Cancer Vaccine Formulation Research Articles

An optically responsive cancer vaccine for inducing robust anti-tumor immunity by apoptotic body carrying nanoadjuvants

A cancer vaccine based on nano-adjuvant fusion represents an effective strategy for anti-tumor immunity. Compared with other cancer vaccine formulations, the ones based on apoptotic bodies not only possess higher load-bearing capacity and biosafety, but also present greater clinical translational potential. In this study, we developed an optically responsive cancer vaccine that induced robust anti-tumor immunity by encapsulating genipin-crosslinked nano-adjuvants in tumor cell apoptotic bodies. The vaccine may potentially be used for prophylactic or therapeutic purposes. As an antigen source and a cargo vehicle, apoptotic bodies can co-deliver antigens and adjuvants simultaneously to antigen-presenting cells to activate T cells. Nano-adjuvants promote the targeted delivery of adjuvants for effective immune responses. Genipin-crosslinked nano-adjuvants enable visual tracking of the tumor vaccine. In addition, the photothermal properties of genipin-crosslinked nano-adjuvants promote lysosomal escape of tumor antigens to enhance cross-presentation. These characteristics enable visualization and controllability of cancer vaccines, thereby avoiding the need for exogenous photosensitive reagents. Apoptotic body-based vaccines can be used to prevent or treat tumors by triggering a strong systemic immune response by promoting T-cell infiltration and improving the tumor immune microenvironment.

Abstract 4955: An analytic pipeline of neoantigen selection for both MHC-I and MHC-II pathways

Abstract Background: Accurate neoantigen selection is pivotal for assessing mutation burden and optimizing cancer vaccine formulation thereby playing a crucial role in cancer immunotherapy. The concurrent activation of CD4+ and CD8+ T cells engenders a robust immune response, which is key to effective treatment. However, currently analytic tools predominantly focus on the MHC-I pathway, typically relying on metrics such as gene expression level and MHC-binding affinity of mutated peptides. To address this gap, our study presents a comprehensive pipeline that encompasses both MHC-I and MHC-II pathways and integrates three critical dimensions of immune response: neoantigen abundance, presentation, and immunogenicity. Methods: We estimated mutated peptide abundance by analyzing genomic allelic frequency, transcriptomic allelic frequency, and RNA expression level. Antigen presentation was assessed using the patient harmonic binding rank (PHBR, a composite binding score for all potential MHC-peptide pairs) and MHC diversity (the variety of MHC alleles involved in peptide binding), with additional consideration for the loss of heterozygosity of MHC-I alleles, potentially indicative of immune escape. For immunogenicity, we factored in agretopicity (comparison of MHC-binding affinity between mutated and wild-type peptides), foreignness (similarity to immunogenic foreign antigens), and dissimilarity (sequence disparity with self-peptides, reflecting T cell negative selection process). Results: Implementing our pipeline on a dataset from Parkhurst et al. (2019), we analyzed immune reactivity in relation to 11,537 somatic mutations from 75 gastrointestinal cancer patients. Key metrics—RNA expression, transcriptomic allelic frequencies, PHBR, and MHC diversity—demonstrated significant correlations with both CD4+ and CD8+ activity (p<0.05). Notably, genomic allelic frequency was a significant factor in CD8+ reactivity, whereas agretopicity was crucial for CD4+ reactivity. Conclusion: Our pipeline elucidates the necessity for implementing both MHC-I and MHC-II pathway analyses to effectively select neoantigen candidates for CD8+ and CD4+ T cells respectively. The study confirms that abundance and antigen presentation metrics are robust indicators of neoantigen immunogenicity, supporting their use as potential markers in crafting more effective cancer vaccines. The findings highlight the critical role of this dual-pathway selection approach in generating a comprehensive immune response through neoantigen vaccination. Citation Format: Ko-Han Lee, Timothy Sears, Andrea Castro, Maurizio Zanetti, Hannah Carter. An analytic pipeline of neoantigen selection for both MHC-I and MHC-II pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4955.

Extracellular vesicles hybrid plasmid-loaded lipid nanovesicles for synergistic cancer immunotherapy

Combination immunotherapy of cancer vaccines with immune checkpoint inhibitors (ICIs) represents a promising therapeutic strategy for immunosuppressed and cold tumors. However, this strategy still faces challenges, including the limited therapeutic efficacy of cancer vaccines and immune-related adverse events associated with systematic delivery of ICIs. Herein, we demonstrate the antitumor immune response induced by outer membrane vesicle from Akkermansia muciniphila (Akk-OMV), which exhibites a favorable safety profile, highlighting the potential application as a natural and biocompatible self-adjuvanting vesicle. Utilizing tumor cell-derived exosome as an antigen source and Akk-OMV as a natural adjuvant, we construct a cancer vaccine formulation of extracellular vesicles hybrid lipid nanovesicles (Lipo@HEV) for enhanced prophylactic and therapeutic vaccination by promoting dendritic cell (DC) maturation in lymph node and activating cytotoxic T cell (CTL) response. The Lipo@HEV is further loaded with plasmid to enable gene therapy-mediated PD-L1 blockade upon peritumoral injection. Meanwhile, it penetrates into lymph node to initiate DC maturation and CTL activation, synergistically inhibiting the established tumor. The fabrication of extracellular vesicles hybrid plasmid-loaded lipid nanovesicles reveals a promising gene therapy-guided and vesicle-based hybrid system for therapeutic cancer vaccination and synergistic immunotherapy strategy.

MuSyC dosing of adjuvanted cancer vaccines optimizes antitumor responses.

With the clinical approval of T-cell–dependent immune checkpoint inhibitors for many cancers, therapeutic cancer vaccines have re-emerged as a promising immunotherapy. Cancer vaccines require the addition of immunostimulatory adjuvants to increase vaccine immunogenicity, and increasingly multiple adjuvants are used in combination to bolster further and shape cellular immunity to tumor antigens. However, rigorous quantification of adjuvants’ synergistic interactions is challenging due to partial redundancy in costimulatory molecules and cytokine production, leading to the common assumption that combining both adjuvants at the maximum tolerated dose results in optimal efficacy. Herein, we examine this maximum dose assumption and find combinations of these doses are suboptimal. Instead, we optimized dendritic cell activation by extending the Multidimensional Synergy of Combinations (MuSyC) framework that measures the synergy of efficacy and potency between two vaccine adjuvants. Initially, we performed a preliminary in vitro screening of clinically translatable adjuvant receptor targets (TLR, STING, NLL, and RIG-I). We determined that STING agonist (CDN) plus TLR4 agonist (MPL-A) or TLR7/8 agonist (R848) as the best pairwise combinations for dendritic cell activation. In addition, we found that the combination of R848 and CDN is synergistically efficacious and potent in activating both murine and human antigen-presenting cells (APCs) in vitro. These two selected adjuvants were then used to estimate a MuSyC-dose optimized for in vivo T-cell priming using ovalbumin-based peptide vaccines. Finally, using B16 melanoma and MOC1 head and neck cancer models, MuSyC-dose–based adjuvating of cancer vaccines improved the antitumor response, increased tumor-infiltrating lymphocytes, and induced novel myeloid tumor infiltration changes. Further, the MuSyC-dose–based adjuvants approach did not cause additional weight changes or increased plasma cytokine levels compared to CDN alone. Collectively, our findings offer a proof of principle that our MuSyC-extended approach can be used to optimize cancer vaccine formulations for immunotherapy.

The synergy of TLR and STING in cancer immunity

Abstract Adjuvants are essential components of cancer vaccine formulations to promote effective immune responses. We initially screened multiple immune-activating targets, including TLR and STING adjuvants using in-vitro assays to test their ability to activate bone marrow DC (BMDC) and showed STING had the most potent activation (i.e., MHC and co-stimulatory molecules) on DCs. Since STING and TLR signaling is non-redundant, TLR and STING adjuvants were combined to enhance DC activation. We utilized the multidimensional synergy of combinations (MuSyc), a novel synergy algorithm, to assess molecular synergy in combining STING and TLR adjuvants, and we noted that the combination of R848 (TLR7-8) plus STING agonists provided a synergistic efficacious and potent response on BMDCs. From this data-set, we generated a MuSYC synergy strategy where we will utilize max signal dose for STING adjuvant and ten times less of max signal dose for R848 (TLR7-8) to determine synergy for other assays. We tested this same MuSYC strategy for activation of human monocytic cell line THP-1 and determined that the synergy strategy induced similar or better activation effects compared to the max signal dose for both adjuvants. Finally, we tested the in-vivo anti-tumor immune response for the B16 melanoma and MOC2 head/neck tumor-bearing vaccination models that resulted in an improved anti-tumor response for the combinatorial STING-TLR adjuvanted vaccines compared to the single agents.

IDENTIFICATION OF MHC PEPTIDES USING MASS SPECTROMETRY FOR NEOANTIGEN DISCOVERY AND CANCER VACCINE DEVELOPMENT.

Immunotherapy with neoantigens presented by major histocompatibility complex (MHC) is one of the most promising approaches in cancer treatment. Using this approach, cancer vaccines can be designed to target tumor-specific mutations that are not found in normal tissues. Clinical trials have demonstrated an increased immune response and eradication of tumors after injecting synthetic peptides selected from the immunopeptidome. Although the sequence of MHC binding peptides can be predicted from genome sequencing and prediction algorithms, this approach results in large numbers of predicted peptides, requiring the confirmation by mass spectrometry (MS) analysis. Identification of MHC peptides by direct MS analysis of immunopeptidome is accurate and sensitive, with tens of thousands of unique peptides potentially identified from either cancer cell line or tumor tissue. Peptides with mutations can also be identified with patient-specific protein databases constructed from genome or transcriptome sequencing data. MS analysis also enables the characterization of the post-translational modifications (PTMs) of those antigens that cannot be predicted. Moreover, PTMs were found to be more efficient in triggering an immune response. In addition to reviewing recent advances in the identification of neoantigens using MS, the techniques for cancer vaccine candidate selection and formulation, vaccine delivery systems, and the potential for use in combination with other therapeutics are also discussed. It is anticipated that MS-based techniques will play an important role in future cancer vaccine development. © 2019 John Wiley & Sons Ltd. Mass Spec Rev 40:110-125, 2021.

An HER2 DNA vaccine with evolution-selected amino acid substitutions reveals a fundamental principle for cancer vaccine formulation in HER2 transgenic mice.

Enhancement of endogenous immunity to tumor-associated self-antigens and neoantigens is the goal of preventive vaccination. Toward this goal, we compared the efficacy of the following HER2 DNA vaccine constructs: vaccines encoding wild-type HER2, hybrid HER2 vaccines consisting of human HER2 and rat Neu, HER2 vaccines with single residue substitutions and a novel human HER2 DNA vaccine, ph(es)E2TM. ph(es)E2TM was designed to contain five evolution-selected substitutions: M198V, Q398R, F425L, H473R and A622T that occur frequently in 12 primate HER2 sequences. These ph(es)E2TM substitutions score 0 to 1 in blocks substitutions matrix (BLOSUM), indicating minimal biochemical alterations. h(es)E2TM recombinant protein is recognized by a panel of anti-HER2 mAbs, demonstrating the preservation of HER2 protein structure. Compared to native human HER2, electrovaccination of HER2 transgenic mice with ph(es)E2TM induced a threefold increase in HER2-binding antibody (Ab) and elevated levels of IFNγ-producing T cells. ph(es)E2TM, but not pE2TM immune serum, recognized HER2 peptide p95 355LPESFDGDPASNTAP369, suggesting a broadening of epitope recognition induced by the minimally modified HER2 vaccine. ph(es)E2TM vaccination reduced tumor growth more effectively than wild-type HER2 or HER2 vaccines with more extensive modifications. The elevation of tumor immunity by ph(es)E2TM vaccination would create a favorable tumor microenvironment for neoantigen priming, further enhancing the protective immunity. The fundamental principle of exploiting evolution-selected amino acid substitutions is novel, effective and applicable to vaccine development in general.

Harnessing Dendritic Cells for Poly (D,L-lactide-co-glycolide) Microspheres (PLGA MS)-Mediated Anti-tumor Therapy.

With emerging success in fighting off cancer, chronic infections, and autoimmune diseases, immunotherapy has become a promising therapeutic approach compared to conventional therapies such as surgery, chemotherapy, radiation therapy, or immunosuppressive medication. Despite the advancement of monoclonal antibody therapy against immune checkpoints, the development of safe and efficient cancer vaccine formulations still remains a pressing medical need. Anti-tumor immunotherapy requires the induction of antigen-specific CD8+ cytotoxic T lymphocyte (CTL) responses which recognize and specifically destroy tumor cells. Due to the crucial role of dendritic cells (DCs) in initiating anti-tumor immunity, targeting tumor antigens to DCs has become auspicious in modern vaccine research. Over the last two decades, micron- or nanometer-sized particulate delivery systems encapsulating tumor antigens and immunostimulatory molecules into biodegradable polymers have shown great promise for the induction of potent, specific and long-lasting anti-tumor responses in vivo. Enhanced vaccine efficiency of the polymeric micro/nanoparticles has been attributed to controlled and continuous release of encapsulated antigens, efficient targeting of antigen presenting cells (APCs) such as DCs and subsequent induction of CTL immunity. Poly (D, L-lactide-co-glycolide) (PLGA), as one of these polymers, has been extensively studied for the design and development of particulate antigen delivery systems in cancer therapy. This review provides an overview of the current state of research on the application of PLGA microspheres (PLGA MS) as anti-tumor cancer vaccines in activating and potentiating immune responses attempting to highlight their potential in the development of cancer therapeutics.

Cancer vaccine formulation dictates synergy with CTLA-4 and PD-L1 checkpoint blockade therapy

Anticancer vaccination is a promising approach to increase the efficacy of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death ligand 1 (PD-L1) checkpoint blockade therapies. However, the landmark FDA registration trial for anti-CTLA-4 therapy (ipilimumab) revealed a complete lack of benefit of adding vaccination with gp100 peptide formulated in incomplete Freund's adjuvant (IFA). Here, using a mouse model of melanoma, we found that gp100 vaccination induced gp100-specific effector T cells (Teffs), which dominantly forced trafficking of anti-CTLA-4-induced, non-gp100-specific Teffs away from the tumor, reducing tumor control. The inflamed vaccination site subsequently also sequestered and destroyed anti-CTLA-4-induced Teffs with specificities for tumor antigens other than gp100, reducing the antitumor efficacy of anti-CTLA-4 therapy. Mechanistically, Teffs at the vaccination site recruited inflammatory monocytes, which in turn attracted additional Teffs in a vicious cycle mediated by IFN-γ, CXCR3, ICAM-1, and CCL2, dependent on IFA formulation. In contrast, nonpersistent vaccine formulations based on dendritic cells, viral vectors, or water-soluble peptides potently synergized with checkpoint blockade of both CTLA-4 and PD-L1 and induced complete tumor regression, including in settings of primary resistance to dual checkpoint blockade. We conclude that cancer vaccine formulation can dominantly determine synergy, or lack thereof, with CTLA-4 and PD-L1 checkpoint blockade therapy for cancer.

An In vivo study: Adjuvant activity of poly-n-vinyl-2-pyrrolidone-co-acrylic acid on immune responses against Melanoma synthetic peptide

ABSTRACTPeptides have been studied as an important class of components in medicine to control many major diseases with vaccination. Polymers as adjuvants are capable of enhancing the vaccine potential against various diseases by improving the delivery of antigens, and they reduce the booster doses of vaccines. In brief, polymers are promising candidates for peptide-based vaccine delivery platforms. The purpose of the present study was to create a possible alternative approach in the treatment of malignant melanoma and/or to prevent metastasis of melanoma. The study was designed as both an experimental and an in vivo study. We prepared a complex and covalent conjugate of MAGE-3 121–134 (L-L-K-Y-R-A-R-E-P-V-T-K-A-E) T-cell epitope as a vaccine candidate for melanoma. These conjugates were able to generate an immune response in mice after a single immunization, without the help of any external adjuvant. The peptide-polymer complexes activated the immune system in the best way and formed the highest antigen specific immune response. These results indicate the adjuvant activity of Poly(N-vinyl-2- pyrrolidone-co-acrylic acid) [P(VP-co-AA)] and the potential use of P(VP-coAA)-peptide based vaccine prototypes for future melanoma cancer vaccine formulations.

The effect of polyanhydride chemistry in particle-based cancer vaccines on the magnitude of the anti-tumor immune response.

Compared to soluble cancer vaccine formulations, tumor antigens encapsulated in biodegradable polymeric particles have been shown to sustain antigen release and provide long-term protection against tumor challenge by improving the immune response towards the antigen. Treatment of mice with cancer vaccines based on different polyanhydride copolymers encapsulating OVA resulted in stimulation of tumor-specific immune responses with different magnitudes. This clearly indicates that polyanhydride chemistry plays a substantial role in stimulating the immune response. Vaccination with 20:80 CPTEG:CPH/OVA, the most hydrophobic formulation, stimulated the strongest cellular and humoral immune responses and provided the longest survival outcome without adding any other adjuvant. The most important finding in this study is that the copolymer composition of polyanhydride particle-based vaccines can have a direct effect on the magnitude of the antitumor immune response and should be selected carefully in order to achieve optimal cancer vaccine efficacy.

Abstract A031: Cancer vaccine formulation dictates synergy with CTLA-4 and PD-L1 checkpoint blockade therapy

Abstract Introduction: Therapeutic blockade of the T cell checkpoint receptors, CTLA-4 and PD-1, can cure some patients with metastatic cancer. Primary resistance to checkpoint blockade therapy is likely due to insufficient spontaneous anti-tumor immunity, and might be overcome by tumor-specific vaccination. However, the same 676-patient landmark study1 that led to FDA approval of anti-CTLA-4 for patients with melanoma showed no added benefit to anti-CTLA-4 monotherapy of concurrent vaccination with gp100 peptide in Incomplete Freund's Adjuvant (IFA), which instead significantly decreased overall response rate and disease control rate through an unknown mechanism1. Experimental Procedure: To understand the parameters that control synergy between checkpoint blockade and anti-cancer vaccination, we modelled vaccination with gp100 peptide in IFA and anti-CTLA-4 therapy in the standard treatment model of established subcutaneous B16 melanoma2. To correct for the fact that B16 melanoma progresses so rapidly that there is no time for multiple cycles of gp100 vaccination as was given to the melanoma patients, we adoptively transferred naive TCR-transgenic pmel-1 CD8+ T cells that specifically recognize the hgp10025-33 epitope. Results: Paralleling what was observed in patients1, gp100/IFA vaccination did not enhance, but significantly decreased, the therapeutic efficacy of anti-CTLA-4 therapy, even though we found high levels of gp100-specific pmel-1 T cells in the circulation. Anti-CTLA-4 monotherapy increased intratumoral localization of Tyrosinase-related protein-2 (TRP-2), p15E and gp100 melanoma antigen-specific CD8+ effector T cells (Teff), while gp100/IFA vaccination-induced, gp100-specific CD8+ Teff accumulated at the inflamed vaccination site. Combination of gp100/IFA vaccination and anti-CTLA-4 therapy caused TRP-2, p15E and gp100-specific Teff to similarly redistribute to the gp100/IFA vaccination site and away from the tumor site. This T cell redistribution was accompanied by reduced tumor control and was mediated by IFN-γ, CXCR3 and ICAM-1. At vaccination sites, ICAM-1 and VCAM-1 expression lacked clear association with the vasculature, and instead was abundant on SSChiCD11bhiLy6GloLy6ChiF4-80+CCR2+ (inflammatory) monocytes. Inflammatory monocytes infiltrating were accompanied by CD8+ Teff recruitment; and, conversely, when CD8+ Teff level were low so were inflammatory monocytes, indicating CCR2/CXCR3 positive feedback loop between CD8+ Teff and inflammatory monocytes resulting in their accumulation at the vaccination site, and consequent local skin inflammation. Non-persistent vaccine formulations do not induce these undesirable effects and potently synergize with anti-CTLA-4 and anti-PD-L1 checkpoint blockade, resulting in markedly increased anti-tumor activity. In a challenging setting of 7-day established tumors where dual checkpoint blockade cured only 10% of the mice, addition of non-persistent Vesicular Stomatitis Virus encoding gp100 (VSV.gp100) resulted in 67% cure (p < 0.0001, >200d). Correspondently, dual checkpoint blockade with gp100/IFA vaccination did not cure any mice. Conclusions: Overall, our results indicate persistent vaccine formulations can fail to increase, or even diminish, the efficacy of CTLA-4 and PD-L1 checkpoint-based cancer therapy through divergent trafficking of checkpoint blockade-induced Teff to the vaccination site. Non-persistent vaccine formulations do not induce these undesirable effects and potently synergize with anti-CTLA-4 and anti-PD-L1 checkpoint blockade, resulting in markedly increased anti-tumor activity. 1. Hodi, F.S., et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363, 711-723 (2010). 2. Fu, T., He, Q. & Sharma, P. The ICOS/ICOSL pathway is required for optimal antitumor responses mediated by anti-CTLA-4 therapy. Cancer Res 71, 5445-5454 (2011). Citation Format: Yared Hailemichael, Tihui Fu, Amber Woods, Jason Roszik, Kimberly S. Schluns, Victor H. Engelhard, Padmanee Sharma, Willem W. Overwijk. Cancer vaccine formulation dictates synergy with CTLA-4 and PD-L1 checkpoint blockade therapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A031.

Enhanced stimulation of anti-breast cancer T cells responses by dendritic cells loaded with poly lactic-co-glycolic acid (PLGA) nanoparticle encapsulated tumor antigens.

BackgroundDeveloping safe and effective cancer vaccine formulations is a primary focus in the field of cancer immunotherapy. Dendritic cells (DC) are currently employed as cellular vaccine in clinical trials of tumor immunotherapy. Recognizing the critical role of DCs in initiating anti-tumor immunity has resulted in the development of several strategies that target vaccine antigens to DCs to trigger anti-tumor T cell responses. To increase the efficiency of antigen delivery systems for anti-tumor vaccines, encapsulation of tumor-associated antigens in polymer nanoparticles (NPs) has been established.MethodsIn this study, the effect of tumor lysate antigen obtained from three stage III breast cancer tissues encapsulated within PLGA NPs to enhance the DC maturation was investigated. The T-cell immune response activation was then fallowed up. Fresh breast tumors were initially used to generate tumor lysate antigens containing poly lactic-co-glycolic acid (PLGA) NP. The encapsulation efficiency and release kinetics were profiled. The efficiency of encapsulation was measured using Bradford protein assays measuring the dissolved NPs. The stability of released antigen from NPs was verified using SDS-PAGE. To evaluate the hypothesis that NPs enhances antigen presentation, including soluble tumor lysate, tumor lysate containing NPs and control NPs the efficiency of NP-mediated tumor lysate delivery to DCs was evaluated by assessing CD3+ T-cell stimulation after T cell/and DCs co-culture.ResultsThe rate of encapsulation was increased by enhancing the antigen concentration of tumor lysate. However, increasing the antigen concentration diminished the encapsulation efficiency. In addition, higher initial protein contenting NPs led to a greater cumulative release. All three patients released variable amounts of IFN-γ, IL-10, IL-12 and IL-4 in response to re-stimulation. T cells stimulated with lysate-pulsed DCs induced a substantial increase in IFN-γ and IL-12 production. We demonstrated that NPs containing tumor lysate can induce maturation and activation of DCs, as antigen alone does.ConclusionPLGA-NPs are attractive vehicles for protein antigen delivery which effectively induce stimulation and maturation of DCs, allowing not only an enhanced antigen processing and immunogenicity or improved antigen stability, but also the targeted delivery and slow release of antigens.

Combination strategies to enhance the potency of monocyte-derived dendritic cell-based cancer vaccines.

Dendritic cells (DCs) are potent inducers of adaptive immunity and their clinical use in cancer vaccine formulations remains an area of active translational and clinical investigation. Although cancer vaccines applied as monotherapies have had a modest history of clinical success, there is great enthusiasm for novel therapeutic strategies combining DC-based cancer vaccines with agents that 'normalize' immune function in the tumor microenvironment (TME). Broadly, these combination vaccines are designed to antagonize/remove immunosuppressive networks within the TME that serve to limit the antitumor action of vaccine-induced T cells and/or to condition the TME to facilitate the recruitment and optimal function and durability of vaccine-induced T cells. Such combination regimens are expected to dramatically enhance the clinical potency of DC-based cancer vaccine platforms.

Effect of cancer vaccine formulation on synergy with anti-CTLA-4 and anti-PD-L1 checkpoint blockade therapy of cancer.

3094Background: A promising avenue to increase the efficacy of anti-CTLA-4 and anti-PD-(L)1 checkpoint blockade therapies is to enhance T cell induction through vaccination. Surprisingly, concurren...

IL-36 is required for the induction of therapeutic immunity by Tbet gene-modified dendritic cells. (TUM10P.1024)

Abstract Our lab has developed a dendritic cell (DC) vaccine as a means of overcoming immune dysfunction in the tumor microenvironment (TME) and generating a therapeutic anti-tumor immune response. As protective Type 1 immune responses are characterized by intrinsic expression of the Tbet transactivator in T cells and DC, we engineered DC with Tbet cDNA (ie, DC.Tbet) and injected these cells into the TME of syngeneic tumor-bearing mice as a therapy. Treatment of MCA205 sarcomas with DC.Tbet promoted the rapid (hours) and sustained (days-weeks) infiltration of Type 1-polarized CD4+ and CD8+ TIL into the TME and led to increased TME expression of factors characteristic of ectopic lymphoid structures (ELS), such as TNFSF14, CCL19, and CCL21. DC.Tbet cells revealed a 60-fold increase in the level of IL-36gamma mRNA over mock-transduced DC and inhibition of signaling through the IL-36 receptor (IL-36R) using natural antagonist IL-1F5 abrogated the ability of DC.Tbet therapy to inhibit tumor growth in vivo, concurrent with reduced recruitment of therapy-induced CD4+ and CD8+ TIL. The efficacy of DC.Tbet therapy was similarly negated in MCA205-bearing IL-36R-/- hosts. These data suggest a mechanism whereby treatment with DC.Tbet leads to recruitment of anti-tumor TIL via an IL-36R signaling-dependent mechanism that supports the development of ELS in the TME. These findings foreshadow the clinical use of IL-36gamma in cancer vaccine formulations as a means to promote therapeutic immunity.

Polymeric nanoparticles for co-delivery of synthetic long peptide antigen and poly IC as therapeutic cancer vaccine formulation

The aim of the current study was to develop a cancer vaccine formulation for treatment of human papillomavirus (HPV)-induced malignancies. Synthetic long peptides (SLPs) derived from HPV16 E6 and E7 oncoproteins have been used for therapeutic vaccination in clinical trials with promising results. In preclinical and clinical studies adjuvants based on mineral oils (such as incomplete Freund's adjuvant (IFA) and Montanide) are used to create a sustained release depot at the injection site. While the depot effect of mineral oils is important for induction of robust immune responses, their administration is accompanied with severe adverse and long lasting side effects. In order to develop an alternative for IFA family of adjuvants, polymeric nanoparticles (NPs) based on hydrophilic polyester (poly(d,l lactic-co-hydroxymethyl glycolic acid) (pLHMGA)) were prepared. These NPs were loaded with a synthetic long peptide (SLP) derived from HPV16 E7 oncoprotein and a toll like receptor 3 (TLR3) ligand (poly IC) by double emulsion solvent evaporation technique. The therapeutic efficacy of the nanoparticulate formulations was compared to that of HPV SLP+poly IC formulated in IFA. Encapsulation of HPV SLP antigen in NPs substantially enhanced the population of HPV-specific CD8+ T cells when combined with poly IC either co-encapsulated with the antigen or in its soluble form. The therapeutic efficacy of NPs containing poly IC in tumor eradication was equivalent to that of the IFA formulation. Importantly, administration of pLHMGA nanoparticles was not associated with adverse effects and therefore these biodegradable nanoparticles are excellent substitutes for IFA in cancer vaccines.

Cationic liposomes loaded with a synthetic long peptide and poly(I:C): a defined adjuvanted vaccine for induction of antigen-specific T cell cytotoxicity.

For effective cancer immunotherapy by vaccination, co-delivery of tumour antigens and adjuvants to dendritic cells and subsequent activation of antigen-specific cytotoxic T cells (CTLs) is crucial. In this study, a synthetic long peptide (SLP) harbouring the model CTL epitope SIINFEKL was encapsulated with the TLR3 ligand poly(inosinic-polycytidylic acid) (poly(I:C)) in cationic liposomes consisting of DOTAP and DOPC. The obtained particles were down-sized to about 140 nm (measured by dynamic light scattering) and had a positive zeta-potential of about 26 mV (according to laser Doppler electrophoresis). SLP loading efficiency was about 40% as determined by HPLC. Poly(I:C) loading efficiency was about 50%, as assessed from the fluorescence intensity of fluorescently labelled poly(I:C). Immunogenicity of the liposomal SLP vaccine was evaluated in vitro by its capacity to activate dendritic cells (DCs) and present the processed SLP to SIINFEKL-specific T cells. The effectiveness of the vaccine to activate CD8(+) T cells was analysed in vivo after intradermal and subcutaneous immunisation in mice, by measuring antigen-specific T cells in blood and spleens and assessing their functionality by cytokine production and in vivo cytotoxicity. The liposomal formulation efficiently delivered the SLP to DCs in vitro and induced a functional CD8(+) T cell immune response in vivo to the CTL epitope present in the SLP. The SLP-specific CD8(+) T cell frequency induced by the poly(I:C)-adjuvanted liposomal SLP formulation showed an at least 25 fold increase over the T cell frequency induced by the poly(I:C)-adjuvanted soluble SLP. In conclusion, cationic liposomes loaded with SLP and poly(I:C) have potential as a powerful therapeutic cancer vaccine formulation.

Optimization of a Method to Prepare Liposomes Containing HER2/Neu- Derived Peptide as a Vaccine Delivery System for Breast Cancer.

The purpose of this study was to optimize a method for the encapsulation of P5 peptide, a new designed peptide containing MHC class I epitopes from rat HER2/neu protein, into liposomes as an approach for breast cancer vaccine formulation. The efficiency of liposomal encapsulation of peptides is generally low and development of an optimized method to increase encapsulation efficiency is a big challenge. In this study, P5 peptide was encapsulated into liposomes using the following three different methods based on film-hydration procedure. In method A, the lipid film containing P5 was hydrated using buffer and then extruded to 100 nm using polycarbonate filter. In method B all the steps were the same as method A, except that the lipid film was hydrated in buffer containing 10% (v/v) of DMSO and P5 peptide. In method C, P5 peptide was added to preformed liposomes (40 mM) in the presence of ethanol (30% v/v) and incubated at 25 ºC for 1h. The highest peptide encapsulation efficiency was achieved using method C (44%). The presence of P5 peptide in purified liposomes was also confirmed using SDS- PAGE analysis. Investigation on the effects of procedure parameters of method C on encapsulation efficiency demonstrated that method is an optimized procedure for encapsulating P5 peptide. Maximal recovery from liposomes for the accurate quantification of peptide was discovered using acidified isopropanol at 1:2 of sample to solvent ratio (v/v). In conclusion, the optimal methods of encapsulation and peptide content determination in liposomes can accelerate the development of liposomal vaccine formulations.

Abstract LB-157: IL-12p70 producing dendritic cell vaccine elicits Tc1 polarized T cells and extends time to progression in metastatic melanoma.

Abstract Systemic administration of IL-12p70 has demonstrated clinical activity in cancer patients but dose-limiting toxicities have hindered its incorporation in vaccine formulations. In this proof-of-concept phase I clinical trial (NCT00683670), we report on the immunological and clinical outcomes upon vaccination of newly diagnosed stage IV melanoma patients with CD40L/IFN-γ matured IL-12p70 producing dendritic cells (mDC). HLA-A*0201+ individuals with treatment naïve metastatic melanoma were vaccinated with mDC pulsed with melanoma gp100-derived peptides, every 3 weeks by intravenous infusion for six doses, after a single dose of cyclophosphamide (300 mg/m2 iv). CT imaging was performed at baseline, week 9 and 18 for clinical assessment using RECIST. PBMC were taken weekly for immune monitoring by tetramer analysis and functional assays. Six of seven treated patients develop sustained T cell immunity to all three melanoma gp100 antigen-derived (G154, G209-2M and G280-9V) peptides, while one patient had transient immunity only to the G209-2M peptide. Three of the six immunological responders developed a radiographic response by RECIST criteria and all three individuals had a time to progression (TTP) >11.5 mo. A Cox regression analysis followed by likelihood-ratio test revealed a positive correlation (p=0.0198) between DC derived IL-12p70 and TTP. Moreover, among clinical responders, vaccine-induced gp100-specific T cells displayed a Tc1 phenotype. In contrast, a selective defect in IL-12p35 transcription was identified in clinical non-responder patient DC and gp100-specific T cells from these patients displayed the Tc2 phenotype. Incorporation of TLR3 and TLR8 agonists into the CD40L/IFN-γ maturation protocol corrected the IL-12p70 production defect in DC derived from clinical non-responder patients. These findings underscore the essential role of IL-12p70 in the development of type-1 immunity in humans with cancer and provide evidence-based rationale for incorporating IL-12p70 into the next generation of cancer vaccine formulations. Citation Format: Beatriz M. Carreno, Michelle Becker-Hapak, Alexander Huang, Megan Chan, Amer Alyasiry, Wen-Rong Lie, Rebecca L. Aft, Lynn A. Cornelius, Katherine M. Trinkaus, Gerald P. Linette. IL-12p70 producing dendritic cell vaccine elicits Tc1 polarized T cells and extends time to progression in metastatic melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-157. doi:10.1158/1538-7445.AM2013-LB-157