugate of claim 1, wherein the mutant SMEZ-2 comprises the mutations W75L and K182Q.

3. The conjugate of claim 1 or 2, wherein the mutant SMEZ-2 comprises the mutations W75L, K182Q, and / or D42C.

4. The conjugate of any one of claims 1 to 3, wherein the mutant SMEZ-2 comprises the mutations W75L, K182Q, and D42C.

5. The conjugate of any one of claims 1 to 4, further comprising a linker.

6. The conjugate of claim 5, wherein the linker is a peptide linker.

7. The conjugate of claim 6, wherein the peptide linker is a glycine-serine linker.

8. The conjugate of claim 6 or 7, wherein the peptide linker comprises the sequence AIA or GGGGS.

9. The conjugate of any one of claims 1 to 4, wherein the at least one target protein is a neoantigen or a tumor-associated antigen (TAA).

10. The conjugate of any one of claims 1 to 4, wherein the at least one target protein is anterior gradient protein 2 (AGR2).

11. The conjugate of claim 10, wherein AGR2 is human AGR2.

12. The conjugate of any one of claims 1 to 4, wherein the at least one target protein is an immune checkpoint protein.

13. The conjugate of claim 12, wherein the immune checkpoint protein is CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR.

14. The conjugate of claim 12, wherein the immune checkpoint protein is CTLA-4, PD-1, or PD-L1.

15. The conjugate of any one of claims 1 to 4, wherein the at least one target protein is CD38.

16. The conjugate of any one of claims 1 to 9, wherein the at least one target protein is CD38, PD-1, PD-L1, CTLA-4, human epidermal growth factor receptor 2 (HER2), prostate-specific membrane antigen (PSMA), melanoma-associated antigen 3 (MAGE-A3), NY-ESO-1, IL-8, or growth / differentiation factor-15 (GDF-1).

17. The conjugate of any one of claims 1 to 9, wherein the at least one target protein is CD33 or a fragment thereof, mesothelin (MSLN), B cell maturation antigen (BCMA), GPRC5D, CD123, CLL-1 (CD371), CD19, CD30, or CD20.

18. The conjugate of claim 17, wherein the CD33 or its fragment comprises one or more amino acid substitutions.

19. The conjugate according to claim 18, wherein the one or more amino acid substitutions are located at D231, D246, C154, and / or C169.

20. The conjugate according to claim 18, wherein the one or more amino acid substitutions are located at D231E, D246E, C154S, and / or C169S.

21. The conjugate according to claim 18 or 19, wherein the CD33 consists of the CD33-IgC domain.

22. The conjugate according to any one of claims 1 to 16, wherein the vaccine conjugate comprises at least a second target protein or a fragment thereof.

23. A pharmaceutical composition comprising the vaccine conjugate according to any one of claims 1 to 22 and an adjuvant.

24. The composition according to claim 23, wherein the adjuvant is incomplete Freund's adjuvant.

25. The composition according to claim 24, wherein the conjugate and incomplete Freund's adjuvant (IFA) are formulated as a 1:1 emulsion.

26. The composition according to claim 23, wherein the conjugate is formulated for intravenous infusion.

27. An expression vector comprising a sequence encoding mutant SMEZ-2 fused to a sequence encoding a target protein or a fragment thereof.

28. The vector according to claim 27, wherein the vector encodes the vaccine conjugate according to any one of claims 1 to 22.

29. A host cell comprising the expression vector according to claim 27 or 28.

30. The host cell according to claim 29, wherein the host cell is Escherichia coli, human embryonic kidney cells (HEK293), or Chinese hamster ovary (CHO) cells.

31. A method for stimulating an immune response in a subject, comprising administering to the subject an effective amount of the vaccine conjugate according to any one of claims 1 to 22 or the pharmaceutical composition according to any one of claims 23 to 25.

32. The method according to claim 31, wherein the immune response is an anti-cancer immune response.

33. The method according to claim 31 or 32, wherein the subject has cancer.

34. The method according to claim 33, wherein the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, urogenital system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer, or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, cholangiocarcinoma, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma tumor, multiple endocrine neoplasia type I and type II tumors, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

35. The method according to claim 33, wherein the cancer is pancreatic cancer.

36. The method according to claim 35, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

37. The method according to claim 33, wherein the cancer is breast cancer.

38. The method according to claim 37, wherein the target protein is CD38, and the cancer is multiple myeloma.

39. The method according to claim 37, wherein the cancer is lymphoma.

40. The method according to claim 39, wherein the lymphoma is T-cell non-Hodgkin lymphoma.

41. The method according to any one of claims 31 to 37, wherein the immune response is an anti-AGR2 specific immune response.

42. The method according to claim 41, wherein the anti-AGR2 specific immune response is detected by measuring an increase in the titer of AGR2-specific immunoglobulin in a blood sample of the subject.

43. The method according to any one of claims 31 to 42, wherein the conjugate is administered by injection.

44. The method according to any one of claims 31 to 43, further comprising administering a second anti-cancer treatment to the subject.

45. The method according to claim 44, wherein the second anti-cancer treatment is immunotherapy, chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy.

46. The method according to claim 44, wherein the second anti-cancer treatment is immunotherapy.

47. The method according to claim 46, wherein the immunotherapy is an immune checkpoint inhibitor.

48. The method according to claim 47, wherein the immune checkpoint inhibitor is selected from inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR.

49. The method according to claim 47, wherein the immune checkpoint inhibitor comprises an anti-PD1 agent.

50. The method according to claim 49, wherein the anti-PD1 agent comprises an anti-PD1 antibody, an anti-PDL1 antibody or an anti-PDL2 antibody.

51. The method according to claim 49, wherein the anti-PD1 agent is nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA or AMP-224.

52. The method according to claim 47, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

53. The method according to claim 52, wherein the anti-CTLA-4 antibody is tremelimumab, or ipilimumab.

54. A method of treating a subject having cancer, comprising administering to the subject the vaccine conjugate according to any one of claims 1 to 22 or the pharmaceutical composition according to any one of claims 23 to 25.

55. The method according to claim 54, wherein the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, urogenital system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, cholangiocarcinoma, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma tumor, multiple neuroendocrine type I and type II tumors, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

56. The method according to claim 54, wherein the cancer is prostate cancer.

57. The method according to claim 56, wherein the prostate cancer is pancreatic ductal adenocarcinoma (PDAC).

58. The method according to claim 54, wherein the cancer is breast cancer.

59. The method according to claim 54, wherein the target protein is CD38, and the cancer is multiple myeloma.

60. The method according to claim 54, wherein the cancer is lymphoma.

61. The method according to claim 60, wherein the lymphoma is T-cell non-Hodgkin lymphoma.

62. The method according to any one of claims 54 to 58, further comprising administering to the subject at least one immune checkpoint inhibitor.

63. The method according to claim 62, wherein administering to the subject at least one immune checkpoint inhibitor comprises administering the at least one immune checkpoint inhibitor before the vaccine conjugate.

64. The method according to claim 62, wherein administering to the subject at least one immune checkpoint inhibitor comprises administering the at least one immune checkpoint inhibitor after or simultaneously with the vaccine conjugate.

65. The method according to any one of claims 62 to 64, wherein the at least one immune checkpoint inhibitor is selected from inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR.

66. The method according to any one of claims 62 to 65, wherein the at least one immune checkpoint inhibitor comprises an anti-PD1 agent.

67. The method according to claim 66, wherein the anti-PD1 agent comprises an anti-PD1 antibody, an anti-PDL1 antibody or an anti-PDL2 antibody.

68. The method according to claim 66, wherein the anti-PD1 agent is nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA or AMP-224.

69. The method according to any one of claims 62 to 68, wherein the at least one immune checkpoint inhibitor is an anti-CTLA-4 antibody.

70. The method according to claim 69, wherein the anti-CTLA-4 antibody is tremelimumab, or ipilimumab.

71. The method according to any one of claims 62 to 70, wherein two immune checkpoint inhibitors are administered to the subject.

72. The method according to claim 71, wherein the two immune checkpoint inhibitors are an anti-PD1 antibody and an anti-CTL4 antibody.

73. The method according to any one of claims 54 to 72, wherein the vaccine conjugate is administered two or more times.

74. The method according to any one of claims 54 to 73, further comprising administering to the subject additional anti-cancer therapy.

75. The method according to claim 74, wherein the additional anti-cancer therapy is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy.

76. The method according to claim 74, wherein the additional anti-cancer therapy comprises a TLR9 agonist and / or a CD40 agonist.

77. The method according to claim 76, wherein the TLR9 agonist is CpG ODN1826.

78. The method according to claim 76, wherein the CD40 agonist is a CD40 agonistic antibody.

79. A medicine box, which contains the vaccine conjugate according to any one of claims 1 to 22 or the pharmaceutical composition according to any one of claims 23 to 25.

80. The medicine box according to claim 79, which further contains an immune checkpoint inhibitor.

81. The medicine box according to claim 80, wherein the immune checkpoint inhibitor is an anti-PD1 antibody or a CTLA-4 antibody.

82. The medicine box according to any one of claims 79 to 81, which further contains a TLR9 agonist.

83. The medicine box according to claim 82, wherein the TLR9 agonist is CpG ODN1826.

84. A composition for treating cancer, which contains the vaccine conjugate according to any one of claims 1 to 22 or the pharmaceutical composition according to any one of claims 23 to 25.

85. The composition according to claim 84, which further contains an immune checkpoint inhibitor.

86. The composition according to claim 85, wherein the immune checkpoint inhibitor is an anti-PD1 antibody or a CTLA-4 antibody.

87. The composition according to any one of claims 84 to 86, which further contains a TLR9 agonist.

88. The composition according to claim 82, wherein the TLR9 agonist is CpG ODN1826.

89. A vaccine conjugate, which contains mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2) conjugated to CD33.

90. The conjugate according to claim 89, wherein the mutant SMEZ-2 contains mutations W75L and K182Q.

91. The conjugate according to claim 89 or 90, wherein the mutant SMEZ-2 contains mutations W75L, K182Q and / or D42C.

92. The conjugate according to any one of claims 89 to 91, wherein the mutant SMEZ-2 contains mutations W75L, K182Q and D42C.

93. The conjugate according to claim 89, wherein CD33 is human CD33.

94. The conjugate according to claim 89, wherein the CD33 or its fragment contains one or more amino acid substitutions.

95. The conjugate according to claim 94, wherein the one or more amino acid substitutions are located at D231, D246, C154 and / or C169.

96. The conjugate according to claim 94, wherein the one or more amino acid substitutions are D231E, D246E, C154S and / or C169S.

97. The conjugate according to claim 94 or 95, wherein the CD33 consists of the CD33-IgC domain.

98. The conjugate according to any one of claims 89 to 93, wherein the conjugate further contains a linker.

99. The conjugate according to claim 98, wherein the linker is a peptide linker.

100. The conjugate according to claim 99, wherein the peptide linker is a glycine-serine linker.

101. The conjugate according to claim 100, wherein the glycine-serine linker is GGGGS.

102. The conjugate according to any one of claims 89 to 104, wherein the vaccine conjugate further comprises a target protein or a fragment thereof that is overexpressed in cancer.

103. A pharmaceutical composition comprising the vaccine conjugate according to any one of claims 89 to 102 and an adjuvant.

104. The composition according to claim 103, wherein the adjuvant is incomplete Freund's adjuvant.

105. The composition according to claim 104, wherein the conjugate and incomplete Freund's adjuvant (IFA) are formulated as a 1:1 emulsion.

106. The composition according to claim 103, wherein the conjugate is formulated for intravenous infusion.

107. An expression vector comprising a sequence encoding a mutant SMEZ-2 fused to CD33 or a fragment thereof.

108. The vector according to claim 107, wherein the vector encodes the vaccine conjugate according to any one of claims 89 to 102.

109. A host cell comprising the expression vector according to claim 107 or 108.

110. The host cell according to claim 109, wherein the host cell is Escherichia coli, human embryonic kidney cells (HEK293) or Chinese hamster ovary (CHO) cells.

111. A method for stimulating an immune response in a subject, comprising administering to the subject an effective amount of the vaccine conjugate according to any one of claims 89 to 102 or the pharmaceutical composition according to any one of claims 103 to 105.

112. The method according to claim 111, wherein the immune response is an anti-cancer immune response.

113. The method according to claim 111 or 112, wherein the subject has cancer.

114. The method according to claim 113, wherein the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, urogenital system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, cholangiocarcinoma, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma tumor, multiple endocrine neoplasia type I and type II tumors, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

115. The method according to claim 113, wherein the cancer is leukemia.

116. The method according to claim 115, wherein the leukemia is acute myeloid leukemia (AML).

117. The method according to any one of claims 111 to 120, wherein the immune response is an anti-CD33 specific immune response.

118. The method according to claim 117, wherein the anti-CD33 specific immune response is detected by measuring an increase in the titer of CD33-specific immunoglobulin in a blood sample of the subject. The method according to any one of claims 111 to 118, wherein the conjugate is administered by injection. The method according to any one of claims 111 to 119, further comprising administering a second anti-cancer treatment to the subject. The method according to claim 120, wherein the second anti-cancer treatment is immunotherapy, chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy. The method according to claim 120, wherein the second anti-cancer treatment comprises a TLR9 agonist. The method according to claim 122, wherein the TLR9 agonist is CpG ODN1826. The method according to claim 120, wherein the second anti-cancer treatment is immunotherapy. The method according to claim 124, wherein the immunotherapy is an immune checkpoint inhibitor. The method according to claim 125, wherein the immune checkpoint inhibitor is selected from inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR. The method according to claim 125, wherein the immune checkpoint inhibitor comprises an anti-PD1 agent. The method according to claim 127, wherein the anti-PD1 agent comprises an anti-PD1 antibody, an anti-PDL1 antibody or an anti-PDL2 antibody.

129. The method according to claim 127, wherein the anti-PD1 agent is nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA or AMP-224. The method according to claim 125, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

131. The method according to claim 130, wherein the anti-CTLA-4 antibody is tremelimumab, or ipilimumab. A method of treating a subject having cancer, comprising administering to the subject the vaccine conjugate according to any one of claims 89 to 102 or the pharmaceutical composition according to any one of claims 103 to 105. The method according to claim 132, wherein the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, urogenital system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, cholangiocarcinoma, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma tumor, multiple endocrine neoplasia type I and type II tumors, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer. The method according to claim 132, wherein the cancer is leukemia. The method according to claim 134, wherein the cancer is acute myeloid leukemia (AML). The method according to claim 132, wherein the cancer is pancreatic cancer or breast cancer. The method according to any one of claims 132 to 136, further comprising administering to the subject at least one immune checkpoint inhibitor.

138. The method according to claim 137, wherein administering to the subject at least one immune checkpoint inhibitor comprises administering the at least one immune checkpoint inhibitor before the vaccine conjugate.

139. The method according to claim 137, wherein administering to the subject at least one immune checkpoint inhibitor comprises administering the at least one immune checkpoint inhibitor after or simultaneously with the vaccine conjugate.

140. The method according to any one of claims 137 to 139, wherein the at least one immune checkpoint inhibitor is selected from inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR.

141. The method according to any one of claims 137 to 140, wherein the at least one immune checkpoint inhibitor comprises an anti-PD1 agent.

142. The method according to claim 141, wherein the anti-PD1 agent comprises an anti-PD1 antibody, an anti-PDL1 antibody or an anti-PDL2 antibody.

143. The method according to claim 141, wherein the anti-PD1 agent is nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA or AMP-224.

144. The method according to any one of claims 137 to 143, wherein the at least one immune checkpoint inhibitor is an anti-CTLA-4 antibody. The method according to claim 144, wherein the anti-CTLA-4 antibody is tremelimumab, or ipilimumab.

146. The method according to any one of claims 137 to 145, wherein two immune checkpoint inhibitors are administered to the subject.

147. The method according to claim 146, wherein the two immune checkpoint inhibitors are an anti-PD1 antibody and an anti-CTL4 antibody.

148. The method according to any one of claims 132 to 147, wherein the vaccine conjugate is administered two or more times.

149. The method according to any one of claims 132 to 148, further comprising administering to the subject an additional anti-cancer treatment.

150. The method according to claim 149, wherein the additional anti-cancer treatment is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy.

151. The method according to claim 149, wherein the additional anti-cancer treatment comprises a TLR9 agonist.

152. The method according to claim 151, wherein the TLR9 agonist is CpG ODN1826.

153. A kit comprising the vaccine conjugate according to any one of claims 89 to 102 or the pharmaceutical composition according to any one of claims 103 to 105.

154. The kit according to claim 153, further comprising an immune checkpoint inhibitor.

155. The kit according to claim 154, wherein the immune checkpoint inhibitor is an anti-PD1 antibody or an anti-CTLA-4 antibody.

156. The kit according to claim 153, further comprising a TLR9 agonist.

157. The kit according to claim 156, wherein the TLR9 agonist is CpG ODN1826.

158. A composition for treating cancer, comprising the vaccine conjugate according to any one of claims 89 to 102 or the pharmaceutical composition according to any one of claims 103 to 105.

159. The composition according to claim 158, further comprising an immune checkpoint inhibitor.

160. The composition according to claim 158 or 159, further comprising a TLR9 agonist and / or a CD40 agonist.

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