Gene-modified Tumor Cells Research Articles

Abstract 6753: Bria-OTS immunotherapy platform: Harnessing gene-modified tumor cells to reinvigorate the cancer immunity cycle for precision anti-tumor responses

Abstract Background: For an immune response to lead to an effective anti-tumor response, a series of stepwise events involving a complex network of iterative cellular and molecular interactions must take place. Cancer, however, often derails this immune cycle, prompting contemporary cancer immunotherapy approaches to seek its reinstatement. Despite significant advances, the inherent disruption of the cancer immunity cycle at multiple junctures, coupled with current therapeutic strategies focusing on isolated stages within this intricate process, has left the attainment of curative therapies as an aspirational goal. We believe that targeting multiple immune processes can restore natural anti-cancer immunity and yield sustained responses. To this end, BriaCell is utilizing gene-modified tumor cells as an immunotherapeutic platform. Our initial version, SV-BR-1-GM, a breast cancer cell line expressing GM-CSF, demonstrates promising clinical outcomes by directly activating CD4+ T-cells in an antigen-specific, HLA-restricted manner. Building on our findings, we hypothesize that tumor cells not only supply antigens but also directly stimulate the immune system. Presenting our second-generation platform, we enhance antigen cross-presentation and direct immune activation through the expression of cytokines, co-stimulatory factors, and HLA alleles. Methods: To achieve this objective, we have developed Bria-OTS cell lines derived from various cancer types with the following genetic modifications: 1) Expression of costimulatory molecules (CD80, CD86, 4-1BBL, CD40) and immunomodulatory cytokines (GM-CSF, IFNα, IL-12 and IL-7). This modification aims to improve the cell's ability to present antigens and activate the host immune system. 2) Expanding HLA alleles to create semi-allogeneic cell lines matching at least one HLA allele across the population aims to enhance the host immune response, enabling recognition of tumor-associated antigens in the context of syngeneic HLA molecules Results: We generated four cell lines for each tumor type, and these cell lines secreted GM-CSF, IFNα, IL12 & IL7, while also expressing CD80, CD86, CD40, and different combinations of both Class I and Class II HLA alleles. In functional assays, our cell lines demonstrated potent antigen presentation, effectively triggering anti-tumor immune responses across diverse immune populations, including T-, NK, and NKT cells. Conclusions: The Bria-OTS cell lines will offer the following key features: 1) multimodal mechanisms of action including thwarting some mechanisms of cancer immune escape. 2) Precision Therapy; Bria-OTS will be precisely matched to individual patients based on their HLA alleles, encompassing over 99% of the U.S. population. 3) rapid, cost-effective treatment as our 'off-the-shelf' cell lines eliminate the need for personalized manufacturing. Citation Format: Miguel A. Lopez-Lago, Vikas Bhardwaj, Pravin Kesarwani, Xiaoyi Zheng, Sagarika Pachhal, Patience Cournoo, Renee Cortez, Charles L. Wiseman, William V. Williams. Bria-OTS immunotherapy platform: Harnessing gene-modified tumor cells to reinvigorate the cancer immunity cycle for precision anti-tumor responses [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 6753.

Abstract 685: Engineering semi-allogeneic whole cancer vaccines with enhanced immunogenicity for the treatment of advanced solid tumors

Abstract Background: Therapeutic cancer vaccines are designed to program a patient’s own immune system to recognize and eliminate tumor cells. We sought to harness gene-modified tumor cells as a vaccine platform and developed cancer vaccines composed of breast cancer cells expressing GM-CSF (SV-BR-1-GM). We have recently reported favorable clinical outcomes in patient populations that match SV-BR-1-GM at one or more HLA alleles. Mechanistically, SV-BR-1-GM cells can directly activate CD4+ T-cells in an antigen-specific HLA-restricted manner, as demonstrated by an in vitro antigen presentation assay (1). All these observations led us to hypothesize that semi-allogeneic cell-based cancer vaccines (partially matching host HLA genotype) would mount an enhanced immune response by forcing the host immune response to recognize tumor-associated antigens in the context of allogeneic HLA-I or II molecules or in proximity of strong non-self-antigens. Therefore, steps were taken to generate semi-allogeneic cancer cell lines expressing an extended repertoire of stimulatory molecules to induce optimal immune activation (Bria-OTS). Methods: SV-BR-1 (Breast cancer), PC-3 (prostate cancer), SK-MEL-24 (Melanoma), and NCI-H2228 (Lung cancer) were selected for vaccine development. To generate semi-allogeneic cell lines matching >99% of the population at least at 1 HLA allele, four cell lines (for each parental tumor cell line) were developed, each carrying two (2) HLA-A and two (2) HLA-DRB3/4/5 alleles, for a total of eight HLA-A and seven HLA-DRB3/4/5 alleles. We transduced selected co-stimulatory molecules and immune-modulatory cytokines. Next, the unique combinations of HLA-A and HLA-DRB3/4/5 alleles were transduced into the cells using lentiviral based vectors. Finally, functionality of the engineered cells was validated by using molecular and cellular based assays. Preliminary data: Four cell lines (for each tumor type) that secreted GM-CSF, IFNα, IL12, IL7 and expressed CD80, CD86, 4-1BBL, and different combinations of both Class I and Class II HLA alleles were selected. Using cell-based assays - including mixed lymphocyte reaction assays -, we demonstrated that the generated cells stimulate naïve T-cells. Evaluation of the cells ability to present antigens in an HLA-restricted antigen-specific manner is also planned. Discussion and conclusions: Different types of cancer vaccines have been developed with moderate success, often hampered by lack of strong immunogenicity and complex manufacturing. BriaCell’s Bria OTS provides a solution by increasing vaccine immunogenicity and decreasing manufacturing costs while personalizing the therapy based on matching the patient’s HLA type.1.Lacher MD et al, Front Immunol. 2018 May 15;9:776 Citation Format: Miguel A. Lopez-Lago, Vikas Bhardwaj, Xiaoyi Zheng, Sagarika pachhal, Renee Cortez, Charles Wiseman, William Williams. Engineering semi-allogeneic whole cancer vaccines with enhanced immunogenicity for the treatment of advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 685.

Роль факторов иммуносупрессии в прогнозе эффективности клеточной иммунотерапии у пациентов с солидными опухолями

Production of different factors with wide immunosuppressive properties by tumor cells is one of the mechanisms that support tumor growth. We think that immunosuppressive action of tumor cells should be taken into account for cellular antitumor therapy optimization. Aim of the study – to evaluate the effectiveness of cellular immunotherapy factors (ISF) for response prediction in patients receiving tag7/PGRP-S gene modified tumor cells (GMV) or autologous dendritic cells vaccine (DCV) for melanoma (Mel), kidney cancer (RCC) and soft tissue sarcoma (STS). Material and methods. We have treated 204 patients with cellular therapy since 2001 till 2016 (88 – GMV, 1 16 – DCV). ISF (MICA, TGF-β, IL-10 и VEGF) were measured in tumor tissue cultures of 88 patients (71 – Mel, 6 – RCC, 11 – STS). Median age of patients was 51 year. Patient were divided as having insufficient response (IR) or sufficient response (SR) for the analysis purpose. Results. ISF concentration in cultures of patients with IR was higher than in patients with SR (р < 0.05). SR could be predicted correctly in 74.4% using ISF. The most important factor was TGF-β1. In the Cox model TGF-β1 and MICA levels were significant predictors of overall survival for Mel patients (р < 0.05). VEGF was the most important factor for time to progression prognosis. Spirman correlations showed moderate significant correlation between ISF (р < 0.05). Conclusions. ISF concentration in cell cultures obtained from the patients with Mel, RCC or STS is important independent predictive factor for time to progression and overall survival. Our data supports rigid selection criteria based on biological properties of tumor cells for patients receiving cellular therapy for solid tumors

Long-term survival in patients (pts) with locally advanced or disseminated malignant melanoma (MM) or renal cell carcinoma (RCC), treated with tag-7 gene-modified (GMV) tumor cells: A phase II trial.

e14022 Background: Immunotherapy may produce long lasting effects on survival.Magnitude of efficacy may be depended from immune factors. We evaluated the overall survival (OS) in pts treated with GMV with biomarker analysis of immunosuppressive factors (ISF) production by their tumor cells. Methods: Since 2001 to 2014 80 pts received GMV: 68 with MM and 12 pts with RCC. Treatment was given in the metastatic setting in 61 pts (MM-51, RCC – 10) and in the adjuvant (after complete cytoreduction) setting in 19 pts (MM-17, RCC-2). Stage 3 was in 26 (33%) pts, stage 4 in 54 (67%). Pt’s tumor samples were transferred to culture, transfected with TAG7and inactivated by radiation. Produced product was injected SC every 3 weeks until progression or 2y of therapy. ISF (MICA, TGF-β1, IL-10 and VEGF) were measured in the supernatants of the tumor cell cultures and used as predictive factors. Results: The 5-yr OS in intention to treat population was 25.1%. There was no differences in MM and RCC OS (Log-rank p = 0.44). Median OS in metastatic setting was 0,7 y, in adjuvant – 3,1 y. Classification trees were built on the basis of ISF production. The median OS was 6.6 y in favorable prognosis (FP) group (MICA level ≤582 pg/ml, n = 15), 4.6 mo in unfavorable (UF) group (MICA level > 582 pg/ml, n = 12) (p < 0.0001). No significant differences were found between classification trees based on the other ISF (TGF-β1, IL-10 and VEGF). In stage 3-4 MM pts with FP median OS was 2,3 y with 31% pts alive in 10y, in UF group – 0,4 y; log-rank p = 1,94E-5. No FP pts received modern immunotherapy. Conclusions: GMV shows high results in carefully selected pts with low ISF production. Method should be further investigated in pts with FP. [Table: see text]

MTE13.02 Basic Immunology for the Clinician

MTE13.02 Basic Immunology for the Clinician

Gene expression profiling of peripheral blood mononuclear cells during treatment with a gene-modified allogeneic tumor cell vaccine in advanced renal cell cancer: tumor-induced immunosuppression and a possible role for NF-κB.

Tumor-induced immunosuppression remains a major challenge for immunotherapy of cancer patients. To further elucidate why an allogeneic gene-modified [interleukin-7 (IL-7)/CD80-cotransfected] renal cell cancer (RCC) vaccine failed to induce clinically relevant TH-1-polarized immune responses, peripheral blood mononuclear cells from enrolled study patients were analyzed by gene expression profiling (GEP) both prior and after vaccination. At baseline before vaccination, a profound downregulation of gene signatures associated with antigen presentation, immune response/T cells, cytokines/chemokines and signaling/transcription factors was observed in RCC patients as compared to healthy controls. Vaccination led to a partial reversion of preexisting immunosuppression, however, GEP indicated that an appropriate TH-1 polarization could not be achieved. Most interestingly, our results suggest that the nuclear factor-kappa B signaling pathway might be involved in the impairment of immunological responsiveness and the observed TH-2 deviation. In summary, our data suggest that GEP might be a powerful tool for the prediction of immunosuppression and the monitoring of immune responses within immunotherapy trials.

Anti-esophageal cancer active immunity induced by FasL/B7-1 gene-modified tumor cells

Anti-esophageal cancer active immunity induced by FasL/B7-1 gene-modified tumor cells

More efficient induction of antitumor T cell immunity by exosomes from CD40L gene-modified lung tumor cells

The incidence of lung cancer increases annually. However, the effects of the present methods for the treatment of lung cancer are extremely poor. It has been reported that exosomes from heat‑stressed 3LL Lewis lung tumor cells effectively elicit systemic antitumor immunity. CD40 signaling is critical in the activation of dendritic cells(DCs), which are important in the induction of antitumor immunity. In the present study, exosomes from CD40 ligand gene‑modified 3LL tumor cells (CD40L‑EXO) were identified to be more immunogenic compared with control‑EXO and lac Z-EXO. CD40L‑EXO induced a more mature phenotype of the DCs and promoted them to secrete high levels of interleukin‑12. CD40L‑EXO‑treated DCs induced a greater proliferation of allogeneic Tcells in the mixed lymphocyte reaction. Moreover, CD40L‑EXO induced robust tumor antigen‑specific CD4+ Tcell proliferation exvivo. CD40L‑EXO were also extremely effective in the protective and therapeutic antitumor tests invivo. These results indicate that CD40L‑EXO may be used as an efficient vaccine for lung cancer immunotherapy.

Antitumor effects obtained by autologous Lewis lung cancer cell vaccine engineered to secrete mouse Interleukin 27 by means of cationic liposome

Interleukin-27 (IL-27), a novel IL-6/IL-12 family cytokine, plays an important role in the early regulation of Th1 responses. The cytokine IL-27 can exert a variety of immune-regulatory functions including cytotoxic T lymphocyte (CTL), CD4+, CD8+ T lymphocytes activation and interferon-γ (IFN-γ) production. In this study, we developed an effective and gene modified tumor cell vaccine. Lewis lung cancer cell LL/2 transfected with the DOTAP:cholesterol cationic liposome could express the mouse IL-27 (mIL-27) gene at a relative high level. The resultant transfectants were then irradiated with X-ray and used as a tumor cell vaccine. This tumor cell vaccine not only contained tumor associated antigen (TAA) of LL/2 cells but also secreted mIL-27 which could induce immune response in mice. The mice vaccinated with LL/2-mIL-27 performed strong tumor inhibiting effect accompanied with a high IFN-γ production. Both CD4+ and CD8+ T lymphocytes were significantly elevated in these mice vaccinated with LL/2-mIL-27 cell vaccine. Moreover, after depletion of CD4+, CD8+ T lymphocytes by injection of antibodies against CD4 and CD8, the vaccinated mice inoculated with autologous LL/2 cells were not protected from tumor challenge. In contrast, vaccinated mice inoculated with autologous LL/2 cells were treated with antibody against natural killer (NK)cells or normal rat IgG still possessed strong antitumor activity. Our data suggested that DOTAP:cholesterol cationic liposome was quite useful in generating an autologous tumor cell vaccine and mIL-27 could be therapeutically used to potentiate the host antitumor immunity.

High Immune Response Rates and Decreased Frequencies of Regulatory T Cells in Metastatic Renal Cell Carcinoma Patients after Tumor Cell Vaccination

Our previously reported phase I clinical trial with the allogeneic gene-modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (T(H)1/T(H)2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies.

Efficacy and safety of cancer vaccine with 4-fold gene-modified allogeneic tumor cells: Results of the phase I/II ASET study in patients with advanced renal cell carcinoma.

4636 Background: MGN1601 was tested in the first-in-man phase I-II clinical study in patients with advanced renal cell carcinoma (RCC) who failed several previous therapy lines and had no further standard therapy available. MGN1601 consists of two active pharmaceutical ingredients in fixed combination: fourfold gene-modified allogeneic tumor cells expressing IL-7, GM-CSF, CD80 and CD154 through MIDGE vectors and a TLR-9 agonist, the immunomodulator dSLIM. Methods: The ASET study is a multicentric, single-arm phase I-II clinical trial. Clinical response was evaluated using CT scans (RECIST 1.1 or immune related Response Criteria, irRC). Efficacy data were evaluated in terms of PFS and OS for the intended to treat and the treated per protocol (TPP) populations, clinical parameters and quality of life. Immune response was determined using DTH to MGN1601, LTT assay, frequency and activation of blood cells, and mRNA, chemokine and cytotoxic T cells analysis as well as tumor tissue evaluation. Results: Nine of 19 included patients completed the TPP, the others discontinued the study earlier due to PD. Median PFS in the TPP group was 12 wks (3 months) and OS (not reached yet) 46 wks (11 months). Three patients achieved disease control (1 PR, 2 SD) after 12 wks. Two patients are continuing treatment in the extension phase and are progression free since 37 and 46 wks, respectively. Re-evaluation of tumor response data using irRC revealed 1 additional patient with a delayed tumor response 4 wks after treatment stop. Herewith, 4 out of 9 TPP patients (45%) achieved disease control. Of 7 patients receiving targeted therapy upon stop of study treatment, 4 had substantial objective responses, providing evidence that the study drug is able to render their tumors more vulnerable to subsequent therapies. Immune analysis showed trends towards increases of T-, NKT-cell and pDC frequencies and other immune parameters in those patients with clinical responses, indicating anti-tumor immunity of study treatment. Conclusions: The therapeutic cancer vaccine MGN1601 shows promising efficacy in late stage mRCC patients. Results warrant further clinical studies with MGN1601.

Abstract LB-233: Immune-related efficacy data of the phase 1-2 study of an with fourfold gene-modified allogeneic tumor cell based vaccine in patients with advanced renal cell carcinoma

Abstract Background. The first-in-man phase 1-2 clinical study (ASET study) of the cell-based therapeutic cancer vaccine MGN1601 included patients with advanced renal cell carcinoma (RCC) who failed previous therapies and had no further standard therapy available. MGN1601 consists of two active pharmaceutical ingredients in fixed combination: fourfold gene-modified allogeneic tumor cells expressing IL-7, GM-CSF, CD80 and CD154 through MIDGE® vectors and a TLR-9 agonist, the DNA-based immunomodulator dSLIM®. Methods. The ASET study is conducted as a multicentric, open, single-arm Phase 1-2 clinical trial. Clinical response (PD, SD, PR, CR) was evaluated using CT scans according to RECIST 1.1 criteria and immune related Response Criteria (irRC). The efficacy data were evaluated in terms of progression-free survival (PFS) and overall survival (OS) for the intended to treat (ITT) and the treated per protocol (TPP) populations of patients.Immune response was determined by the following parameters: ELISA of serum cytokines and chemokines, DTH to MGN1601, LTT assay to standard antigens, frequency and activation of plasmacytoid dendritic cells (pDC), myeloid dendritic cells (mDC), monocytes, natural killer cells (NK), NKT-, B-, and T cells. Blood mRNA and frequency of cytotoxic T cells by ELISPOT as well as tumor tissue were analyzed in a patient subgroup. Results. Nineteen patients, who had advanced stage RCC and failed up to 7 previous therapy lines (median 3 lines), were included. Nine of these patients completed the treatment phase per protocol (TPP), the others discontinued the study earlier due to PD. Currently, one patient is still in treatment phase. Median PFS in the TPP group was 12 weeks (3 months) and OS (not reached yet) 45 weeks (11 months). Three patients achieved disease control (one PR and two SD) after 12 weeks according to RECIST 1.1 criteria. Two patients are continuing treatment in the extension phase and are progression free since 37 and 46 weeks, respectively. Re-evaluation of tumor response data using irRC revealed one additional patient, who had a delayed tumor response 4 weeks after stop of treatment. Herewith, 4 out of 9 TPP patients (45%) achieved disease control. Of six patients receiving a targeted therapy following progress, four had substantial objective responses, suggesting that the study drug rendered their tumors more vulnerable to subsequent therapies. Immune analysis of the cell populations after 12 weeks showed trends towards increases of T cell, NKT-cell and pDC frequencies in those patients with clinical responses to the therapy, indicating anti-tumor immunity due to the study treatment. Conclusions. The therapeutic cancer vaccine MGN1601 shows promising efficacy in late stage mRCC patients. Results warrant further clinical studies with MGN1601. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-233. doi:1538-7445.AM2012-LB-233

Autologous Versus Allogeneic Cell-Based Vaccines?

Devitalized tumor cells either autologous or allogeneic have been used as anti-cancer vaccines with the purpose of facilitating the induction of an immune response able to destroy growing tumor cells since the identification of tumor antigens was deemed not to be necessary, particularly in the autologous system. Such vaccines were tested first in animal models and then in the clinics as unmodified tumor cells or after insertion of genes coding for factors known to increase the immune response against tumors. These vaccines were usually given by subcutaneous injections along with different immunological adjuvants. Such immunization approaches were found to be effective in mice when carried out in a tumor preventive setting but significantly less in the therapeutic context, that is, in the presence of an established tumor. By analyzing several clinical trials of vaccination using either autologous or allogeneic unmodified and gene-modified tumor cells published in the last 10 to 15 years, we conclude for a lack of sufficient evidence for efficacy of this strategy in inducing both a strong immune response and a therapeutic response. A potential variant of this strategy is the direct intratumoral injection of immunostimulatory genes delivered by vectors in vivo. But even this approach failed to provide a statistically significant clinical benefit for the cancer patients.We also point out the inherent drawbacks of the tumor cell-based vaccine strategy that include (a) a limited frequency by which human tumor lines can be obtained from clinical samples, (b) the low number of available cells for vaccination, (c) the release of immune-suppressive factors by tumor cells, and (d) the cost and time necessary for standardization and collecting/expanding a number of cells according to the approved regulatory requirements. Thus, taking into consideration the new developments in cancer vaccines, we believe that tumor cell-based vaccines should be dismissed as anti-cancer vaccines unless a clear benefit could be demonstrated by the few ongoing trials of combination with new immunomodulating reagents (eg, anti-CTLA4, PD-1, chemotherapy).

Glycosyl-phosphatidylinositol-anchored interleukin-2 expressed on tumor-derived exosomes induces anti-tumor immune response

To prepare IL-2-anchored and tumor-derived exosomes vaccine, and investigate the antitumor efficiency of the special cytotoxic T-lymphocytes induced by Ex/GPI-IL-2. To construct pEGFP-N1-IL2gpi plasmid coding a fusion gene of a DNA oligo encoding GPI-anchor signal sequence attaching to human IL-2 cDNA. Then T24 cell lines stably expressing GPI-IL-2 proteins (T24/GPI-IL-2) were established. Ex/GPI-IL-2 were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and the morphology and molecule markers were analyzed. The mixed lymphocyte reaction study and cytotoxic study were performed to determine the proliferative effect of T lymphocytes and the cytotoxicity induced by Ex/GPI-IL-2. The pEGFP-N1-IL2gpi plasmid was successfully constructed, and cell lines stably expressing GPI-IL-2 fusion proteins were established. Ex/GPI-IL-2 were small vesicular and saucer-shaped in diameter of 30-90 nm, containing heat shock protein 70, intercellular adhesion molecule-1, MAGE-1 and GPI-IL-2. Ex/GPI-IL-2-pulsed could dendritic cells induce proliferation of T cells and cytotoxic immune response more efficiently (P<0.05). GPI-IL-2 gene-modified tumor cells can make the exosomes containing GPI-IL-2 with an increased anti-tumor effect. Our study provides a feasible approach for exosome-based tumor immunotherapy of bladder transitional cell tumors.

Immuno-Therapy with Anti-CTLA4 Antibodies in Tolerized and Non-Tolerized Mouse Tumor Models

Monoclonal antibodies specific for cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA4) are a novel form of cancer immunotherapy. While preclinical studies in mouse tumor models have shown anti-tumor efficacy of anti-CTLA4 injection or expression, anti-CTLA4 treatment in patients with advanced cancers had disappointing therapeutic benefit. These discrepancies have to be addressed in more adequate pre-clinical models. We employed two tumor models. The first model is based on C57Bl/6 mice and syngeneic TC-1 tumors expressing HPV16 E6/E7. In this model, the HPV antigens are neo-antigens, against which no central tolerance exists. The second model involves mice transgenic for the proto-oncogen neu and syngeneic mouse mammary carcinoma (MMC) cells. In this model tolerance to Neu involves both central and peripheral mechanisms. Anti-CTLA4 delivery as a protein or expression from gene-modified tumor cells were therapeutically efficacious in the non-tolerized TC-1 tumor model, but had no effect in the MMC-model. We also used the two tumor models to test an immuno-gene therapy approach for anti-CTLA4. Recently, we used an approach based on hematopoietic stem cells (HSC) to deliver the relaxin gene to tumors and showed that this approach facilitates pre-existing anti-tumor T-cells to control tumor growth in the MMC tumor model. However, unexpectedly, when used for anti-CTLA4 gene delivery in this study, the HSC-based approach was therapeutically detrimental in both the TC-1 and MMC models. Anti-CTLA4 expression in these models resulted in an increase in the number of intratumoral CD1d+ NKT cells and in the expression of TGF-β1. At the same time, levels of pro-inflammatory cytokines and chemokines, which potentially can support anti-tumor T-cell responses, were lower in tumors of mice that received anti-CTLA4-HSC therapy. The differences in outcomes between the tolerized and non-tolerized models also provide a potential explanation for the low efficacy of CTLA4 blockage approaches in cancer immunotherapy trials.

82. A Phase 1-2 Clinical Study of MGN1601, a Tumor Vaccine Comprising Allogeneic, Gene-Modified and Irradiated Tumor Cells in Combination with an Immunomodulator, in Patients with Metastatic Renal Cell Carcinoma (ASET Study) – Preliminary Results

82. A Phase 1-2 Clinical Study of MGN1601, a Tumor Vaccine Comprising Allogeneic, Gene-Modified and Irradiated Tumor Cells in Combination with an Immunomodulator, in Patients with Metastatic Renal Cell Carcinoma (ASET Study) – Preliminary Results

Preliminary safety results of an ongoing phase I/II clinical study of MGN1601, a tumor vaccine comprising allogeneic, gene-modified, and irradiated tumor cells in combination with an immunomodulator in patients with metastatic renal cell carcinoma (ASET study).

392 Background: MGN1601 is a cell-based RCC tumor vaccine MGN1601 consisting of two active pharmaceutical ingredients: genetically modified allogeneic (human) cells transfected with four different MIDGE vectors encoding IL-7, GM-CSF, CD80 and CD154 and a synthetic DNA-based immunomodulator dSLIM-30L1, a TLR-9 agonist. The vaccine is being developed for treatment of patients with advanced RCC. Its prophylactic and therapeutic anti-tumor activity has been shown in several in-vivo models. A good safety profile of MGN1601 was shown in a wide program of acute and chronic toxicity studies. Based on these promising data, this phase 1/2 study was started in patients with advanced RCC. Methods: This multicentric open clinical study for the assessment of safety and efficacy of MGN1601 in patients with advanced RCC was initiated in October 2010. A total of 24 patients have to be recruited into the study. The treatment consists of 8 MGN1601 treatments administered as follows: the first 3 treatments are administered on a weekly basis, and the consecutive 5 treatments on a bi-weekly basis. The treatment dose contains 107 transfected tumor cells and 5 mg dSLIM per administration, which has been proven to be safe with a high safety margin in repeated toxicity studies. The efficacy and safety of the study treatment will be evaluated based on extensive immunological tests, radiological assessment, safety laboratory results and assessments of the quality of life. Here, the first safety data are presented. [Table: see text]

Allogeneic gene-modified tumor cells (RCC-26/IL-7/CD80) as a vaccine in patients with metastatic renal cell cancer: a clinical phase-I study

Despite novel targeted agents, prognosis of metastatic renal cell cancer (RCC) remains poor, and experimental therapeutic strategies are warranted. Transfection of tumor cells with co-stimulatory molecules and/or cytokines is able to increase immunogenicity. Therefore, in our clinical study, 10 human leukocyte antigen (HLA)-A(*)0201(+) patients with histologically-confirmed progressive metastatic clear cell RCC were immunized repetitively over 22 weeks with 2.5-40 × 10(6) interleukin (IL)-7/CD80 cotransfected allogeneic HLA-A(*)0201(+) tumor cells (RCC26/IL-7/CD80). Endpoints of the study were feasibility, safety, immunological and clinical responses. Vaccination was feasible and safe. In all, 50% of the patients showed stable disease throughout the study; the median time to progression was 18 weeks. However, vaccination with allogeneic RCC26/IL-7/CD80 tumor cells was not able to induce TH1-polarized immune responses. A TH2 cytokine profile with increasing amounts of antigen-specific IL-10 secretion was observed in most of the responding patients. Interferon-γ secretion by patient lymphocytes upon antigen-specific and non-specific stimulation was substantially impaired, both before and during vaccination, as compared with healthy controls. This is possibly due to profound tumor-induced immunosuppression, which may prevent induction of antitumor immune responses by the gene-modified vaccine. Vaccination in minimal residual disease with concurrent depletion of regulatory cells might be one strategy to overcome this limitation.

Preclinical efficacy data of MGN1601, a tumor vaccine comprising 4-fold gene-modified and irradiated allogeneic tumor cells in combination with a DNA-based immunomodulator for the treatment of metastatic renal carcinoma.

e15067 Background: MGN1601 is a cell-based tumor vaccine for the treatment of mRCC. MGN1601 consists of two active pharmaceutical ingredients. One consists of allogeneic, human tumor cells, derived from RCC tissue, and transfected in-vitro with four different MIDGE vectors for transient expression of human IL-7, GM-CSF, CD80 and CD154. The other ingredient is the DNA-based immunomodulator dSLIM-30L1, mainly a TLR-9 agonist. Methods: Murine tumor models were used to evaluate the efficacy of a murine homologue of MGN1601 (mMGN1601). mMGN1601 was set up from Renca cells and transfected with 4 different MIDGE vectors for transient expression of mouse IL-7, GM-CSF, CD80 and CD154. For tumor inoculation in syngenic BALB/c mice Renca cells were used. For efficacy of the vaccine in a prophylactic setting, mice were vaccinated with mMGN1601 and thereafter challenged with Renca cells. In therapeutic settings Balb/c mice first received naïve Renca cells and were subsequently vaccinated four times with different test items, either the complete mMGN1601 or one out of three lacking different components. To address the mode of action, modulations of the immune system were monitored in NMRI mice (allogeneic setting). Results: Prophylactic setting: None of the vaccinated mice showed tumor growth, whereas all of the tumor-bearing mice of the control group died due to the tumor growth. Efficacy evaluation in the therapeutic setting revealed, that the complete tumor vaccine clearly achieved the most pronounced effects in tumor reduction, showing a survival of 92%, compared to 25% survival in the control group. The incomplete derivatives led to less pronounced therapeutic efficacy. The immunomodulatory effects of mMGN1601 in allogeneic NMRI mice generated a more than 3-fold increase of the cytotoxic activity of spleen cells, an augmented T-cell proliferation (> 6-fold) and an increase of CD4+, CD8+ and CD86+ cells at the site of vaccination (up to 20-fold). Conclusions: The murine homologue of MGN1601 showed excellent antitumor efficacy and significant pharmacological effects in murine models. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration MOLOGEN AG MOLOGEN AG MOLOGEN AG

Glycosyl-Phosphatidylinositol-Anchored Interleukin-2 Expressed on Tumor-Derived Exosomes Induces Antitumor Immune Response in Vitro

Tumor-derived exosomes (TEXs) have been considered as a new kind of cancer vaccine, but the antitumor effects are not satisfactory. In order to improve the efficacy of TEXs, we investigated whether exosomes derived from glycosyl-phosphatidylinositol-anchored interleukin 2 (GPI-IL-2) gene-modified bladder cancer cells can increase the antitumor effects. We transfected melanoma antigen-1 (MAGE-1)-expressing T24 tumor cells with a plasmid encoding GPI-IL-2 and prepared the TEXs. Exosomes expressing GPI-IL-2 were characterized by electron microscope and Western blot analysis. IL-2 was present on the cell surface in the GPI-anchored form as demonstrated by fluorescent microscope and ELISA analyses. Exosomes expressing GPI-IL-2 naturally contained bioactive GPI-IL-2 and tumor-associated antigen MAGE-1. Moreover, exosomes expressing GPI-IL-2-pulsed dendritic cells could induce the proliferation of T cells and the antigen-specific cytotoxic T-lymphocyte immune response more efficiently. GPI-IL-2 gene-modified tumor cells can make the TEXs contain GPI-IL-2, resulting in increased antitumor effects. Our study provided a feasible approach for exosome-based tumor immunotherapy.