Induction of Anti-Tumour Immune Responses by Dendritic Cells Generated with Flt3-Ligand

<p>Dendritic cells (DCs) are potent antigen presenting cells that are crucial for the initiation of an immune response. Due to this property, DCs have been used as the basis of cancer vaccines in immunotherapy. In clinical trials, DCs used for vaccination are commonly generated by culturing monocytes from each patients' blood with the growth factors GM-CSF and IL-4 (GMCSF/IL-4 DCs). The DCs generated are reportedly similar to those that arise in vivo during inflammation and trials using these DCs have been met with some success. A recently developed method of generating mouse or human DCs in vitro, involves the culture of bone marrow (BM) precursors with the cytokine Flt3-Ligand (Flt3L-DCs). Flt3L-DCs differ substantially in phenotype from GMCSF/IL-4 DCs and more closely resemble steady-state DCs in vivo. This thesis investigated the suitability of Flt3L-DCs for cancer immunotherapy. Murine BM cells cultured in Flt3L generated three DC subsets. These consisted of plasmacytoid DCs (pDCs) that were CD11c⁺B220⁺, and conventional DCs (cDCs) that were CD11c⁺B220⁻ and could be further subdivided into CD11bhigh and CD24high populations. We observed that cDCs responded to stimulation with a variety of Tolllike receptor (TLR) agonists, as evaluated by the up-regulation of activation markers. However pDCs responded to the agonist CpG at a higher extent compared to all other agonists used. In addition, combining TLR agonists could further enhance the activation of Flt3L-DCs. Among all combinations tested, Pam3Cys/Poly I:C was the most optimal at inducing the secretion of inflammatory cytokines IL-12p70 and TNF-α. Furthermore, Pam3Cys/Poly I:C stimulated Flt3L-cDCs exhibited a greater ability at inducing CD4⁺ T cell proliferation and cross-presentation of soluble antigen to CD8⁺ T cells, compared to Flt3L-cDCs activated with the respective individual agonists. Studies have shown that GM-CSF DCs are highly reliant on glycolytic metabolism during activation in order to up-regulate activation markers. Therefore, we also characterised Flt3L-cDCs for their ability to up-regulate activation markers following stimulation with the agonist LPS and treatment with the glycolysis inhibitor 2-Deoxy-D-glucose (2-DG). In line with previous reports, DCs generated in culture with GMCSF/IL-4 were unable to up-regulate activation markers at all the 2-DG concentrations used. In contrast, Flt3L-cDCs appeared to have a threshold level where only high concentrations of 2-DG inhibited their ability to up-regulate activation markers. This result indicates that steady-state and inflammatory DCs preferentially use different metabolic pathways upon activation. The ability of optimally activated Flt3L-cDCs and GMCSF/IL-4 DCs to confer tumour protection was also examined. While unstimulated Flt3L-cDCs or GMCSF/IL-4 DCs could protect mice from tumour growth, vaccination with activated DCs from either population was required for complete tumour protection. Furthermore, we found that even in optimal conditions of activation, 1x10⁵ Flt3LcDCs were required for maximal tumour protection, whereas 1x10⁴ GMCSF/IL-4 DCs provided sufficient protection. These findings indicate that Flt3L-cDCs can be used as the basis of a therapeutic cancer vaccine, but are not superior to GMCSF/IL- 4 DCs. Further studies are required to establish conditions that can enhance the efficacy of Flt3L-cDCs.</p>

Induction of Anti-Tumour Immune Responses by Dendritic Cells Generated with Flt3-Ligand

<p>Dendritic cells (DCs) are potent antigen presenting cells that are crucial for the initiation of an immune response. Due to this property, DCs have been used as the basis of cancer vaccines in immunotherapy. In clinical trials, DCs used for vaccination are commonly generated by culturing monocytes from each patients' blood with the growth factors GM-CSF and IL-4 (GMCSF/IL-4 DCs). The DCs generated are reportedly similar to those that arise in vivo during inflammation and trials using these DCs have been met with some success. A recently developed method of generating mouse or human DCs in vitro, involves the culture of bone marrow (BM) precursors with the cytokine Flt3-Ligand (Flt3L-DCs). Flt3L-DCs differ substantially in phenotype from GMCSF/IL-4 DCs and more closely resemble steady-state DCs in vivo. This thesis investigated the suitability of Flt3L-DCs for cancer immunotherapy. Murine BM cells cultured in Flt3L generated three DC subsets. These consisted of plasmacytoid DCs (pDCs) that were CD11c⁺B220⁺, and conventional DCs (cDCs) that were CD11c⁺B220⁻ and could be further subdivided into CD11bhigh and CD24high populations. We observed that cDCs responded to stimulation with a variety of Tolllike receptor (TLR) agonists, as evaluated by the up-regulation of activation markers. However pDCs responded to the agonist CpG at a higher extent compared to all other agonists used. In addition, combining TLR agonists could further enhance the activation of Flt3L-DCs. Among all combinations tested, Pam3Cys/Poly I:C was the most optimal at inducing the secretion of inflammatory cytokines IL-12p70 and TNF-α. Furthermore, Pam3Cys/Poly I:C stimulated Flt3L-cDCs exhibited a greater ability at inducing CD4⁺ T cell proliferation and cross-presentation of soluble antigen to CD8⁺ T cells, compared to Flt3L-cDCs activated with the respective individual agonists. Studies have shown that GM-CSF DCs are highly reliant on glycolytic metabolism during activation in order to up-regulate activation markers. Therefore, we also characterised Flt3L-cDCs for their ability to up-regulate activation markers following stimulation with the agonist LPS and treatment with the glycolysis inhibitor 2-Deoxy-D-glucose (2-DG). In line with previous reports, DCs generated in culture with GMCSF/IL-4 were unable to up-regulate activation markers at all the 2-DG concentrations used. In contrast, Flt3L-cDCs appeared to have a threshold level where only high concentrations of 2-DG inhibited their ability to up-regulate activation markers. This result indicates that steady-state and inflammatory DCs preferentially use different metabolic pathways upon activation. The ability of optimally activated Flt3L-cDCs and GMCSF/IL-4 DCs to confer tumour protection was also examined. While unstimulated Flt3L-cDCs or GMCSF/IL-4 DCs could protect mice from tumour growth, vaccination with activated DCs from either population was required for complete tumour protection. Furthermore, we found that even in optimal conditions of activation, 1x10⁵ Flt3LcDCs were required for maximal tumour protection, whereas 1x10⁴ GMCSF/IL-4 DCs provided sufficient protection. These findings indicate that Flt3L-cDCs can be used as the basis of a therapeutic cancer vaccine, but are not superior to GMCSF/IL- 4 DCs. Further studies are required to establish conditions that can enhance the efficacy of Flt3L-cDCs.</p>

A Novel Combined Conjugate Therapeutic Cancer Vaccine, Recombinant EGF-CRM197, in Patients With Advanced Solid Tumors: A Phase I Clinical Study

IntroductionThe therapeutic cancer vaccine recombinant Epidermal Growth Factor (EGF)-CRM197 is a novel combined conjugate EGF with CRM197 as a carrier protein. Immunization with the EGF-CRM197 vaccine can induce high levels of neutralizing anti-EGF antibodies that inhibit EGF/EGFR signaling and thereby suppress growth of tumors that rely on this signaling pathway. Herein, we characterize the humoral immune responses elicited by the recombinant EGF-CRM197 vaccine in patients with advanced solid tumors in a phase I clinical trial and assess the safety, tolerability, and immunogenicity of this vaccine (CTR20190473).MethodsA total of 16 subjects were enrolled in this study. Under 6 + 3 design, patients in each dosing cohort were administrated subcutaneously at a dosage of 0.4 mg, 0.8 mg, and 1.6 mg, respectively. The patients received vaccinations for immune induction (once a week for 4 consecutive weeks) and booster vaccinations (once every 4 weeks). Safety evaluation was performed 1 week after the immune induction. Booster vaccination was given until the occurrence of disease progression, intolerance, withdrawal of informed consent by the patient, or negative result of anti-EGF test after two booster vaccinations.ResultsVaccination with EGF-CRM197 is safe and well-tolerated in patients with advanced solid tumors. Adverse reactions at the injection site were the most common adverse events (AEs) in recipients. No severe adverse reactions post vaccination were observed in the present study. Vaccinated patients developed a robust neutralizing antibody response triggered by EGF-CRM197 that significantly reduced the levels of EGF in serum. For lung cancer patients who were super good antibody responders (sGAR) to EGF-CRM197, the median progress-free survival (PFS) was 4.83 months, significantly longer than that of the good antibody responder (GAR) patients with lung cancer whose median PFS was 2.10 months (P=0.0018). The median overall survival (OS) of GAR lung cancer patients was 10.67 months while the OS) for sGAR lung cancer patients was not reached until analysis was performed. The median follow-up of the sGAR lung cancer patients was 14.6 months.ConclusionOur study demonstrates that the recombinant EGF-CRM197 therapeutic cancer vaccine can induce a good immune response in patients with advanced solid tumors and is safe and well tolerated, which ensures further clinical development of the vaccine for extending the survival time of EGF-CRM197 sensitive patients with advanced solid tumors.Clinical Trial Registrationhttp://www.chinadrugtrials.org.cn, identifier CTR20190473, EGF-CRM197.

860 The anti-tumor activity of HSP-90 therapeutic cancer vaccine (AST-021p) combine with TLR2/3 agonist in a MMTV-neu transgenic model

BackgroundAST-021p, which is derived from HLA class II binding epitopes of human HSP90 protein, is an investigational therapeutic cancer vaccine for the malignant neoplasms. AST-021p is designed to demonstrate the immunologic efficacy by activating antigen-specific CD4+ Th1 cell in humans. Due to their ability to link the innate with the adaptive immune response, Toll-like receptor (TLR) agonists are highly promising as adjuvants in vaccines against life-threatening and complex diseases such as cancer, AIDS and malaria. In this study, AST-021p was investigated to evaluate the immunogenicity and tumor growth inhibitory effect under the condition of combining with various immune adjuvants derived from TLR agonists, using in-vivo model.MethodsThree different agonists of TLR (TLR-4, TLR-2/3, TLR-7/8) were assigned to investigate the immunogenicity in each group (4 FVB mice/group, total 4 groups). AST-021p was intradermally injected 3 times with different TLR-agonists and the immunogenicity was assessed from mouse splenocyte by HSP90-specific IFN-γ ELISpot method. We also examined the efficacy of AST-021p and selected TLR-agonist in MMTVneu Tg mice (4 mice/group, conducted twice and A total 8 mice was assigned to each group). The combination of AST-021p and TLR-2/3 agonist (AST-021p plus TLR-2/3 agonist) was injected 3 times every 10 days to mice followed by inoculated mouse mammary cancer cell line. The tumor volume change and immunogenicity were evaluated.ResultsThe most effective TLR-agonist as a potent immune adjuvant was a TLR-2/3 agonist (L-pampoTM, supplied by CHA Vaccine Institute). In MMTV-Neu transgenic mice, AST-021p (100 μg) plus TLR-2/3 agonist significantly enhanced immunogenicity by increasing up to 130±10 HSP-90 epitope specific T cells per 1x105 splenocytes (P<0.001). AST-021p plus TLR-2/3 agonist also showed higher tumor growth inhibitory effect (170±108 mm3) on post-implantation 35th day by suppressing mouse mammary cancer cell line (5x105)-derived tumor growth, compared with a TLR-2/3 agonist alone (1031±450 mm3).ConclusionsCombination regimen of AST-021p and TLR-2/3 agonist (as immune adjuvant) demonstrated significant immunogenicity and tumor prevention effect in in-vivo study. These data supported the clinical study of AST-021p combined with TLR-2/3 agonist as active immune adjuvant in certain tumor types, and phase 1/2 clinical program would be expected to be initiated.AcknowledgementsNot applicableTrial RegistrationNot applicableReferencesCsermely P, Schnaider T, Soti C, Prohaszka Z, Nardai G. The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol Ther 1998;79,129–168.Wang H, Lu M, Yao M, Zhu W. Effects of treatment with an Hsp90 inhibitor in tumors based on 15 phase II clinical trials. Mol Clin Oncol 2016;5,326–334.Ramalingam S, Goss G, Rosell R. Schmid-Bindert G, Zaric B, Andric Z, Bondarenko I, Komov D, Ceric T, Khuri F. A randomized phase II study of ganetespib, a heat shock protein 90 inhibitor, in combination with docetaxel in second-line therapy of advanced non-small cell lung cancer (GALAXY-1). Ann Oncol Off J Eur Soc Med Oncol 2015,26,1741–1748.Ethics ApprovalAll experimental procedures involving mice were performed with the guidance protocols approved by the Institutional Animal Care and Use Committee of Korea University (IACUC, Approval number: KOREA-2019-129)ConsentIt is not an abstract containing sensitive or identifiable information.

776 The anti-tumor activity of HER-2/neu ICD therapeutic cancer vaccine (AST-301, pNGVL3-hICD) in Her2-expressed gastric cancer xenograft model

BackgroundThe HER-2/neu, potent oncogenic protein, has several characteristics that make it a good antigen to serve as the model for developing pDNA-based tumor vaccine strategies.1–3 The observation that immunity co-exists with antigen-positive cancer cells indicates that HER-2 is immunogenic. AST-301 (pNGVL3-hICD) is a plasmid DNA-based therapeutic cancer vaccine encoding HER2 ICD sequence. The clinical efficacy and safety of AST-301 were already proven in HER2-positive breast cancer population, and long-term immunogenicity and survival were demonstrated well via phase 1 study (PN 109, NCT00436254) In this in-vivo study, AST-301 was investigated to evaluate the efficacy in HER2/neu-expressed gastric cancer xenograft model.MethodsA HER2-expressed gastric cancer xenograft model was established with NCI-N87 cell line inoculation in athymic-nude mice. Treatment groups were assigned as AST-301 alone (AST-301, 0.1 mg/head, i.d.), Trastuzumab (TZM, 20 mg/kg, i.p.) or AST-301 combining with Trastuzumab (AST-301+TZM) respectively. To evaluate tumor protective effect of drugs, mice were immunized 3 times in every week. Immunization of AST-301 or AST-301+TZM was completed, followed by tumor cell line inoculation. In another study to verify the anti-tumor effect of them, the administration of drugs was started when the tumor volumes reached approximately 150 mm3. AST-301 was immunized 3 times in every week to post-implantation 32nd day and TZM was injected 5 times per week. The tumor volumes were estimated and the percentage of tumor growth inhibition was calculated.ResultsIn our two in vivo efficacy studies, there was no significant specific safety issue in all groups. Tumor protective effect was observed in AST-301 group (1506.7±1603.0 mm3) compared with control group (GM-CSF, 1266.3±862.5 mm3) as an immune adjuvant (figure 1). However, AST-301+TZM group (1533.0±1186.3 mm3) did not show the tumor protective effect. The groups of AST-301 and AST-301+TZM were significantly higher to the anti-tumor activity than control group, and AST-301 was more effective than AST-301+TZM or TZM alone. On day 33 (32 days after starting treatment), tumor growth inhibition rate were 38.3 % (2503.4±1034.6 mm3), 10.9 % (1545.0±599.9 mm3) and 23.6 % (1912.9±97.1 mm3) in groups of AST-301, TZM and AST-301+TZM compared with control group, respectively.ConclusionsTumor protective and tumor therapeutic effect of AST-301 were demonstrated well in various doses and regimens on HER2/neu positive gastric cancer xenograft model. These data would be supporting a proof of concept (PoC) clinical study of HER-2/neu ICD therapeutic cancer vaccine in certain type of HER2/neu-expressed gastric cancer patient.ReferencesDisis ML. Enhancing cancer vaccine efficacy via modulation of the tumor microenvironment. Clin Cancer Res 2009;15:6476–6478.Disis ML, Wallace DR, Gooley TA, et al. Concurrent trastuzumab and HER2/neu-specific vaccination vaccination in patients with metastatic breast cancer. J Clin Oncol 2009;27:4685–4692.Disis ML, Schiffman K, Guthrie K, et al. Effect of dose on immune response in patients vaccinated with an her-2/neu intracellular domain protein–based vaccine. J Clin Oncol 2004;22:1916–1925.Ethics ApprovalThis experiment was conducted ethically with the approval of the Institutional Animal Care and Use Committee (KBIO-IACUC-2021-038) in the Osong Medical Innovation Foundation Experimental Animal Center.ConsentN/AAbstract 776 Figure 1Tumor protective and tumor therapeutic effect of AST-301 on HER2/neu positive gastric cancer xenograft model

Development of the T-ALLiPSC-based Therapeutic Cancer Vaccines for T-cell acute Lymphoblastic Leukemia

PurposeThe T-cell acute lymphoblastic leukemia (T-ALL) is a kind of hematological malignancy in children. Despite the significant improvement in the cure rate of T-ALL upon treatment with chemotherapy regimens, steroids, and allotransplantation there are relapses. This study focuses on the tumor-specific therapeutic vaccines derived from the induced pluripotent stem cells (iPSC) to address the issue of T-ALL recurrence.MethodsPatient-derived tumor cells were reprogrammed into the iPSCs and the RNA-seq data of the T-ALL-iPSCs and H-iPSCs were analyzed. In vitro, the whole cell lysate antigens of iPSCs were prepared to induce the dendritic cells (DC) maturation, which in turn stimulated the tumor-specific T cells to kill the T-ALL tumor cells (Jurkat, CCRF-CEM, MOLT-4).ResultsBoth T-ALL-iPSCs and H-iPSCs were highly related to the tumor-related genes. The transcriptome analysis showed the T-ALL-iPSCs to be similar to the T-ALL tumor cells. The cytotoxic T lymphocyte (CTL) stimulated by the DC-loaded T-ALL-iPSC-derived antigens showed specific cytotoxicity against the T-ALL cells in vitro.ConclusionsThe T-ALL-iPSC-based therapeutic cancer vaccine can elicit a specific anti-tumor effect on T-ALL.

Protective and Therapeutic Effects of an IL-15:IL-15Rα-Secreting Cell-Based Cancer Vaccine Using a Baculovirus System

This study reports the use of the BacMam system to deliver and express self-assembling IL-15 and IL-15Rα genes to murine B16F10 melanoma and CT26 colon cancer cells. BacMam-based IL-15 and IL-15Rα were well-expressed and assembled to form the biologically functional IL-15:IL-15Rα complex. Immunization with this IL-15:IL-15Rα cancer vaccine delayed tumor growth in mice by inducing effector memory CD4+ and CD8+ cells and effector NK cells which are tumor-infiltrating. It caused strong antitumor immune responses of CD8+ effector cells in a tumor-antigen specific manner both in vitro and in vivo and significantly attenuated Treg cells which a control virus-infected cancer vaccine could induce. Post-treatment with this cancer vaccine after a live cancer cell injection also prominently delayed the growth of the tumor. Collectively, we demonstrate a vaccine platform consisting of BacMam virus-infected B16F10 or CT26 cancer cells that secrete IL-15:IL-15Rα. This study is the first demonstration of a functionally competent soluble IL-15:IL-15Rα complex-related cancer vaccine using a baculovirus system and advocates that the BacMam system can be used as a secure and rapid method of producing a protective and therapeutic cancer vaccine.