Superantigen vaccine conjugates for the treatment of cancer - Patent Application 20070122999

Abstract

The present disclosure provides compositions comprising vaccine conjugates with SMEZ-2 carriers. Further provided are methods for treating cancer, comprising administering the vaccine conjugates provided herein. TIFF2025535082000005.tif73148

Description

[Technical Field]

[0001] Priority claim This application claims the benefit of priority to U.S. Provisional Application Nos. 63 / 413,457 and 63 / 413,468, filed October 5, 2022, the entire contents of both of which are incorporated herein by reference.

[0002] background 1. Field The present disclosure relates generally to the field of molecular biology. More specifically, the present disclosure relates to compositions comprising vaccine conjugates for the treatment of cancer. [Background technology]

[0003] 2. Related Technologies Pancreatic ductal adenocarcinoma (PDAC) accounted for 56,700 new cancer cases and 45,700 deaths in 2019, with the lowest 5-year survival rate of all cancers at only 9% (Siegel et al., 2019). Treatment options are currently limited and remain largely unchanged, with surgery, chemotherapy, gemcitabine, or FOLFIRINOX being the first-line treatment options (Oettle et al., 2013; Oettle et al., 2007; Conroy et al., 2011; Conroy et al., 2018). Vaccine therapy has previously been used in clinical trials for PDAC patients, but with very limited success. A phase I / II trial using a synthetic KRAS-derived peptide demonstrated detectable immune responses when administered with GM-CSF, and this is currently undergoing clinical trials with checkpoint blockade (NCT04117087) (Gjertsen et al., 1995; Gjertsen et al., 2001). A telomerase 16aa peptide vaccine (GV1001) designed to bind to multiple MHC molecules showed clear efficacy in phase I / II, but phase III PrimoVax and TeloVac trials failed to demonstrate a survival benefit (Bernhardt et al., 2006; Gunturu et al., 2013). Clinical trials of both a gastrin-based vaccine (G17DT) and a poxvirus-based vaccine encoding carcinoembryonic antigen and mucin 1 (PANVAC-V) failed to demonstrate improved survival (Gilliam et al., 2012; Arlen et al., 2007). GVAX® is an allogeneic GM-CSF-expressing irradiated pancreatic cancer cell line vaccine currently in clinical trials (with checkpoint blockade) alongside CRS-207, an attenuated mesothelin-expressing Listeria-based vaccine administered in a prime / boost fashion (NCT03190265) (Tsujikawa et al., 2020). However, the Phase IIb ECLIPSE study using this vaccine combination alone failed to demonstrate a survival benefit over chemotherapy (Le et al., 2019).In addition to the overall failure of vaccine therapy for PDAC, there have been few objective responses to other immunotherapies. Thus, the lack of response in PDAC to existing immunotherapeutic options highlights the need for new treatment options that can overcome these barriers.

[0004] Although the use of cancer vaccines to treat various malignancies in patients through targeted immune stimulation is attractive, objective clinical responses have been largely limited (Rosenberg et al., 2004). The lack of response is a complex issue, primarily due to thymic and T cell tolerance to self-antigens, which are upregulated in various cancers (Melief et al., 2015). Neoantigens and tumor-associated antigens (TAAs) have recently become increasingly popular, as their expression is typically limited or absent in non-malignant cells but aberrantly increased in cancer cells. In silico methods are commonly used to predict peptide epitopes of either TAA or neoantigens, both of which are immunogenic and effectively bind to MHC molecules for recognition by T cells (Nat Biotechnol., 2017). These methods are still largely theoretical, and due to the complexity of multiple factors (peptide processing prediction, epitope binding to MHC molecules, mutation orientation to T cells, different MHC alleles in patients, etc.), there is a wide gap to be filled before these software-based algorithms can have clinical utility.

[0005] Currently, sipuleucel-T is the only approved cancer vaccine for the treatment of metastatic castration-resistant prostate cancer. Sipuleucel-T is an autologous dendritic cell vaccine prepared by extracting peripheral blood mononuclear leukocytes from patients via leukapheresis to generate mature antigen-presenting cells (APCs), sending them to a manufacturing facility, and repeatedly pulsing them with recombinant fusion proteins (prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GM-CSF)). These activated cells are then reinfused into the patient to generate an immune response. Overall, sipuleucel-T has been shown to improve median survival by 4.1 months, but it is a complex and expensive procedure with significant manufacturing hurdles (Kantoff et al., 2010, Hammerstrom et al., 2011). To date, numerous other cancer vaccine strategies have been used in various cancer types, but have been largely unsuccessful (Melero et al., 2014). Recent Phase I data from an RNA vaccine candidate in melanoma, combined with checkpoint blockade, showed promising results that revitalized the clinical utility of vaccination against TAAs (Sahin et al., 2020). However, there remains an unmet need for cancer vaccines for the treatment of cancers such as PDAC.

[0006] While significant progress has been made in the treatment of pediatric acute lymphoblastic leukemia (pALL), effective treatments for pediatric acute myeloid leukemia (pAML) have lagged behind. pAML is rarer than pALL, accounting for approximately 20% of childhood leukemia cases (Morais et al., 2020). However, it is also more lethal, with a 5-year survival rate of only 67–68% (Siegel et al., 2022). One challenge in treating pAML is the heterogeneity of the disease. The French-American-British (FAB) classification of AML divides the disease into eight subtypes, M0–M7. Each subtype is characterized by a specific hematopoietic lineage and maturation stage. While adult AML (aAML) is thought to be caused by the accumulation of somatic mutations, these mutations are relatively rare in pAML. Pediatric patients are far more likely to exhibit cytogenetic abnormalities that lead to de novo AML (Bolouri et al., 2017). These characteristics make it difficult to develop targeted therapeutics that are effective in the majority of patients. Furthermore, the primary focus for AML treatment has been on aAML, with the hope that it will also be effective in pAML. However, the differences between aAML and pAML suggest that a therapy may be effective in one but not the other.

[0007] Currently, one CD33-targeted therapy has been approved by the FDA: pAML-gemtuzumab ozogamicin (GO), an antibody-drug conjugate (ADC). GO consists of an anti-CD33 monoclonal antibody linked to a cytotoxic calicheamicin payload. After endocytosis of the ADC, the linker is cleaved, and the calicheamicin induces cell death through double-stranded DNA breaks. However, GO has several drawbacks that suggest this may not be the best method for targeting CD33. One major weakness is that the binding epitope of GO is located on the immunoglobulin-like V (IgV) domain. 50% of CD33-positive AML patients express the single nucleotide polymorphism (SNP) rs12459419 C>T, which causes a CD33 splice variant lacking the IgV domain (Lamba et al., 2017). This renders GO ineffective in half of the patients eligible for its use because the CD33 protein they express lacks the gemtuzumab-binding epitope.

[0008] Furthermore, premature cleavage of the linker between gemtuzumab and the calicheamicin payload may result in off-target effects. Another ADC, inotuzumab ozogamicin, is a CD22-targeting agent that also utilizes calicheamicin. The use of these drugs has been shown to increase the risk of hepatic veno-occlusive disease (VOD) in patients undergoing hematopoietic stem cell transplantation (HSCT) (Ladha et al., 2020). Another potential issue associated with GO is resistance to calicheamicin. Previous studies have shown that P-glycoprotein (Pgp) expression in AML blasts can cause drug efflux, reducing the efficacy of GO (Linenberger et al., 2001).

[0009] Finally, in a clinical trial comparing pAML patients receiving standard chemotherapy with those receiving chemotherapy supplemented with GO, GO demonstrated only modest improvements. Patients receiving GO had significantly improved event-free survival (EFS) compared with patients receiving standard chemotherapy, but there was no difference in overall survival (OS). When patients were stratified into low-, intermediate-, and high-risk groups, none of them demonstrated a significant increase in event-free survival (EFS) (Gamis et al., 2014). Thus, there remains a significant need for new pAML treatments, and CD33 has yet to reach its full potential as a target. Summary of the Invention

[0010] overview In a first aspect, 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 fragment thereof, wherein the at least one target protein is overexpressed in cancer.

[0011] 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 certain aspects, the mutant SMEZ-2 comprises the mutations W75L, K182Q, and D42C.

[0012] In some aspects, the conjugate comprises a linker, for example, between SMEZ-2 and 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.

[0013] In certain aspects, at least one target protein is a neoantigen or tumor-associated antigen (TAA). In a specific aspect, the target protein is anterior gradient 2 (AGR2), for example, the AGR2 is 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 comprises 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, the one or more amino acid substitutions are at D231, D246, C154, and / or C169. In some aspects, the one or more amino acid substitutions are D231E, D246E, C154S, and / or C169S.

[0014] In certain aspects, the vaccine conjugate comprises at least a second target protein or fragment thereof, hi some aspects, at least one target protein is CD38.

[0015] In some aspects, at least one target protein is an immune checkpoint protein. In certain aspects, the immune checkpoint protein is CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR.

[0016] Further embodiments provide pharmaceutical compositions comprising a vaccine conjugate of this embodiment (e.g., a vaccine conjugate comprising mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2) conjugated to at least one target protein or fragment thereof, wherein the at least one target protein is overexpressed in cancer) and an adjuvant. In some aspects, the adjuvant is Freund's incomplete adjuvant. In certain aspects, the conjugate is formulated as a 1:1 emulsion with Freund's incomplete adjuvant (IFA). In some aspects, the conjugate is formulated for intravenous infusion, subcutaneous injection, or intramuscular injection.

[0017] Another embodiment provides an expression vector comprising a sequence encoding a mutant SMEZ-2 fused to a sequence encoding a target protein or fragment thereof. In some aspects, the target protein is a neoantigen or a TAA. In certain aspects, the vector encodes a vaccine conjugate of this embodiment (e.g., a vaccine conjugate comprising mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2) conjugated to at least one target protein or fragment thereof). Host cells comprising the expression vector of this embodiment, such as Escherichia coli (E. coli), human embryonic kidney cells (HEK293), or Chinese hamster ovary (CHO) cells, are also provided herein.

[0018] In yet another embodiment, a method is provided for stimulating an immune response in a subject, comprising 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 at least one target protein or fragment thereof, wherein the at least one target protein is overexpressed in cancer) or a pharmaceutical composition of this embodiment.

[0019] In some aspects, the immune response is an anti-cancer immune response. In certain aspects, the subject has cancer. In specific aspects, the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, digestive cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, multiple neuroendocrine tumors 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 certain 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.

[0020] 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 increased titer of AGR2-specific immunoglobulin in a sample of the subject's blood. In other aspects, the immune response is an anti-CD38, anti-PD1, anti-CTLA4, or anti-PDL1 response, and is measured by measuring an increased titer of CD38, PD1, CTLA4, or PDL1-specific immunoglobulin in a sample of the subject's blood.

[0021] In some aspects, the conjugate is administered by injection, such as intravenous infusion.

[0022] In a further aspect, the method further comprises administering a second anti-cancer therapy to the subject. In some aspects, the second anti-cancer therapy is immunotherapy, chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or cytokine therapy. In some aspects, the second anti-cancer therapy is immunotherapy, such as an immune checkpoint inhibitor. For example, the immune checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In a specific aspect, 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, KEYTRUDA®, 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, YERVOY®, or ipilimumab. In some aspects, the additional anti-cancer therapy includes a TLR9 agonist such as CpG ODN1826 and / or a CD40 agonist such as a CD40 agonist antibody. In certain aspects, the subject is administered an immune checkpoint inhibitor and a TLR9 agonist.

[0023] A further embodiment provides a method of treating a subject having cancer, comprising 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 at least one target protein or fragment thereof, wherein the at least one target protein is overexpressed in cancer) or a pharmaceutical composition of this embodiment.

[0024] In some aspects, the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, multiple neuroendocrine tumors type I and 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 a specific aspect, 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.

[0025] In a further aspect, the method further comprises administering at least one immune checkpoint inhibitor to the subject. In some aspects, administering at least one immune checkpoint inhibitor to the subject comprises administering the at least one immune checkpoint inhibitor before the vaccine conjugate. In other aspects, administering at least one immune checkpoint inhibitor to the subject comprises administering the at least one immune checkpoint inhibitor after or simultaneously with the vaccine conjugate. For example, the immune checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In a specific aspect, 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, KEYTRUDA®, 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, YERVOY®, or ipilimumab. In some aspects, the subject is administered two immune checkpoint inhibitors. In certain 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 therapy includes a TLR9 agonist, such as CpG ODN1826, and / or a CD40 agonist, such as a CD40 agonist antibody. In certain aspects, the subject is administered an immune checkpoint inhibitor and a TLR9 agonist.

[0026] In a further aspect, the method further comprises administering to the subject an additional anti-cancer therapy, hi some aspects, the additional anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or cytokine therapy.

[0027] Another embodiment provides a kit comprising a vaccine conjugate of this embodiment (e.g., a vaccine conjugate comprising mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2)) or a 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 a CTLA-4 antibody.

[0028] Further embodiments provide compositions comprising a vaccine conjugate of this embodiment (e.g., a vaccine conjugate comprising mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2)) or a pharmaceutical composition of this embodiment for use in treating cancer. In some aspects, the composition further comprises an immune checkpoint inhibitor.

[0029] In another aspect, the present disclosure provides a vaccine conjugate comprising mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2) conjugated to CD33 or a fragment thereof.

[0030] 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 certain aspects, the mutant SMEZ-2 comprises the mutations W75L, K182Q, and D42C. In certain aspects, the vaccine conjugate comprises at least a second target protein or a fragment thereof.

[0031] 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 includes 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, the one or more amino acid substitutions are at D231, D246, C154, and / or C169. In certain aspects, the one or more amino acid substitutions are D231E, D246E, C154S, and / or C169S.

[0032] In certain aspects, the linker is a peptide linker, such as a glycine serine linker, particularly a GGGGS linker.

[0033] Further embodiments provide pharmaceutical compositions 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 Freund's incomplete adjuvant. In certain aspects, the conjugate is formulated as a 1:1 emulsion with Freund's incomplete adjuvant (IFA). In some aspects, the conjugate is formulated for intravenous infusion, subcutaneous, or intramuscular injection.

[0034] Another embodiment provides an expression vector comprising a sequence encoding a mutant SMEZ-2 fused to a sequence encoding CD33 or a fragment thereof. Also provided herein are host cells, such as E. coli, human embryonic kidney (HEK293) cells, or Chinese hamster ovary (CHO) cells, comprising the expression vector of this embodiment.

[0035] In yet another embodiment, a method is provided for stimulating an immune response in a subject, comprising 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.

[0036] In some aspects, the immune response is an anti-cancer immune response. In certain aspects, the subject has cancer. In a specific aspect, the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, multiple neuroendocrine tumors type I and 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 a specific aspect, the cancer is acute myeloid leukemia.

[0037] 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 increased titer of CD33-specific immunoglobulins in a sample of the subject's blood.

[0038] In some aspects, the conjugate is administered by injection, such as intravenous infusion.

[0039] In a further aspect, the method further comprises administering a second anti-cancer therapy to the subject. In some aspects, the second anti-cancer therapy is immunotherapy, chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or cytokine therapy. In some aspects, the second anti-cancer therapy is immunotherapy, such as an immune checkpoint inhibitor. For example, the immune checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In a specific aspect, 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, KEYTRUDA®, 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, YERVOY®, or ipilimumab.

[0040] A further embodiment provides a method of treating a subject having cancer, comprising 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.

[0041] In some aspects, the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, multiple neuroendocrine tumors type I and 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 a specific aspect, the cancer is acute myeloid leukemia.

[0042] In a further aspect, the method further comprises administering at least one immune checkpoint inhibitor to the subject. In some aspects, administering at least one immune checkpoint inhibitor to the subject comprises administering the at least one immune checkpoint inhibitor before the vaccine conjugate. In other aspects, administering at least one immune checkpoint inhibitor to the subject comprises administering the at least one immune checkpoint inhibitor after or simultaneously with the vaccine conjugate. For example, the immune checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In a specific aspect, the immune checkpoint inhibitor comprises an anti-PD1 / PDL 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, KEYTRUDA®, 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, YERVOY®, or ipilimumab. In some aspects, the subject is administered two immune checkpoint inhibitors. In certain 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.

[0043] In a further aspect, the method further comprises administering to the subject an additional anti-cancer therapy, hi some aspects, the additional anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or cytokine therapy.

[0044] Another embodiment provides a kit comprising a vaccine conjugate of this embodiment (e.g., a vaccine conjugate comprising mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2)) or a 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 a CTLA-4 antibody.

[0045] Further embodiments provide compositions comprising a vaccine conjugate of this embodiment (e.g., a vaccine conjugate comprising mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2)) or a pharmaceutical composition of this embodiment for use in treating cancer. In some aspects, the composition further comprises an immune checkpoint inhibitor.

[0046] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, for example, a compound synthesized by one method can be used in the preparation of a final compound according to a different method.

[0047] In the appended claims and / or this specification, the use of the words "a" or "an" when used in conjunction with the word "comprising" may mean "one," but is also consistent with the meanings "one or more," "at least one," and "one or more than one." The word "about" means plus or minus 5% of the specified number.

[0048] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating 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 become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0049] The accompanying drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0050] [Figure 1] Mechanism of antigen presentation promoted by mutant SMEZ-2. [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). [Figure 3] Engineered construct of AGR2-SMEZ-2 conjugate expressed in E. coli . [Figure 4] ELISA data for anti-AGR2 IgG in plasma of C57BL / 6 mice vaccinated with 100 μg of AGR2-SMEZ2 or equimolar amounts of the indicated controls. Proteins were administered by intramuscular injection. [Figure 5] Figures 5A-5B: C57BL / 6 mice were treated according to the schedule shown in Figure 5A. Mice were injected with 100 μg of AGR2-SMEZ or an equimolar amount of AGR2 (Figure 5A), 50 μg of ODN 1826 (CpG, C), and then implanted with 100,000 AGR2-expressing KPC cells (T). Proteins were administered subcutaneously in IFA. ODN 1826 was given intramuscularly. (Figure 5B) Tumor volumes were measured 16 days after implantation. [Figure 6] Figures 6A-6B: Recombinant murine CD38, SMEZ2, and CD38-SMEZ2 conjugates were expressed and purified from mammalian cells. (Figure 6A) SDS-PAGE analysis is shown. (Figure 6B) Plasma was collected from Balb / c mice 10 days after two subcutaneous injections of 50 μg mCD38-SMEZ in IFA (or an equimolar control). Serum was diluted 1:10,000, and anti-CD38 IgG was measured by ELISA. [Figure 7] Figures 7A-7C: SDS-PAGE of purified proteins. (Figure 7A) Mouse CTLA-4 and CTLA-4-SMEZ. (Figure 7B) Mouse PD-1 and mouse PD1-SMEZ. (Figure 7C) Mouse PD-L1 and PDL1-SMEZ. [Figure 8] Figures 8A-8B: In vivo activity of SMEZ-PD1, SMEZ-PD-L1, and SMEZ-CTLA4. (Figure 8A) C57BL / 6 mice were injected three times with a cocktail consisting of SMEZ conjugates of murine PD-1, PDL1, and CTLA-4. Blood was collected, and IgG specific for each antigen was quantified by ELISA. (Figure 8B) Splenocytes were collected 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 cocktail. [Figure 9] Figures 9A-9C: Preclinical efficacy of CD33-SMEZ-2 in a mouse model of AML. (Figure 9A) The indicated proteins were expressed and purified from Expi293 HEK cells. Western blots of each protein expressing a 6x His tag for affinity purification are shown. (Figure 9B) C57BL / 6 mice were subcutaneously injected with 40 pmol of the indicated protein in IFA emulsion. Blood was collected after three doses, 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 treatment of C57BL / 6 mice with CD33-SMEZ-2, the mice were challenged with 1 x 10 C1498-hCD33 cells by intravenous injection. Survival data are shown. [Figure 10]Figures 10A-10C: (Figure 10A) C57BL / 6 mice were orthotopically injected with 15,000 KPC cells derived from syngeneic pancreatic tumors originating from LSL-KrasG12D; LSL-Trp53R172H; Pdx1-cre mice overexpressing hAGR2. KPC cells were injected into the head of the pancreas on day 0. Mice were treated with 50 μg of SMEZ-AGR2 or SMEZ-MSLN (mesothelin) (subcutaneously; emulsified in IFA) twice before and twice after the orthotopic injection (days -24, -10, 4, and 18). Mice were treated with 50 μg ODN 1826 (IM) and 250 μg α-mPD-1 (IM; BioXCell; clone RMP1-14) three times a week for 3 weeks (days 3, 5, 7, 10, 12, 14, 17, 19, and 21), starting on day 3. Control mice received vehicle for all three treatments. Mice were sacrificed on day 25, and tumors were excised (FIG. 10B), photographed, and weighed (FIG. 10C). [Figure 11]Figures 11A-11C: (Figure 11A) Protein structures of various CD33-SMEZ constructs are shown. The top panel shows full-length CD33-SMEZ, which contains the two extracellular domains of CD33, IgV and IgC, connected to the engineered SMEZ-2 superantigen. The bottom left model illustrates CD33IgC-SMEZ, which contains only the IgC domain of CD33 conjugated to engineered SMEZ-2. Finally, the bottom right shows CD33IgC-SMEZ-3Mut, an optimized version of CD33IgC-SMEZ with three amino acid substitutions that the inventors hypothesize will stabilize the protein, reduce aggregation, and increase its developability. (Figure 11B) Western blots (left panel) show the original and modified versions of CD33IgC and CD33IgC-SMEZ under non-reducing conditions. Proteins were visualized by blotting a 6xHis affinity tag engineered into all proteins. Both CD33 IgC and CD33 IgC-SMEZ exhibited protein aggregation, which was reduced in the 3Mut candidate after the introduction of three amino acid mutations. (Right panel) Protein aggregation was quantified using the Proteostat protein aggregation assay, confirming significantly reduced aggregation in both CD33 IgC and CD33 IgC-SMEZ (referred to herein as M2T) compared with the 3Mut version 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 vehicle control (PBS). One week after the fourth dose, α-CD33 antibody titers were measured in mouse plasma by enzyme-linked immunosorbent assay (ELISA). As expected, the SMEZ-conjugated protein induced higher levels of anti-IgC IgG in mice compared with the IgC protein alone. DETAILED DESCRIPTION OF THE INVENTION

[0051] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Anterior gradient 2 (AGR2) is a member of the protein disulfide isomerase (PDI) family, which is involved in the formation of disulfide bonds for newly synthesized proteins in the endoplasmic reticulum (ER). Like other proteins in the PDI family, AGR2 possesses an N-terminal ER leader sequence and a C-terminal ER retention signal (Patel et al., 2013). Despite these sequence characteristics that localize and retain AGR2 in the ER, it is also observed extracellularly, where it interacts with the extracellular matrix and is associated with increased invasiveness, proliferation, survival, and metastasis in several types of human cancer, including PDAC (Fessart et al., 2016; Moidu et al., 2020; Di Maro et al., 2014; Ma et al., 2017; Wang et al., 2008). Indeed, significantly increased extracellular AGR2 was observed in both immunohistochemical (IHC) staining and tissue microarrays of PDAC, but not in normal pancreatic tissue from patient samples. Consequently, the elevated expression levels of AGR2, together with its pro-tumorigenic features, led to the hypothesis that AGR2 is an attractive TAA immunotherapy target in PDAC.

[0052] Therefore, in certain embodiments, the present invention provides a vaccine conjugate, such as the detoxified AGR2-SMEZ-2 conjugate, which can stimulate a robust immune response against the PDAC that expresses high AGR2.This conjugate utilizes the MHC II binding ability of SMEZ-2 to enable APC to efficiently take up and present AGR2 peptide.This conjugate can lead to a robust anti-PDAC immune response and tumor eradication.

[0053] This vaccine approach offers several advantages over monoclonal antibody (mAb) therapy, including adaptability to conjugation with other tumor types and different TAAs, lower manufacturing costs, and greater durability. Furthermore, because single AGR2 mutations are less likely to escape the immune response, the polyclonal response elicited is less likely to lead to treatment resistance. This technology overcomes the current challenges presented by peptide-based cancer vaccines, where the high-affinity binding properties of SMEZ-2 eliminate the need for in silico simulations of peptide binding to MHC molecules. Therefore, further embodiments provide vaccine conjugates for other TAAs and neoantigen-based vaccine candidates.

[0054] In certain aspects, bacterial superantigen conjugates are provided herein by cloning a tumor-associated antigen or immune target (i.e., AGR2, CD38, PD-1, PD-L1, CTLA-4) in cis into a mutant form of the SMEZ-2 bacterial superantigen. The mutant superantigen has high affinity for MHC class II molecules, thereby promoting and enhancing antigen presentation to immune effector cells and stimulating an immune response against target cells expressing the antigen linked to SMEZ2. The present data demonstrate that AGR2-SMEZ2, CD38-SMEZ, PD-1-SMEZ, PDL1-SMEZ, and CTLA4-SMEZ conjugates induce robust humoral responses, with high titers of specific immunoglobulins detected in the plasma of treated mice.

[0055] 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 through the binding of sialylated glycans. CD33 consists of two extracellular domains, the immunoglobulin-like V (IgV) domain and the immunoglobulin-like C (IgC) domain, a transmembrane domain, an immunoreceptor tyrosine-based inhibitory motif (ITIM) domain, and an ITIM-like domain. The signaling function and role of CD33 are not fully understood, but it is thought to inhibit cell activation and proliferation upon ligand binding. CD33 is normally expressed on myeloid cells and is present in approximately 85–90% of both pAML and aAML cases. It is notably not expressed on lymphocytes or erythrocytes.

[0056] Streptococcal mitogenic exotoxin Z-2 (SMEZ-2) is a streptococcal superantigen (SAg) that crosslinks MHC class II and T cell receptors (TCRs), leading to mature T cell proliferation, cytokine overproduction, and ultimately T cell anergy (Deacy et al., 2021). Radcliff et al. previously demonstrated that a modified form of SMEZ-2, termed SMEZ-2 M1, can prevent TCR binding and thereby abrogate the subsequent cytokine storm. SMEZ-2 M1 retains its ability to bind to MHC class II, enhancing the presentation of conjugated antigens to the immune system (Radcliff et al., 2012).

[0057] Thus, in certain embodiments, vaccine conjugates, such as detoxified CD33-SMEZ-2 conjugates, are provided herein. In this 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 high affinity for MHC class II molecules, thereby promoting and enhancing the presentation of CD33 peptides to immune effector cells and stimulating anti-CD33-specific immune responses against AML. Data showed that the CD33-SMEZ-2 conjugate induced robust anti-CD33 humoral responses, and high titers of CD33-specific immunoglobulins were detected in the plasma of treated mice. Further animal studies demonstrated that these effects were sufficient to reduce AML burden and significantly prolong survival in mice.

[0058] Another embodiment provides a CD33 vaccine conjugate of this embodiment (e.g., a vaccine conjugate with mutant streptococcal mitogenic exotoxin Z-2 (SMEZ-2)) in which strategic amino acid substitutions have been made to the full-length or truncated CD33 protein. In some aspects, full-length CD33 was modified at predicted deamidation sites, including asparagine 98, to improve stability and prevent protein aggregation and to generally improve the drug suitability of the candidate. In some aspects, truncated forms of CD33 consisting only of the CD33-IgC domain were further modified by mutation of amino acids predicted to be high-risk sites for isomerization, including aspartic acid 231, or free cysteines, including cysteine ​​154, to improve stability and prevent protein aggregation and to generally improve the drug suitability of the candidate.

[0059] I. Cancer Vaccine Conjugates The compositions and methods can include cancer vaccines as a form of active immunotherapy in which an antigenic peptide, polypeptide, or protein, or an autologous or allogeneic tumor cell composition or "vaccine" is administered to a subject. Vaccines can be administered systemically, for example, by intravenous, intradermal, or intramuscular injection. Vaccines can also be administered multiple times to enhance the immune response to the administered antigen.

[0060] The term "vaccine" is used according to its ordinary and common meaning in medicine and immunology and refers to a composition comprising an antigenic component (e.g., an antigenic protein) for administration to a subject (e.g., a human) to elicit an immune response against the antigenic component (e.g., 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 (e.g., an aluminum adjuvant). A liquid vaccine is a vaccine in liquid form, which may be, for example, a solution, suspension, emulsion, or dispersion of the antigenic component (e.g., an antigenic protein) of the vaccine, and may optionally include other ingredients. A dry vaccine is a vaccine that contains 5% or less water.

[0061] A vaccine is a preparation used to enhance immunity against a specific disease. Vaccines contain an agent that is used to induce a response from the subject's immune system. Various agents typically 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.

[0062] The term "prime-boost" or "prime-boost" when applied to vaccine administration methodologies 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 point (e.g., several hours, days, weeks, or months later) (boost). The first and second vaccines may be the same or different, but are intended to elicit an immune response useful in treating or preventing 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.

[0063] The term "associated" or "related to" when used herein to describe a disease (e.g., a viral or bacterial disease) means that the disease is caused or the symptoms of the disease are caused by something described as being associated with or associated with the disease. As used herein, something described as being associated with a disease can be a target for treating the disease, even if it is a causative agent.

[0064] The vaccine antigens described herein may be chemically linked to a carrier or recombinantly expressed using an immunogenic carrier peptide or polypeptide (e.g., an antigen-carrier fusion peptide or polypeptide) to enhance the immune response. Means for conjugating a polypeptide or peptide to an immunogenic carrier protein are well known in the art and include, for example, glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine. In certain embodiments, the carrier is mutant SMEZ-2.

[0065] A. SMEZ-2 bacterial superantigen Streptococcus pyogenes-derived streptococcal mitogenic exotoxin Z-2 (SMEZ-2) is a bacterial superantigen (SAg) and the most immunogenic SAg discovered to date (Kamezawa et al., 1997; Proft et al., 2000). Wild-type SMEZ-2 binds with high affinity to both MHC class II molecules and T cell receptors (TCRs), indiscriminately activating T cells and stimulating up to 20% of the body's T cell pool (Li et al., 1999). This leads to the development of a nonspecific immune response, which, at microgram doses, results in massive cytokine release and toxic shock syndrome (Alouf et al., 2003). The T cell binding and mitogenic (and toxic) effects of SMEZ-2 can be abrogated by mutation of key residues in the TCR Vβ-binding interface (W75L, K182Q, and D42C), modifications that generate a protein that maintains 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 of mutant SMEZ-2 provides an efficient carrier system for directly targeting antigens to antigen-presenting cells (APCs). This effectively "hijacks" the function of SMEZ-2 through its detoxification, providing a protein "carrier" for efficient presentation of conjugated antigens (Dickgreber et al., 2009).

[0066] B. Target Protein Some target proteins of the antigens targeted by the vaccine conjugates are expressed in the context of a disease, condition, or cell type targeted by the vaccine conjugate. Among these diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, such as cancers and tumors, including hematological cancers and cancers of the immune system, such as lymphomas, leukemias, and / or myelomas, including B, T, and myeloid leukemias, lymphomas, and multiple myeloma. In some embodiments, the antigen is selectively expressed or overexpressed in cells of the disease or condition, e.g., tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed in normal cells and / or engineered cells. Any suitable antigen may be used in the present methods. Exemplary antigens include, but are not limited to, antigenic molecules from infectious agents, autoantigens, tumor / cancer-associated antigens, and tumor neoantigens.

[0067] As used herein, the terms "tumor-associated antigen," "tumor antigen," and "cancer cell antigen" are used interchangeably. In each case, these terms refer to a protein, glycoprotein, or carbohydrate that is specifically or preferentially expressed by cancer cells.

[0068] Tumor-associated antigens can be of any type, as long as they are expressed on the cell surface of tumor cells.Tumor-associated antigens can be derived from cancers such as prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, renal cancer, mesothelioma, ovarian cancer, sarcoma, or melanoma.Exemplary tumor-associated antigens or tumor cell-derived antigens 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.These non-limiting examples of tumor antigens are expressed in a wide range of tumor types, such as 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).

[0069] Exemplary embodiments of tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigens, melanoma-associated antigens, mutant p53, mutant ras, etc. In certain aspects, antigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and CEA. In certain aspects, antigens for 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 and include, for example, 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_009272.1), and the like. The following genes were identified: CA-125 (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).

[0070] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. In addition, tumor antigens can be self-peptide hormones, such as full-length gonadotropin-releasing hormone (GnRH), a short peptide of 10 amino acids that is useful in the treatment of many cancers.

[0071] Tumor antigens include tumor antigens derived from cancers characterized by tumor-associated antigen expression, such as HER-2 / neu expression. Tumor-associated antigens of interest include lineage-specific tumor antigens, such as the melanocyte-melanoma lineage antigen MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase, and tyrosinase-related proteins. Exemplary tumor-associated antigens include p53, Ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., 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, -catenin / m, caspase-8 / m, CEA, CDK-4 / m, ELF2M, GnT-V, G2 50, 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 signaling factor 1 (TACSTD1), TACSTD2, receptor tyrosine kinase kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor kappa B (NF-B),Notch receptors (e.g., Notch1-4), c-Met, mammalian target 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), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2), ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelial, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, 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 tumor antigens derived from or including any one or more of the following:

[0072] Antigens can include epitope regions or epitope peptides derived from genes mutated or differentially transcribed in tumor cells compared to normal cells, such as telomerase enzyme, survivin, mesothelin, mutant ras, bcr / abl rearrangements, Her2 / neu, mutant 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 myeloma and B-cell lymphoma; tumor antigens containing epitope regions or epitope peptides derived from oncoviral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; and non-mutated oncofetal proteins with tumor-selective expression, such as carcinoembryonic antigen and alphafetoprotein.

[0073] In other embodiments, antigens are obtained or derived from pathogenic microorganisms such as viruses, fungi, parasites, and bacteria, or from opportunistic pathogenic microorganisms (also referred to herein as infectious disease microorganisms). In certain embodiments, antigens derived from such microorganisms include full-length proteins.

[0074] C. Vaccine Conjugate Production A variety of commercially available vectors and expression systems can be used to produce the vaccine conjugates, including those designed for mammalian cells, insect (Spodoptera; baculovirus-delivered) cells, and bacterial cells. In some aspects, purification is performed using E. coli, e.g., E. coli BL21(DE3) cells, HEK cells, or CHO cells.

[0075] In certain embodiments, the vaccine conjugate is purified. As used herein, the term "purified" is intended to refer to a composition that can be isolated from other components, and the protein is purified to any degree compared to its naturally occurring state. Thus, a purified protein also refers to a protein that is free from the environment in which it may naturally occur. When the term "substantially purified" is used, this expression refers to a composition in which the protein or peptide forms a major component of the composition, for example, 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.

[0076] In certain embodiments, the plasmid vector is intended for use in transforming host cells. Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are generally used in connection with these hosts. The vector usually carries a replication site and a marking sequence capable of providing phenotypic selection in transformed cells. As a non-limiting example, E. coli is often transformed using a derivative of pBR322, a plasmid derived from E. coli. pBR322 contains genes for ampicillin resistance and tetracycline resistance, thus providing an easy means for identifying transformed cells. pBR plasmids, or other microbial plasmids or phages, must also contain or be modified to contain, for example, a promoter that the microorganism can use to express its own proteins. In some aspects, the vaccine conjugate is cloned into pET15TEV_NESG, which expresses a protein with an N-terminal 6x histidine tag cleavable by tobacco etch virus (TEV) protease. The vaccine conjugate was expressed in BL21(DE3) Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). To avoid nonspecific immunogenicity or unexpected side effects, the 6x histidine tag was excised by overnight digestion with TEV and purified by additional rounds of IMAC, where the flow-through contained the purified protein.

[0077] Additionally, phage vectors containing replicon and control sequences compatible with host microorganisms can be used as transformation vectors in connection with these hosts. For example, phage lambda GEM™ 11 can be utilized to generate recombinant phage vectors that can be used to transform host cells such as E. coli LE392.

[0078] Additional useful plasmid vectors include the pIN vectors (Inouye et al., 1985), and the pGEX vectors for use in generating glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and isolation or cleavage. Other suitable fusion proteins are those with -galactosidase, ubiquitin, etc.

[0079] Bacterial host cells, e.g., E. coli, containing the expression vector are grown in any of a number of suitable media, such as LB. As will be appreciated by those skilled in the art, expression of the recombinant protein in a particular vector can be induced by contacting the host cells with an agent specific for the particular promoter, e.g., by adding IPTG to the medium, or by switching the incubation to a higher temperature. After culturing the bacteria for an additional period, usually 2 to 24 hours, the cells are harvested by centrifugation and washed to remove residual medium.

[0080] Protein purification techniques are well known to those skilled in the art. At one level, these techniques involve crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions. After separating the polypeptide from other proteins, the polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of pure peptides include ion exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. Other methods for protein purification include precipitation using ammonium sulfate, PEG, antibodies, etc., or heat denaturation followed by centrifugation; gel filtration, reverse-phase, hydroxylapatite, 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. The polypeptide can be purified from other cellular components using an affinity column that binds to the tagged portion of the polypeptide. As is generally known in the art, it is contemplated that the order in which the various purification steps are performed may be varied or certain steps may be omitted and still result in a method suitable for the preparation of a substantially purified protein or peptide.

[0081] In light of the present disclosure, various methods for quantifying the degree of purification of a protein or peptide will be known to those of skill in the art. 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 and compare it to the specific activity of the initial extract, thereby calculating the degree of purity. The actual units used to express the amount of activity will, of course, depend on the particular assay technique chosen to follow purification and whether the expressed protein or peptide exhibits detectable activity. It is known that polypeptide migration can vary, sometimes significantly, with different conditions of SDS / PAGE (Capaldi et al., 1977). Therefore, it will be understood that the apparent molecular weight of purified or partially purified expression products may vary under different electrophoretic conditions.

[0082] Those skilled in the art will be well-versed in constructing vectors by standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference) for expression of the antigen receptors of the present disclosure. Vectors include plasmids, cosmids, viruses (bacteriophages, 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.), adenoviral (Ad) vectors, including replication-competent, replication-deficient, and gutless forms, adeno-associated viruses, and the like. These include, but are not limited to, 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, mouse 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.

[0083] 1. Regulatory Elements The expression cassette contained in the vector useful in the present disclosure specifically includes (in the 5' to 3' direction) a eukaryotic transcriptional promoter operably linked to a protein-coding sequence, a splice signal including intervening sequences, and a transcription termination / polyadenylation sequence. The promoters and enhancers that control the transcription of protein-coding genes in eukaryotic cells are composed of multiple genetic elements. The cellular machinery can collect and integrate the regulatory information conveyed by each element, thereby allowing different genes to evolve distinct, often complex, patterns of transcriptional regulation. Promoters used in the context of the present disclosure include constitutive promoters, inducible promoters, and tissue-specific promoters.

[0084] 2. Promoter / Enhancer The expression constructs provided herein include a promoter that drives the expression of an antigen receptor. Promoters generally contain sequences that function to position the start site for RNA synthesis. The most well-known example of this is the TATA box, but in some promoters that lack a TATA box, such as the promoters of the mammalian terminal deoxynucleotidyl transferase gene and the SV40 late gene, individual elements overlying the start site themselves serve to fix the start location. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but many promoters have been shown to contain functional elements downstream of the start site. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates DNA transcription and promotes expression of the encoded RNA.

[0085] Spacing between promoter elements is often flexible, allowing elements to be inverted or moved relative to one another and still retain promoter function. In the tk promoter, spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. In some promoters, individual elements appear to function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0086] A promoter can be one naturally associated with a nucleic acid sequence, obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer can be naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages can be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers can 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 different transcriptional regulatory regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA construction include the lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences can be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR™, in combination with the compositions disclosed herein. Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, etc., can be similarly employed.

[0087] Naturally, it will be important to employ a promoter and / or enhancer that effectively directs expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. The use of promoter, enhancer, and cell type combinations for protein expression is generally known to those skilled in the art of molecular biology (see, e.g., Sambrook et al. 1989, incorporated herein by reference). The promoter employed may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions for directing high-level expression of the introduced DNA segment, which is advantageous, for example, in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0088] Furthermore, any promoter / enhancer combination (e.g., from the Eukaryotic Promoter Database EPDB) can be used to drive expression. The use of T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if an appropriate bacterial polymerase is provided as part of the delivery complex or as an additional gene expression construct.

[0089] Non-limiting examples of promoters include early or late viral promoters such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate early promoter, or the Rous sarcoma virus (RSV) early promoter; eukaryotic promoters such as the beta-actin promoter, the GADPH promoter, and the metallothionein promoter; and tethered response element promoters such as the cyclic AMP response element promoter (cre), the serum response element promoter (sre), the phorbol ester promoter (TPA), and the minimal TATA box proximal response element promoter (tre). Human growth hormone promoter sequences (e.g., the human growth hormone minimal promoter described in Genbank, accession number X05244, nucleotides 283-341) or the mouse mammary tumor promoter (available from the American College of Cancer, catalog number ATCC 45007) can also be used. In particular embodiments, the promoter is a CMV IE, Dectin-1, Dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, beta actin, MHC class I or MHC class II promoter, although any other promoter useful for driving expression of therapeutic genes is applicable to the practice of the present disclosure.

[0090] In certain aspects, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that have the potential to increase promoter activity and act in cis and regardless of their orientation, even over relatively long distances (up to several kilobases from the target promoter). However, enhancer function is not necessarily limited to such long distances and can also function in the immediate vicinity of a given promoter.

[0091] 3. Initiation signal and linked expression Specific initiation signals may also be used in the expression constructs provided herein for efficient translation of the coding sequence. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. Those skilled in the art will be able to easily determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in-frame" with the reading frame of the desired coding sequence. Exogenous translational control signals and initiation codons may be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcriptional enhancer elements.

[0092] In certain embodiments, internal ribosome entry site (IRES) elements are used to create multiple genes, or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and initiate translation at internal sites. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) and IRESs from mammalian messages have been described. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together, creating polycistronic messages. Thanks to the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0093] Furthermore, specific 2A sequence elements can be used to achieve linked or co-expression of genes in the constructs provided herein. For example, cleavage sequences can be used to link open reading frames to form a single cistron, allowing genes to be co-expressed. Exemplary cleavage sequences are F2A (foot-and-mouth disease virus 2A) or "2A-like" sequences (e.g., Thosea asigna virus 2A; T2A).

[0094] 4. Origin of replication To propagate a vector in a host cell, it may contain one or more origins of replication sites (often referred to as "ori"), which are specific nucleic acid sequences at which replication is initiated, such as a nucleic acid sequence corresponding to the EBV oriP described above, or an engineered oriP with a similar or enhanced function in programming. Alternatively, the replication origins or autonomously replicating sequences (ARS) of other extrachromosomally replicating viruses described above may be used.

[0095] 5. Selectable and Screenable Markers 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 can confer an identifiable change to the cell, allowing for easy identification of cells containing the expression vector. Generally, a selectable marker confers a selectable characteristic. A positive selectable marker is one whose presence allows for its selection, while a negative selectable marker is one whose presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0096] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants; for example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows for the differentiation of transformants based on the realization of a condition, other types of markers are contemplated, including screenable markers such as GFP, which rely on colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be used as negative selection markers. Those skilled in the art will likely know how to use immunological markers in conjunction with FACS analysis. The marker used is not believed to be important, as long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection markers and screenable markers are well known to those skilled in the art.

[0097] D. Formulation and Administration The present disclosure provides pharmaceutical compositions comprising mutant SMEZ-2 conjugated to a target protein. Such compositions comprise a prophylactically or therapeutically effective amount of the target protein or a fragment thereof or a peptide immunogen and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more specifically, in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, e.g., those of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a specific carrier when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0098] The compositions may also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations can contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences." Such compositions may contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with an appropriate amount of carrier to provide the proper dosage form for the patient. The formulation should be suitable for the mode of administration, which may be oral, intravenous, intraarterial, buccal, intranasal, nebulized, bronchial inhalation, or mechanical ventilation delivery.

[0099] The active vaccine can be formulated for parenteral administration, for example, by intradermal, intravenous, intramuscular, subcutaneous, or even intraperitoneal injection. Intradermal and intramuscular administration are contemplated. Alternatively, the vaccine can be administered topically, for example, via nasal drops, inhalation, or a nebulizer, directly to the mucosa. Pharmaceutically acceptable salts include acid salts and those formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed with free carboxyl groups may be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron(III), and organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0100] Generally, the components of the compositions of the present disclosure are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. When the composition is administered by injection, it can be dispensed using 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 prior to administration.

[0101] The compositions of the present disclosure can be formulated as 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.

[0102] As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of nonspecific stimulators of the immune response, known as adjuvants. Adjuvants have been used experimentally to promote a general increase in immunity to poorly immunogenic antigens (e.g., U.S. Pat. No. 4,877,611). Adjuvants have been used to stimulate responses in immunization protocols for many years, and as such, adjuvants are well known to those skilled in the art. Some adjuvants affect the way antigens are presented. For example, adsorption of protein antigens to alum increases the immune response. Furthermore, emulsification of antigens prolongs the duration of antigen presentation and initiates the innate immune response. Suitable molecular adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins, or synthetic compositions.

[0103] The term "adjuvant" is used according to its ordinary meaning in immunology and refers to a substance commonly used as a component of an immunogenic composition. When administered to a subject together with one or more specific antigens as part of an immunogenic composition, an adjuvant can enhance the antigen-specific immune response in the subject. In some embodiments, an adjuvant accelerates the immune response to the antigen. In some embodiments, an adjuvant prolongs the immune response to the antigen. In some embodiments, an adjuvant enhances the immune response to the antigen.

[0104] Those skilled in the art will recognize different types of adjuvants approved for human use for experimental use that can be conjugated to vaccines in accordance with the present disclosure. These include, among others, alkyl lysophospholipids (ALPs), BCG, and biotin (including biotinylated derivatives). Specific adjuvants specifically contemplated for use include Gram-positive adjuvants, BCG-positive adjuvants, BCG-positive adjuvants, BCG-negative ... - These are teichoic acids derived from bacterial cells. These include lipoteichoic acid (LTA), ribitol teichoic acid (RTA), and glycerol teichoic acid (GTA). The active forms of their synthetic counterparts can also be used in connection with the compositions of the present disclosure (Takada et al., 1995).

[0105] In some aspects, the compositions described herein may further comprise an adjuvant. While alum is an approved adjuvant for humans, adjuvants used in experimental animals include Freund's complete adjuvant (a nonspecific stimulator of the immune response containing killed Mycobacterium tuberculosis), Freund's incomplete adjuvant, and aluminum hydroxide adjuvant. Other adjuvants that may be used in animals, and sometimes in humans, include interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, interferon, Bacillus Calmette-Guerin (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). Also contemplated is RIBI, which contains three components extracted from bacteria: MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene / Tween 80 emulsion. MHC antigens may also be used.

[0106] E. Treatment Methods In particular, compositions that can be used to treat cancer in subjects (e.g., human subjects) are disclosed herein. The compositions are preferably administered to mammals (e.g., rodents, humans, non-human primates, dogs, cattle, sheep, horses, cats, etc.) in an effective amount, i.e., an amount that can produce the desired results in the treated subject (e.g., cause apoptosis of cancer cells or kill bacterial cells). The toxicity and therapeutic efficacy of the compositions used in the disclosed methods can be determined by standard pharmaceutical procedures. As is well known in the fields of medicine and veterinary medicine, the dosage for any one animal depends on many factors, including the subject's size, body surface area, weight, age, the specific composition administered, the time and route of administration, general health, clinical symptoms of infection or cancer, and other drugs administered simultaneously. The compositions described herein are typically administered at a dosage that inhibits bacterial cell growth or proliferation, inhibits biofilm growth, or induces cancer cell death (e.g., induces apoptosis of cancer cells), as assayed by identifying a reduction in hematological parameters (complete blood count (CBC)) or cancer cell growth or proliferation.

[0107] As used herein, the term "subject" 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 prenatal and postnatal forms. In many embodiments, a subject is a human. A subject may be a patient, which refers to a person who visits a medical provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be suffering from or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0108] As used herein, the term "therapeutically effective amount" or "effective dosage" refers to the dosage or concentration of a drug that is effective for treating a disease or condition. For example, in relation to the use of the monoclonal antibody or antigen-binding fragment thereof disclosed herein to treat cancer, a therapeutically effective amount is a dosage or concentration of the monoclonal antibody or antigen-binding fragment thereof that can reduce tumor volume, eradicate all or part of the tumor, inhibit or delay tumor growth or cancer cell invasion into other organs, inhibit the growth or proliferation of cells that mediate the cancerous condition, inhibit or delay the metastasis of tumor cells, improve any symptoms or markers associated with a tumor or cancerous condition, prevent or delay the onset of a tumor or cancerous condition, or any combination thereof.

[0109] As used herein, "treating" a condition or "treatment" includes preventing or alleviating the condition, delaying the onset or rate of progression of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or any combination thereof.

[0110] Therapeutic methods (including prophylactic treatments) of the present disclosure generally involve administering a therapeutically effective amount of a composition described herein to a subject, e.g., a mammal, particularly a human, in need thereof. Such treatments are suitably administered to subjects, particularly humans, who are suffering from, have, are susceptible to, or are at risk for a disease, disorder, or symptom thereof. The determination of a subject being "at risk" can be made by diagnostic tests or any objective or subjective determination by the subject or the opinion of a health care provider (e.g., genetic testing, enzyme or protein markers, markers (as defined herein), family history, etc.).

[0111] In one embodiment, the present disclosure provides a method for monitoring the progress of a treatment. The method involves determining the level of changes in hematological parameters and / or cancer stem cell (CSC) analysis using cell surface proteins as diagnostic markers (e.g., CD34, CD38, CD90, and CD117, but not limited to) or diagnostic measurements (e.g., screening assays) in a subject suffering from or susceptible to a cancer-related disorder or its symptoms (e.g., leukemia) and administered a therapeutic amount of a composition described herein. To determine the subject's disease status, the level of the marker determined in this method can be compared to known levels of the marker in either healthy controls or other affected patients. In preferred embodiments, a second level of the marker in the subject is determined at a time later than the determination of the first level, and the two levels are compared to monitor the course of the disease or the effectiveness of a therapy. In certain preferred embodiments, a pre-treatment level of the marker in the subject is determined before initiating treatment according to the methods described herein, and the pre-treatment level of the marker can then be compared to the level of the marker in the subject after treatment has begun to determine the effectiveness of the treatment.

[0112] F. Combination Therapy It is contemplated that the vaccine conjugates described herein can be used in combination therapy with additional anti-cancer agents or compounds that alleviate one or more of the side effects of the disease or therapy experienced by the patient. The following is a general discussion of therapies that can be used in conjunction with the therapies 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 the composition of the present disclosure and at least one other therapy. These therapies are provided in combined amounts effective to achieve a reduction in one or more disease parameters. This process can include contacting cells / subjects with both agents / therapies simultaneously, for example, using a single composition or pharmacological formulation containing both agents, or by simultaneously contacting the cells / subjects with two separate compositions or formulations, one containing the compound and the other containing the other agent. Alternatively, the composition of the present disclosure can precede or follow the other treatment by intervals ranging from minutes to weeks. Generally, it is ensured that a significant period of time does not lapse between each delivery time point so that the therapies can still exert their beneficial combined effect on the cells / subject. In such cases, it is contemplated that the cells will be contacted with both modalities within about 12-24 hours of each other, within about 6-12 hours of each other, or with a delay of only about 12 hours. In some circumstances, it may be desirable to extend the period of treatment significantly, where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between each administration.

[0113] It is contemplated that multiple administrations of either the compound or the other therapy may be desired. As exemplified below, various combinations may be employed where the vaccine conjugate is "A" and the other therapy is "B." TIFF2025535082000002.tif18128

[0114] The following are examples of standard anti-cancer therapies that can be used in combination with the compositions and methods of the present application.

[0115] 1. Chemotherapy The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. These agents or drugs are classified by their mode of action within the cell, for example, whether and at what stage they affect the cell cycle. Alternatively, agents may be characterized based on their ability to directly cross-link DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.

[0116] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metuledopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (particularly cryptophycin) cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, e.g., enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma l I and calicheamicin ω1 Idynemicins, including dynemicin A, uncialamycin, and its derivatives; bisphosphonates such as clodronate; esperamycin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (mo morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate and 5-fluorouracil (5-F antimetabolites such as denopterin, methotrexate, pteropterin, trimetrexate; folic acid analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; aminoglutethimide, mitotane, and tocopherol; Antiadrenal agents such as rilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziconazole; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanol; nitraelin; pentostatin; fenamet;Pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin. complex); vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, metoprolol flufenaconazole, chlorambucil, busulfan, nitrosoureas, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, paclitaxel, docetaxel, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatin, 5-fluorouracil, vincristine, vinblastine, and methotrexate, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0117] 2. Radiation therapy Radiotherapy, also known as radiation therapy, is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation provides energy that injures or destroys cells in the treated area by damaging their genetic material, making it impossible for these cells to continue to grow. Radiation damages both cancer cells and normal cells, but the latter are able to repair themselves and function properly.

[0118] Radiation therapy used in accordance with the present disclosure may include, but is not limited to, the use of -rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA-damaging agents, such as microwave and UV radiation, are also contemplated. All of these agents are highly likely to induce widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dose ranges range from 12.9-51.6 mC / kg for prolonged periods (3-4 weeks) in daily doses to 0.516-1.55 mC / kg in single doses. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0119] Radiation therapy can involve the use of radiolabeled antibodies to deliver doses of radiation directly to the cancer site (radioimmunotherapy). Antibodies are highly specific proteins produced by the body in response to the presence of antigens (substances 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 produced in the laboratory and attached to radioactive materials (a process known as radiolabeling). When injected into the body, the antibodies actively seek out cancer cells, which are then destroyed by the cell-killing (cytotoxic) effects of the radiation. This approach minimizes the risk of radiation damage to normal cells. Conformal radiation therapy uses the same radiation therapy equipment as conventional radiation therapy treatments, a linear accelerator, but places a metal block in the path of the x-ray beam to change its shape to match the shape of the cancer. This ensures a higher radiation dose is delivered to the tumor. Surrounding normal cells and nearby structures receive a lower dose of radiation, thereby reducing the potential for side effects. A device called a multi-leaf collimator has been developed and can be used as an alternative to a metal block. A multi-leaf collimator consists of multiple metal sheets fixed to a linear accelerator. Each layer can be adjusted to allow the radiation therapy beam to be shaped to the treatment area without the need for metal blocks. Accurate positioning of the radiation therapy equipment is critical to conformal radiation therapy treatments, and special scanning equipment can be used to verify the position of internal organs at the start of each treatment.

[0120] High-resolution intensity-modulated radiation therapy also uses a multi-leaf collimator, whose layers are moved during treatment while the treatment is being performed. This method likely achieves even more accurate shaping of the treatment beam, allowing for a consistent radiation therapy dose throughout the treatment area.

[0121] Research studies have shown that conformal and intensity-modulated radiation therapy can reduce the side effects of radiation therapy treatment, but this precise shaping of the treatment area can stop microscopic cancer cells just outside the treatment area from being destroyed, meaning these specialized radiation therapy techniques may increase the risk of cancer recurrence in the future.

[0122] Scientists are also looking for ways to improve the effectiveness of radiation therapy. Two types of investigational drugs are being studied for their effect on cells receiving radiation: radiosensitizers, which increase the likelihood of tumor cells being damaged, and radioprotectors, which protect normal tissue from the effects of radiation. Hyperthermia, which uses heat, is also being studied for its effectiveness in making tissue more sensitive to radiation.

[0123] 3. Immunotherapy In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Trastuzumab (Herceptin™) is one example. The immune effector can be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody may act alone as the therapeutic effector or may recruit other cells to actually affect cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and simply act as targeting agents. Alternatively, the effector can be a lymphocyte bearing a surface molecule that interacts directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic modalities, i.e., direct cytotoxic activity and ErbB2 inhibition or reduction, is believed to provide therapeutic benefit in the treatment of ErbB2-overexpressing cancers.

[0124] In one aspect of immunotherapy, tumor cells must have some marker that is easy to target, i.e., not present on the majority of other cells. Many tumor markers exist, any of which may 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, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immunostimulatory effects. Immune stimulatory molecules also exist, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, and -IFN; chemokines such as MIP-1, MCP-1, and IL-8; and growth factors such as FLT3 ligand. Combining immune stimulatory molecules, either as proteins or using gene delivery in combination with tumor suppressors, has been shown to enhance antitumor effects (Ju et al., 2000). Furthermore, antibodies against any of these compounds can be used to target the anticancer drugs discussed herein.

[0125] Examples of immunotherapies currently under investigation or in use include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patent Nos. 5,801,005 and 5,739,169); cytokine therapy, such as interferon alpha, and IL-1, GM-CSF, and TNF; gene therapy, such as TNF, IL-1, IL-2, and p53 (U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-ganglioside GM2, anti-HER-2, and anti-p185 (U.S. Patent No. 5,824,311).

[0126] In active immunotherapy, antigenic peptides, polypeptides, or proteins, or autologous or allogeneic tumor cell compositions or "vaccines," are administered, generally with a separate bacterial adjuvant (Ravindranath and Morton, 1991; Morton et al., 1992; Mitchell et al., 1990; Mitchell et al., 1993).

[0127] In adoptive immunotherapy, the patient's circulating or tumor-infiltrating lymphocytes are isolated in vitro, activated with lymphokines such as IL-2 or transduced with genes for tumor necrosis, and readministered.

[0128] Checkpoint inhibitors are an emerging class of immunotherapeutic drugs. Checkpoint inhibitor therapy is a form of cancer immunotherapy currently under investigation. This therapy targets immune checkpoints, which are key regulators of the immune system that stimulate or inhibit its action, allowing tumors to protect themselves from immune system attack. Checkpoint therapy can block inhibitory checkpoints, thereby restoring immune system function. The first anticancer drug targeting immune checkpoints was ipilimumab, a CTLA4 blocker approved in the United States in 2011.

[0129] Currently approved checkpoint inhibitors target the molecules CTLA4, PD-1, and PD-L1. PD-1 is a transmembrane programmed cell death 1 protein (also known as PDCD1 and CD279) that interacts with PD-L1 (PD-1 ligand 1, or CD274). PD-L1 on the cell surface binds to PD-1 on the surface of immune cells and inhibits immune cell activity. Among the functions of PD-L1 is its important regulatory role on T cell activity. Upregulation of PD-L1 on the cell surface (through cancer) appears to inhibit T cells that would otherwise attack. Antibodies that bind to either PD-1 or PD-L1, thereby blocking the interaction, may enable T cells to attack tumors.

[0130] The first checkpoint antibody approved by the FDA was ipilimumab, approved in 2011 for the treatment of melanoma. It blocks the immune checkpoint molecule CTLA-4. Clinical trials have also shown some benefit of anti-CTLA-4 therapy, especially in combination with other drugs, for lung or pancreatic cancer.

[0131] However, patients treated with checkpoint blockade (especially CTLA-4 blocking antibodies) or combinations of checkpoint blocking antibodies are at high risk of suffering from immune-related adverse events, such as cutaneous, gastrointestinal, endocrine, or hepatic autoimmune reactions, most likely due to the broad range of T cell activation induced by anti-CTLA-4 antibodies when administered into the bloodstream by injection.

[0132] Using a mouse model of bladder cancer, researchers found that local injection of low doses of anti-CTLA-4 in the tumor area had the same tumor-inhibiting ability as delivering the antibody into the bloodstream. At the same time, circulating antibody levels were lower, suggesting that local administration of anti-CTLA-4 therapy resulted in fewer adverse events.

[0133] The first clinical trial results with the IgG4 PD1 antibody nivolumab (trade name Opdivo, developed by Bristol-Myers Squibb) were published in 2010. It was approved in 2014. Nivolumab is approved to treat melanoma, lung cancer, kidney cancer, bladder cancer, head and neck cancer, and Hodgkin's lymphoma.

[0134] Pembrolizumab (brand name Keytruda) is another PD1 inhibitor approved by the FDA in 2014 and was the second checkpoint inhibitor approved in the U.S. Keytruda is approved to treat melanoma and lung cancer and is manufactured by Merck.

[0135] Spartalizumab (PDR001) is a PD-1 inhibitor currently being developed by Novartis for the treatment of both 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 endogenous checkpoint blockade, such as CISH.

[0136] Checkpoint inhibitors can cause immunological side effects. Altering checkpoint inhibition can have diverse effects on most organ systems of the body. The exact mechanisms are unclear but differ in some respects based on the molecule targeted.

[0137] 4. Surgery Approximately 60% of cancer patients will undergo some type of surgery, including preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatments of the present disclosure, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0138] Curative surgery includes resection, which physically removes, excises, and / or destroys all or part of the cancerous tissue. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical procedures include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery). Furthermore, it is contemplated that the present disclosure may be used in conjunction with the removal of superficial cancers, pre-cancers, or incidental amounts of normal tissue.

[0139] When cancer cells, tissues, or tumors are partially or completely removed, a cavity may be formed in the body.Treatment can be achieved by perfusion, direct injection, or local application of additional anti-cancer therapy to the area.Such treatment 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 be various dosages.

[0140] In some specific embodiments, adjuvant treatment with compounds of the present disclosure after tumor removal is believed to be particularly effective in reducing tumor recurrence. Additionally, compounds of the present disclosure can also be used in neoadjuvant settings.

[0141] 5. Other agents It is also contemplated that other agents may be used in conjunction with the present disclosure. These additional agents include immunomodulators, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing factors, or other biological agents. Immunomodulators include tumor necrosis factors; 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. Furthermore, it is contemplated that upregulation of cell surface receptors or their ligands, such as Fas / Fas ligand, DR4, or DR5 / TRAIL (Apo-2 ligand), enhances the apoptosis-inducing ability of the present disclosure by establishing autocrine or paracrine effects on hyperproliferative cells. Increased intercellular signaling by increasing the number of GAP junctions is believed to increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatics or differentiation agents can be used in combination with the present disclosure to improve the anti-hyperproliferative effect of treatment. It is contemplated that cell adhesion inhibitors can improve the effect of the present disclosure. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, can be used in combination with the present disclosure to improve the effect of treatment.

[0142] Many advances in cancer therapy have been made since the introduction of cytotoxic chemotherapy drugs. However, one of the consequences of chemotherapy is the development / acquisition of a drug-resistant phenotype and the development of multidrug resistance. The development of drug resistance remains a major obstacle in the treatment of such tumors, and therefore, alternative approaches, such as gene therapy, are clearly needed. Another form of therapy for use in combination with chemotherapy, radiation therapy, or biological therapy includes hyperthermia, a procedure in which a patient's tissues are exposed to high temperatures (up to 106°F). Local, regional, or systemic application of hyperthermia may require external or internal heating devices. Local hyperthermia involves the application of heat to a small area, such as a tumor. Heat can be generated externally by radiofrequency radiation targeted to the tumor from a device outside the body. Internal heating may involve a sterile probe containing a heated thin wire or hollow tube filled with warm water, an implanted microwave antenna, or a radiofrequency electrode.

[0143] Regional therapy involves heating a patient's organs or limbs, which is accomplished using devices that generate high energy, such as magnets. Alternatively, a portion of the patient's blood may be withdrawn, heated, and then perfused into an area to be heated internally. Whole-body heating may also be used if the cancer has spread throughout the body. Warm water blankets, hot wax, induction coils, and thermal chambers may be used for this purpose.

[0144] Those skilled in the art are guided by "Remington's Pharmaceutical Sciences," Vol. 15, Chapter 33, especially pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration, in any event, will determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet the standards of sterility, pyrogenicity, general safety, and purity required by the standards of the FDA's Office of Biologics.

[0145] It should also be noted that any of the aforementioned therapies may prove useful in their own right in the treatment of cancer.

[0146] II. Kit In various aspects of this embodiment, kits containing therapeutic agents and / or other therapeutic agents and delivery agents are contemplated. In some embodiments, the present disclosure contemplates kits for preparing and / or administering the vaccine compositions of this embodiment. The kits may include one or more sealed vials containing any of the pharmaceutical compositions of this embodiment. The kits may include, for example, a vaccine conjugate and reagents for preparing, formulating, and / or administering the components of this embodiment or for performing one or more steps of the methods of the invention. In some embodiments, the kits may also include suitable containers, such as Eppendorf tubes, assay plates, syringes, bottles, or tubes, that do not react with the components of the kit. The containers may be made of a sterilizable material, such as plastic or glass.

[0147] The kit may include one or more reagents for a biotechnology product or assay. The kit may further include reagents for an in vitro assay such as Western blot, flow cytometry, immunoprecipitation, ELISA, or immunofluorescence.

[0148] The kit may further include instructions outlining the procedural steps of the methods described herein, following substantially the same procedures as described herein or known to those of skill in the art. The instructional information may be in a computer-readable medium containing machine-readable instructions that, when executed using a computer, result in the display of an actual or hypothetical procedure for delivering a pharmaceutically effective amount of a therapeutic agent. [Example]

[0149] III. Working Example The following examples are included to demonstrate preferred embodiments of the present disclosure. It will be understood by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the present disclosure, and therefore can be considered preferred modes for that practice. However, those skilled in the art will, in light of the present disclosure, understand that many changes can be made in the specific embodiments that are disclosed and still obtain a similar or similar result, without departing from the spirit and scope of the present disclosure.

[0150] Example 1 – Development of SMEZ-2 vaccine conjugate We developed a detoxified AGR2-SMEZ-2 conjugate that stimulates a robust immune response against PDAC with high extracellular AGR2 expression. This 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 current challenges presented by peptide-based cancer vaccines, as the high-affinity binding properties of SMEZ-2 obviate the need for in silico simulations of peptide binding to MHC molecules. Therefore, this method provides an AGR2-based PDAC vaccine as well as other TAA- and neoantigen-based vaccine candidates.

[0151] A candidate vaccine conjugate was synthesized by fusing the DNA sequence of human AGR2 (21-175) to the sequence of a detoxified and T cell receptor-binding-deficient mutant SMEZ-2 (W75L, K182Q, D42C) using the AsiSI restriction site. This fusion was cloned into pET15TEV_NESG, which expresses a protein with an N-terminal 6x histidine tag cleavable 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 nonspecific immunogenicity or unexpected side effects, the 6x histidine tag was excised by overnight digestion with TEV and purified by an additional 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 individually cloned, expressed, and purified using the same methods as the conjugates. The proteins were highly soluble in E. coli, with yields of 33 mg / L for AGR2, 11 mg / L for SMEZ-2 (W75L, K182Q, D42C), and 4 mg / L for AGR2-SMEZ-2 (W75L, K182Q, D42C). As an alternative to expression in E. coli, these proteins can also be produced in other cell types, such as HEK cells and CHO cells.

[0152] The immune responses elicited by unconjugated AGR2, unconjugated SMEZ2, a mixture of unconjugated AGR2 and SMEZ2, 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 containing a complete phosphorothioate backbone containing one or more CpG dinucleotides. Mice (n=10) were injected every two weeks with a total volume of 100 μL containing 100 μg AGR2-SMEZ-2 or an equimolar amount of other proteins. Anti-AGR2-specific IgG from mouse plasma was analyzed by ELISA. Data in Figure 4 were obtained from blood samples 10 days after the second injection. The results showed that 10 days after the second vaccination, anti-AGR2 antibody titers in the AGR2-SMEZ-2 treatment group were significantly higher than those in mice treated with AGR2 alone or AGR2 co-injected with SMEZ, suggesting that the SMEZ2 carrier can enhance the presentation of tumor-associated antigens such as AGR2 to the immune system.

[0153] Next, we evaluated the antitumor potential of this therapeutic approach in a preclinical mouse model of pancreatic ductal adenocarcinoma (PDAC). Specifically, we used PDAC cells harvested from pancreatic tumors in LSL-KrasG12D; LSL-Trp53R172H; Pdx1-cre (KPC) mice. KPC32908 cells were engineered to express high levels of human AGR2, which shares over 95% sequence identity with mouse AGR2. Next, 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, mice were challenged with KPC cells via subcutaneous injection into the hind flank. This model rapidly develops aggressive KPC PDAC tumors that are resistant to standard treatments, chemotherapy and checkpoint inhibitor immunotherapy. However, the combination of AGR2-SMEZ2 and the TLR9 agonist CpG ODN1826 significantly reduced tumor growth rate (Figure 5B), thereby demonstrating tumor control of pancreatic tumors in a highly invasive model system.

[0154] Next, we evaluated the activity of the SMEZ2 conjugate system for other tumor-associated antigens and tumor types / models. CD38 is a viable therapeutic target for 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 immune presentation of tumor-associated antigens and induce natural polyclonal humoral responses, we constructed a murine CD38-SMEZ2 conjugate, expressed it in a mammalian expression system, and purified it. The purified protein product is shown in Figure 6A. Next, wild-type BALB / C mice were treated subcutaneously in IFA with two doses of 50 μg mCD38-SMEZ or an equimolar control. Plasma was collected 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 with the AGR2-SMEZ2 protein, significantly higher levels of CD38-specific IgG were measured in the blood of mice administered the CD38-SMEZ2 conjugate than in cohorts administered 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.

[0155] Next, we evaluated the SMEZ2 platform against additional targets, including immune cell-localized antigens that regulate antitumor T cell responses. Specifically, we generated constructs against programmed cell death protein-1 (PD-1, also known as CD279) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), two inhibitory immune checkpoint molecules that suppress T cell function

[35] . Antagonistic antibodies or immune checkpoint inhibitors (ICIs) against these molecules have been clinically active in a variety of tumor types, leading to FDA approval of several ICIs in the field, including pembrolizumab / KETRUDA (anti-PD-1), nivolumab / OPDIVO (anti-PD-1), and ipilimumab / YERVOY (anti-CTLA-4) [36-40]. Monoclonal antibodies against programmed cell death ligand 1 (PD-L1), which binds to PD-1 to activate the PD-1 immune checkpoint and transmit inhibitory T cell signals, have also shown clinical utility. Atezolizumab / TECENTRIQ was the first anti-PD-L1 antibody approved by the FDA

[41] . In addition to the role that the immune checkpoint molecules CTLA-4 and PD-1 play in suppressing antitumor T cell immunity, they are also highly expressed on 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's lymphoma, such as cutaneous T cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), and NK / T cell lymphoma, in which wild-type or gene fusion expression of PD-1 and CTLA-4 is elevated compared to normal T cells [42-44]. Therefore, immune checkpoints may be actionable targets for driving antitumor immune responses in certain tumor types and attractive therapeutic targets for T-cell malignancies.

[0156] To explore the possibility of targeting immune checkpoints using the SMEZ2 platform, we generated, expressed, and purified murine PD1-SMEZ, CTLA4-SMEZ, and PDL1-SMEZ constructs in a mammalian cell culture system (Figure 7). We then evaluated the ability of the superantigen constructs to induce PD-1 / PD-L1 / CTLA-4-specific immune responses in vivo. The results in Figure 8 demonstrate that a cocktail containing all three conjugates was able to induce a potent polyclonal humoral response against murine PD-1, PD-L1, and CTLA-4 (Figure 8A). Although it was hypothesized that antibodies raised against these antigens could result in immune checkpoint blockade and enhance T cell viability, proliferation, and activity, the results demonstrate that the cocktail actually depleted T cell populations in the spleen (Figure 8B). This suggests that the response was primarily an anti-T cell response rather than a T cell-supportive effect. These results further prove the concept of the SMEZ platform and provide evidence of its versatility against multiple tumor- and disease-specific antigens. This further demonstrates that SMEZ checkpoint constructs or cocktails may be useful for depleting T cell populations in pathological settings such as T cell non-Hodgkin's lymphoma or autoimmune disorders.

[0157] Example 2 – Development of a CD33-SMEZ-2 vaccine conjugate To evaluate the potential of the SMEZ-2 superantigen to drive immune responses against CD33+ AML, a full-length human CD33-SMEZ-2 conjugate protein was cloned, produced, and purified in a mammalian cell culture system (Figure 9A). The purified protein was then tested in vivo using an immunocompetent syngeneic mouse model of AML in C57BL / 6 mice. The protein was injected subcutaneously as an emulsion in Freund's incomplete adjuvant (IFA).

[0158] At a dose of 40 pmol injected at 2-week intervals, the CD33-SMEZ-2 conjugate was able to induce a significantly more potent humoral response against CD33, as determined by ELISA measurement of anti-hCD33 IgG in the plasma of inoculated mice (Fig. 9B ).

[0159] To evaluate the anti-AML effect of CD33-SMEZ-2 therapeutic agent, a syngeneic AML cell model of spontaneously arising C1498 AML cells in C57BL / 6 mice was used. Mice were treated with two doses of CD33-SMEZ-2, and two weeks later, 1x10 6H-cells transduced with the human CD33 gene were added. 6 C1498 cells were injected intravenously. This treatment protocol significantly extended median survival (p<0.05 N=5) and cured 60% of treated mice, where "cure" was defined as survival beyond 100 days.

[0160] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods described herein, and in the steps or sequence of steps of the methods, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would 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.

[0161] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2025535082000003.tif189128TIFF2025535082000004.tif97128

Claims

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

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

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

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

8. 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 said at least one target protein is anterior gradient 2 (AGR2).

11. 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. 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. 13. 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 said at least one target protein is CD38.

16. 10. 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. 10. 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. 18. The conjugate of claim 17, wherein the CD33 or fragment thereof comprises one or more amino acid substitutions.

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

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

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

22. The conjugate of 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 of any one of claims 1 to 22 and an adjuvant.

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

25. 25. The composition of claim 24, wherein the conjugate is formulated as a 1:1 emulsion with Freund's incomplete adjuvant (IFA).

26. 24. The composition of claim 23, wherein the conjugate is formulated for intravenous infusion.

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

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

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

30. 30. The host cell of claim 29, which is an Escherichia coli (E. coli), a human embryonic kidney cell (HEK293), or a Chinese hamster ovary (CHO) cell.

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

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

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

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

35. 34. The method of claim 33, wherein the cancer is pancreatic cancer.

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

37. 34. The method of claim 33, wherein the cancer is breast cancer.

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

39. 38. The method of claim 37, wherein the cancer is lymphoma.

40. 40. The method of claim 39, wherein the lymphoma is T-cell non-Hodgkin's lymphoma.

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

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

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

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

45. 45. The method of claim 44, wherein the second anticancer therapy is immunotherapy, chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, antiangiogenic therapy, or cytokine therapy.

46. 45. The method of claim 44, wherein the second anti-cancer therapy is an immunotherapy.

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

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

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

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

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

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

53. 53. The method of claim 52, wherein the anti-CTLA-4 antibody is tremelimumab, YERVOY®, or ipilimumab.

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

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

56. 55. The method of claim 54, wherein the cancer is prostate cancer.

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

58. 55. The method of claim 54, wherein the cancer is breast cancer.

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

60. 55. The method of claim 54, wherein the cancer is lymphoma.

61. 61. The method of claim 60, wherein the lymphoma is T-cell non-Hodgkin's lymphoma.

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

63. 63. The method of claim 62, wherein said step of administering at least one immune checkpoint inhibitor to said subject comprises administering said at least one immune checkpoint inhibitor prior to said vaccine conjugate.

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

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

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

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

68. 67. The method of claim 66, wherein said anti-PD1 agent is nivolumab, pembrolizumab, pidilizumab, KEYTRUDA®, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA, or AMP-224.

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

70. 70. The method of claim 69, wherein the anti-CTLA-4 antibody is tremelimumab, YERVOY®, or ipilimumab.

71. The method of any one of claims 62-70, wherein the subject is administered two immune checkpoint inhibitors.

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

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

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

75. 75. The method of claim 74, wherein the additional anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or cytokine therapy.

76. 75. The method of claim 74, wherein the additional anticancer therapy comprises a TLR9 agonist and / or a CD40 agonist.

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

78. 77. The method of claim 76, wherein the CD40 agonist is a CD40 agonist antibody.

79. A kit comprising a vaccine conjugate according to any one of claims 1 to 22 or a pharmaceutical composition according to any one of claims 23 to 25.

80. 80. The kit of claim 79, further comprising an immune checkpoint inhibitor.

81. The kit of claim 80, wherein the immune checkpoint inhibitor is an anti-PD1 antibody or a CTLA-4 antibody.

82. 82. The kit of any one of claims 79-81, further comprising a TLR9 agonist.

83. 83. The kit of claim 82, wherein the TLR9 agonist is CpG ODN1826.

84. 26. A composition comprising the vaccine conjugate of any one of claims 1 to 22 or the pharmaceutical composition of any one of claims 23 to 25 for use in the treatment of cancer.

85. 85. The composition of claim 84, further comprising an immune checkpoint inhibitor.

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

87. The composition of any one of claims 84-86, further comprising a TLR9 agonist.

88. 83. The composition of claim 82, wherein the TLR9 agonist is CpG ODN1826.

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

90. 90. The conjugate of claim 89, wherein the mutant SMEZ-2 comprises the mutations W75L and K182Q.

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

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

93. 90. The conjugate of claim 89, wherein the CD33 is human CD33.

94. 90. The conjugate of claim 89, wherein said CD33 or fragment thereof comprises one or more amino acid substitutions.

95. 95. The conjugate of claim 94, wherein said one or more amino acid substitutions are at D231, D246, C154 and / or C169.

96. 95. The conjugate of claim 94, wherein said one or more amino acid substitutions are D231E, D246E, C154S and / or C169S.

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

98. 94. The conjugate of any one of claims 89 to 93, further comprising a linker.

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

100. 100. The conjugate of claim 99, wherein the peptide linker is a glycine serine linker.

101. 101. The conjugate of claim 100, wherein said glycine serine linker is GGGGS.

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

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

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

105. 105. The composition of claim 104, wherein the conjugate is formulated as a 1:1 emulsion with Freund's incomplete adjuvant (IFA).

106. 104. The composition of 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. 108. The vector of claim 107, encoding the vaccine conjugate of any one of claims 89 to 102.

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

110. 110. The host cell of claim 109, which is an E. coli, a human embryonic kidney cell (HEK293), or a Chinese hamster ovary (CHO) cell.

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

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

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

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

115. 114. The method of claim 113, wherein the cancer is leukemia.

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

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

118. The method of claim 117, wherein the anti-CD33 specific immune response is detected by measuring an increased titer of CD33 specific immunoglobulins in a sample of the subject's blood.

119. 119. The method of any one of claims 111-118, wherein the conjugate is administered by injection.

120. 120. The method of any one of claims 111-119, further comprising administering to the subject a second anti-cancer therapy.

121. 121. The method of claim 120, wherein the second anticancer therapy is immunotherapy, chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, antiangiogenic therapy, or cytokine therapy.

122. 121. The method of claim 120, wherein said second anticancer therapy comprises a TLR9 agonist.

123. 123. The method of claim 122, wherein the TLR9 agonist is CpG ODN1826.

124. 121. The method of claim 120, wherein the second anticancer therapy is an immunotherapy.

125. 125. The method of claim 124, wherein the immunotherapy is an immune checkpoint inhibitor.

126. 126. The method of claim 125, wherein said immune checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR.

127. 126. The method of claim 125, wherein said immune checkpoint inhibitor comprises an anti-PD1 agent.

128. The method of claim 127, wherein the anti-PD1 agent comprises an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-PDL2 antibody.

129. 128. The method of claim 127, wherein said anti-PD1 agent is nivolumab, pembrolizumab, pidilizumab, KEYTRUDA®, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA, or AMP-224.

130. The method of claim 125, wherein said immune checkpoint inhibitor is an anti-CTLA-4 antibody.

131. 131. The method of claim 130, wherein the anti-CTLA-4 antibody is tremelimumab, YERVOY®, or ipilimumab.

132. 104. A method of treating a subject having cancer, comprising administering to said subject a vaccine conjugate of any one of claims 89 to 102 or a pharmaceutical composition of any one of claims 103 to 105.

133. 133. The method of claim 132, wherein the cancer is oral cavity cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, multiple neuroendocrine tumors type I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

134. 133. The method of claim 132, wherein the cancer is leukemia.

135. 135. The method of claim 134, wherein the cancer is acute myeloid leukemia (AML).

136. 133. The method of claim 132, wherein the cancer is pancreatic cancer or breast cancer.

137. The method of any one of claims 132-136, further comprising administering to said subject at least one immune checkpoint inhibitor.

138. 138. The method of claim 137, wherein said step of administering at least one immune checkpoint inhibitor to said subject comprises administering said at least one immune checkpoint inhibitor prior to said vaccine conjugate.

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

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

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

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

143. 142. The method of claim 141, wherein said anti-PD1 agent is nivolumab, pembrolizumab, pidilizumab, KEYTRUDA®, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA, or AMP-224.

144. 144. The method of any one of claims 137-143, wherein said at least one immune checkpoint inhibitor is an anti-CTLA-4 antibody.

145. 145. The method of claim 144, wherein said anti-CTLA-4 antibody is tremelimumab, YERVOY®, or ipilimumab.

146. The method of any one of claims 137-145, wherein the subject is administered two immune checkpoint inhibitors.

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

148. 148. The method of any one of claims 132-147, wherein said vaccine conjugate is administered more than once.

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

150. 150. The method of claim 149, wherein the additional anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or cytokine therapy.

151. 150. The method of claim 149, wherein the additional anticancer therapy comprises a TLR9 agonist.

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

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

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

155. The kit of claim 154, wherein said immune checkpoint inhibitor is an anti-PD1 antibody or a CTLA-4 antibody.

156. 154. The kit of claim 153, further comprising a TLR9 agonist.

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

158. 106. A composition comprising the vaccine conjugate of any one of claims 89 to 102 or the pharmaceutical composition of any one of claims 103 to 105 for use in the treatment of cancer.

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

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

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