Background CV8102 is a non-coding, non-capped RNA complexed with a carrier peptide activating the innate (via TLR7/8, RIG-I) and adaptive immune system.1, 2 An ongoing expansion of a phase I trial is investigating the intratumoral (i.t.) administration of CV8102 in patients with advanced cutaneous melanoma (cMEL), either as a single agent or in combination with systemic anti-PD-1 antibody therapy. In the initial dose escalation part, the recommended dose for subsequent cohort expansion and phase II was defined as 600 mg. Here we report preliminary immune profiling results. Methods For the expansion cohorts, 30 anti-PD-1 refractory Melanoma patients received CV8102 in combination with Pembrolizumab or Nivolumab and 10 patients as single agent. Eight i.t. injections of CV8102 were administered over a 12-week period, with optional continuation in case of clinical benefit. Blood samples for flow cytometry-based immune cell phenotyping and next-generation RNA sequencing (RNAseq) were collected at baseline and multiple time points during the treatment period. For characterization of the tumor microenvironment (TME), core needle biopsies of injected and/or non-injected lesions were taken before, during and after treatment. Changes on various tumor-infiltrating immune cells were assessed by multiplex immunofluorescence (MultiOmyx < sup >TM) and immune-related gene expression profiling using nCounter® Pan Cancer IO360< sup >TM panel (NanoString). Results Blood RNAseq analysis showed increase in several transcripts related to systemic immune response activation like interferon alpha and gamma after the first dose. Paired serial biopsies showed changes in the TME, not only in the immune cell infiltration but also in gene expression profiling. Comprehensive results of blood RNAseq, immune cell phenotyping, and tissue analysis for the expansion cohorts will be presented, including examples of individual patients. Conclusions Intratumoral injection of CV8102 activated several pathways of immune response detectable in peripheral blood after the first dose and showed immunological changes in the tumor microenvironment, with increased parameters of immune infiltration in a group of patients. Trial Registration NCT03291002 References [1] Ziegler A, Soldner C, Lienenklaus S, Spanier J, Trittel S, Riese P, Kramps T, Weiss S, Heidenreich R, Jasny E, Guzmán CA, Kallen KJ, Fotin-Mleczek M, Kalinke U. A New RNA-Based Adjuvant Enhances Virus-Specific Vaccine Responses by Locally Triggering TLR- and RLH-Dependent Effects. J Immunol. 2017;198(4):1595–1605. doi: 10.4049/jimmunol.1601129 [2] Heidenreich R, Jasny E, Kowalczyk A, Lutz J, Probst J, Baumhof P, Scheel B, Voss S, Kallen KJ, Fotin-Mleczek M. A novel RNA-based adjuvant combines strong immunostimulatory capacities with a favorable safety profile. Int J Cancer. 2015;137(2):372–84. doi: 10.1002/ijc.29402. Ethics Approval The study was approved by central or local ethics committees depending on the country: In Germany: Central Ethics Committees in Tuebingen, Germany under 785/2016AMG1. In France: COMITE DE PROTECTION DES PERSONNES SUD-EST I under 2019-49, approval dated 17-May-2019. In Spain: CEC COMITÉ DE ÉTICA DE INVESTIGACIÓN CLÍNICA CON MEDICAMENTOS del Hospital Universitari Vall d'Hebron, Barcelona, approval date 28-Nov-2019 under the EUdraCT number. In Austria: Central Ethics Committee in Graz under 31-426 ex 18/19 approved on 19-Sep-2019. In the Russian Federation: ETHICS COMMITTEE AT FSBI "NMRC OF ONCOLOGY n.a. N.N. BLOKHIN" OF THE MINISTRY OF HEALTHCARE OF THE RUSSIAN FEDERATION, INTERDISCIPLINARY ETHICS COMMITTEE of Omsk Region, Local Ethics Committee at FSAEI HE "I.M. Sechenov First MSMU" of the Ministry of Healthcare of Russia (Sechenov University, Extract from Minutes № 03-21 of the scheduled meeting of the Local Ethics Committee dated 03 February 2021), BIOMEDICAL ETHICS COMMITTEE at N.I. PIROGOV CLINIC OF HIGH MEDICAL TECHNOLOGIES (IN-PATIENT AND OUTPATIENT FACILITIES), ST. PETERSBURG STATE UNIVERSITY (EXTRACT FROM MINUTES № 02/21 of the meeting of the Biomedical Ethics Committee), Ethics Committee at Federal State Budgetary Institution "National Medical Research Center of Oncology named after N. N. Petrov′′ of the Ministry of Health of the Russian Federation (Extract No. 5/130 from the Minutes of the regular session No. 8 of the Ethics Committee). Consent Written informed consent from the patient was obtained for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Background CV8102 is a non-coding, non-capped RNA complexed with a carrier peptide activating the innate (via TLR7/8, RIG-I) and adaptive immune system.1 2 An ongoing phase I trial is investigating the intratumoral (i.t.) administration of CV8102 in patients with advanced cutaneous melanoma (cMEL), squamous cell carcinoma of the skin (cSCC) or head and neck (hnSCC) and adenoid cystic carcinoma (ACC), either as a single agent or in combination with systemic anti-PD-1 antibodies. Preliminary immune profiling results will be reported. Methods An open-label, cohort-based, dose escalation and expansion study in patients with advanced cMEL, cSCC, hnSCC or ACC is ongoing investigating CV8102 i.t. as single agent and in combination with anti-PD-1 antibodies. Eight i.t. injections of CV8102 were administered over a 12 week period with optional continuing treatment in case of clinical benefit. In the initial dose escalation part, the recommended phase II dose for subsequent cohort expansion was defined. Blood samples for immune cell phenotyping, RNA sequencing (RNAseq) and serum cytokine/chemokine analysis were collected at baseline and multiple time points during the treatment period. For characterization of the tumor microenvironment (TME), optional core needle biopsies of injected and/or non-injected lesions were taken before, during and after treatment. Changes on various tumor-infiltrating immune cells were assessed by multiplex immunofluorescence (MultiOmyx < sup >TM ) and immune-related gene expression profiling using nCounter® Pan Cancer IO360 < sup >TM panel (NanoString). Results During the dose escalation part, 33 patients received CV8102 (dose range of 25–900 µg) as single agent and 25 patients received CV8102 in combination with an anti-PD-1 antibody. A dose of 600 µg was selected as recommended phase II dose. Serum cytokine/chemokine and blood RNAseq analysis showed transient increases in several markers like interferons alpha and gamma after the first dose. First analyses of paired biopsies showed changes in the TME of injected and non-injected lesions. Complete results of cytokine and chemokine analysis in serum and blood RNAseq for the dose escalation cohorts will be presented. Multiplex immunofluorescence and gene expression profiling from paired biopsies from individual patients will be also included. Conclusions Intratumoral injection of CV8102 activated several cytokine/chemokine pathways in the peripheral blood and showed immunological changes in the tumor microenvironment of injected and non-injected lesions. Trial Registration NCT03291002 References Ziegler A, Soldner C, Lienenklaus S, Spanier J, Trittel S, Riese P, Kramps T, Weiss S, Heidenreich R, Jasny E, Guzmán CA, Kallen KJ, Fotin-Mleczek M, Kalinke U. A New RNA-Based Adjuvant Enhances Virus-Specific Vaccine Responses by Locally Triggering TLR- and RLH-Dependent Effects. J Immunol 2017;198(4):1595–1605. doi: 10.4049/jimmunol.1601129. Heidenreich R, Jasny E, Kowalczyk A, Lutz J, Probst J, Baumhof P, Scheel B, Voss S, Kallen KJ, Fotin-Mleczek M. A novel RNA-based adjuvant combines strong immunostimulatory capacities with a favorable safety profile. Int J Cancer 2015 Jul 15;137(2):372–84. doi: 10.1002/ijc.29402. Ethics Approval The study was approved by the Central Ethics Committees in Tuebingen, Germany under 785/2016AMG1, in France by the COMITE DE PROTECTION DES PERSONNES SUD-EST I under 2019–49, approval dated 17-May-2019, in Barcelona, Spain by the CEC COMITÉ DE ÉTICA DE INVESTIGACIÓN CLÍNICA CON MEDICAMENTOS del Hospital Universitari Vall d'Hebron, approval date 28-Nov-2019 under the EUdraCT number, in Austria by the Central Ethics Committee in Graz under 31–426 ex 18/19 approved on 19-Sep-2019. Consent Written informed consent from the patient was obtained for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

CV9201 is an RNActive®-based cancer immunotherapy encoding five non-small cell lung cancer-antigens: New York esophageal squamous cell carcinoma-1, melanoma antigen family C1/C2, survivin, and trophoblast glycoprotein. In a phase I/IIa dose-escalation trial, 46 patients with locally advanced (n = 7) or metastatic (n = 39) NSCLC and at least stable disease after first-line treatment received five intradermal CV9201 injections (400-1600 µg of mRNA). The primary objective of the trial was to assess safety. Secondary objectives included assessment of antibody and ex vivo T cell responses against the five antigens, and changes in immune cell populations. All CV9201 dose levels were well-tolerated and the recommended dose for phase IIa was 1600 µg. Most AEs were mild-to-moderate injection site reactions and flu-like symptoms. Three (7%) patients had grade 3 related AEs. No related grade 4/5 or related serious AEs occurred. In phase IIa, antigen-specific immune responses against ≥ 1 antigen were detected in 63% of evaluable patients after treatment. The frequency of activated IgD+CD38hi B cells increased > twofold in 18/30 (60%) evaluable patients. 9/29 (31%) evaluable patients in phase IIa had stable disease and 20/29 (69%) had progressive disease. Median progression-free and overall survival were 5.0 months (95% CI 1.8-6.3) and 10.8 months (8.1-16.7) from first administration, respectively. Two- and 3-year survival rates were 26.7% and 20.7%, respectively. CV9201 was well-tolerated and immune responses could be detected after treatment supporting further clinical investigation.

CV9201 is an RNActive®-based cancer immunotherapy encoding five non-small cell lung cancer-antigens: New York esophageal squamous cell carcinoma-1, melanoma antigen family C1/C2, survivin, and trophoblast glycoprotein. In a phase I/IIa dose-escalation trial, 46 patients with locally advanced (n = 7) or metastatic (n = 39) NSCLC and at least stable disease after first-line treatment received five intradermal CV9201 injections (400–1600 µg of mRNA). The primary objective of the trial was to assess safety. Secondary objectives included assessment of antibody and ex vivo T cell responses against the five antigens, and changes in immune cell populations. All CV9201 dose levels were well-tolerated and the recommended dose for phase IIa was 1600 µg. Most AEs were mild-to-moderate injection site reactions and flu-like symptoms. Three (7%) patients had grade 3 related AEs. No related grade 4/5 or related serious AEs occurred. In phase IIa, antigen-specific immune responses against ≥ 1 antigen were detected in 63% of evaluable patients after treatment. The frequency of activated IgD+CD38hi B cells increased > twofold in 18/30 (60%) evaluable patients. 9/29 (31%) evaluable patients in phase IIa had stable disease and 20/29 (69%) had progressive disease. Median progression-free and overall survival were 5.0 months (95% CI 1.8–6.3) and 10.8 months (8.1–16.7) from first administration, respectively. Two- and 3-year survival rates were 26.7% and 20.7%, respectively. CV9201 was well-tolerated and immune responses could be detected after treatment supporting further clinical investigation.

Background Intratumoral injection of immunomodulating agents is a promising approach to prime antitumor immune responses and to control tumor growth while avoiding major systemic toxicities. Intratumoral CV8102, a TLR7/8/RIG-1 agonist based on noncoding single stranded RNA showed dose dependent single agent activity and synergism with PD-1 blockade in preclinical models. Trial design CV-8102-008 (NCT03291002) is a phase I, open-label, dose escalation and expansion study of intratumoral CV8102 in patients with advanced, inoperable cutaneous melanoma (cMEL), squamous cell carcinoma of the skin (cSCC) or head and neck (hnSCC) or adenoid cystic carcinoma (ACC). Eight intratumoral injections of CV8102 are provided over a 12 weeks period, unless disease progression requiring initiation of next-line therapy or unacceptable toxicity occurs. Patients showing evidence of clinical benefit on single agent CV8102 are eligible for further treatment. Currently the first part of the trial, with escalating doses of single agent CV8102 and CV8102 in combination with anti-PD-1 antibodies to determine the maximum tolerated/ recommended dose (MTD/RCD) guided by a Bayesian logistic regression model with overdose control. and flexible cohort sizes is ongoing. Safety, biological and tumor response according to RECIST 1.1/ir RECIST of CV8102 alone/in combination with anti-PD-1 antibodies will be further characterized in expansion cohorts including anti-PD-1 naive and refractory patients. Clinical trial identification NCT03291002. Legal entity responsible for the study CureVac AG. Funding CureVac AG. Disclosure J. Krauss: Advisory / Consultancy: Vaccibody; Advisory / Consultancy: Zelluna; Research grant / Funding (self): Heidelberg ImmunoTherapeutics. T. Eigentler: Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: Novartis; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pierre Fabre; Advisory / Consultancy, Speaker Bureau / Expert testimony: Bristol-Myers Squibb. C. Weishaupt: Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self): BMS; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: CureVac; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: La Roche Posay; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Leo Pharma; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Novartis; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Pierre Fabre; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Sanofi; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Takeda. P. Terheyden: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: BMS; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Pierre-Fabre; Honoraria (self), Advisory / Consultancy: Roche; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Merck Serono; Honoraria (self): CureVac. L. Heinzerling: Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Amgen; Advisory / Consultancy: MSD; Advisory / Consultancy: BMS; Advisory / Consultancy: Roche; Advisory / Consultancy: CureVac; Advisory / Consultancy: Pierre-Fabre; Advisory / Consultancy: Sanofi. P. Mohr: Advisory / Consultancy, Shareholder / Stockholder / Stock options: Pierre Fabre; Advisory / Consultancy, Research grant / Funding (institution), Shareholder / Stockholder / Stock options: Merck; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Roche; Advisory / Consultancy, Shareholder / Stockholder / Stock options: GSK; Advisory / Consultancy, Research grant / Funding (institution), Shareholder / Stockholder / Stock options: BMS; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Novartis; Advisory / Consultancy: LEO; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Amgen. B. Weide: Advisory / Consultancy: CureVac GmbH; Advisory / Consultancy, Research grant / Funding (self): BMS; Advisory / Consultancy, Research grant / Funding (self): MSD. S. Ochsenreither: Honoraria (self), Advisory / Consultancy: MSD; Honoraria (self), Advisory / Consultancy: BMS; Honoraria (self), Advisory / Consultancy: Merck; Honoraria (self), Advisory / Consultancy: CureVac; Honoraria (self), Advisory / Consultancy: Glenmark; Honoraria (self), Advisory / Consultancy: Incyte. R. Gutzmer: Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): JohnsonJ Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Novartis; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Amgen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Merck-Serono; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Roche Pharma; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Bristol-MyersSquibb; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Almirall-Hermal; Honoraria (self), Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: SUN; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Pierre-Fabre; Honoraria (self), Advisory / Consultancy: LEO; Honoraria (self), Advisory / Consultancy: 4SC; Honoraria (self), Advisory / Consultancy: Incyte; Honoraria (self), Advisory / Consultancy: Takeda. J.C. Becker: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Merck Serono; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Pfizer; Honoraria (self), Advisory / Consultancy: CureVac; Honoraria (self), Advisory / Consultancy: eTheRNA; Honoraria (self), Advisory / Consultancy: Lytix; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy: Rigontec; Honoraria (self), Advisory / Consultancy: Takeda; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: 4SC; Research grant / Funding (institution): Alcedis; Research grant / Funding (institution): Boehringer Ingelheim; Research grant / Funding (institution): Bristol-Myers Squibb; Travel / Accommodation / Expenses: Incyt. F. Funkner: Full / Part-time employment: CureVac AG. R. Heidenreich: Full / Part-time employment: CureVac AG. S. Kays: Full / Part-time employment: CureVac AG. U. Klinkhardt: Full / Part-time employment, Former: CureVac; Full / Part-time employment: Boehringer Ingelheim. U.S. Gnad-Vogt: Full / Part-time employment, Officer / Board of Directors: CureVac AG. B. Scheel: Full / Part-time employment: CureVac AG. O. Schonborn-Kellenberger: Full / Part-time employment, consultant to CureVac AG: Cogitars GmbH. T. Seibel: Full / Part-time employment: CureVac AG. All other authors have declared no conflicts of interest.

Preclinical studies demonstrate synergism between cancer immunotherapy and local radiation, enhancing anti-tumor effects and promoting immune responses. BI1361849 (CV9202) is an active cancer immunotherapeutic comprising protamine-formulated, sequence-optimized mRNA encoding six non-small cell lung cancer (NSCLC)-associated antigens (NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and MUC-1), intended to induce targeted immune responses.

Abstract CV8102 comprises a single-stranded non-coding RNA complexed with a cationic peptide. It acts as an agonist to TLR-7/-8 and RIG-I (Ziegler 2018) to stimulate the innate and adaptive immune system. CV8102 was shown to induce an upregulation of inflammatory cytokines, chemokines and IFN-γ related genes at the injection site along with an activation of T, NK, NKT and migratory dendritic cells in the draining lymph nodes (Heidenreich 2015). Intratumoral (IT) CV8102 demonstrated dose-dependent anti-tumor activity and synergized with systemic PD-1 inhibition in preclinical models. Methods This Phase I study investigates IT CV8102 as single agent and in combination with systemic anti-PD-1 antibodies (as per product label). Patients (pts) with advanced inoperable melanoma (MEL), cutaneous/head and neck squamous cell or adenoid cystic carcinoma (cSCC, SCCHN, ACC) are eligible for single agent CV8102, pts with MEL and SCCHN who did not respond or slowly progressed on anti-PD-1 therapy are eligible for the combination. CV8102 is administered for up to 8 IT injections into a single accessible tumor lesion over a 12-week period. A Bayesian logistic regression model with overdose control is used for the dose escalation parts. Response is assessed by RECIST 1.1/irRECIST (injected and non-injected target lesions). Pre- and on-treatment samples are collected for biomarker analyses. Results A total of 20 pts have been treated with either CV8102 alone (N=15: 6 MEL, 2 SCCHN, 5 ACC, 2 cSCC) or CV8102 in combination with anti-PD-1 (N=5: 4 MEL, 1 SCCHN). Dose cohorts with doses up to 150 µg (CV8102 alone) and 100µg (CV8102+anti-PD-1) have been completed. Most common AEs were mild to moderate flu-like symptoms and injection site reactions. No dose limiting toxicities (DLT) were observed during the DLT period of the first two weeks of treatment. 12 pts treated with CV8102 alone were evaluable for response assessment. 1 MEL pt treated at 150µg experienced a complete regression of injected and non-injected lesions. This patient also experienced a marked increase of IL-6 and CRP at 6 and 24 hours after the first injection, respectively. 7 pts achieved stable disease, with two pts treated at 100 µg (SCCHN pt) and 200µg (MEL pt who had developed acquired resistance to previous anti-PD-1 therapy) showing regression of non-injected lymph node lesions. Dose escalation parts are continuing, updated safety and efficacy results will be presented. Conclusion Intratumoral single agent CV8102 appears well tolerated and showed preliminary evidence of clinical efficacy with shrinkage of injected and non-injected lesions. Citation Format: Thomas Eigentler, Juergen Krauss, Jutta Schreiber, Carsten Weishaupt, Patrick Terheyden, Lucie Heinzerling, Peter Mohr, Benjamin Weide, Ralf Gutzmer, Juergen C. Becker, Felix Kiecker, Angelika Daehling, Fatma Funkner, Regina Heidenreich, Sarah-Katharina Kays, Ute Klinkhardt, Birgit Scheel, Oliver Schoenborn-Kellenberger, Tobias Seibel, Claudia Stosnach, Tanja Strack, Ulrike Gnad-Vogt. Intratumoral RNA-based TLR-7/-8 and RIG-I agonist CV8102 alone and in combination with anti-PD-1 in a Phase I dose-escalation and expansion trial in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-021.

Background: Local activation of innate immune signaling pathways, such as Toll-like Receptors (TLRs) or RIG-I-like Receptors (RLRs), is a promising cancer immunotherapeutic approach to overcome the tumor immunosuppressive microenvironment and to induce/restore anti-tumor immunity. CV8102 consists of a single-stranded, non-coding RNA complexed with a cationic peptide and acts as an agonist to human TLR-7, -8 and RLRs. Local administration of CV8102 was shown to induce upregulation of inflammatory cytokines, chemokines and IFN-y related genes at the injection site followed by activation of T, NK, NKT cells and migratory dendritic cells in the draining lymph node (Heidenreich 2015; Ziegler 2017). In a syngeneic murine model, intratumoral (IT) delivery of CV8102 induced dose-dependent anti-tumor activity and synergized with systemic anti-PD1 antibody treatment. Trial design: This phase I study is evaluating IT injection of CV8102 alone or in combination with a systemic anti-PD-1 antibody in patients with advanced solid tumors. Patients with advanced inoperable melanoma, cutaneous/head and neck squamous cell or adenoid cystic carcinoma and at least one lesion accessible for IT injection are eligible. After determination of the maximum tolerated/ recommended dose of CV8102 alone and in combination with an anti-PD1 antibody, expansion cohorts are planned in different indications. A Bayesian logistic regression model with overdose control will be used to guide the dose escalation. Patients in each cohort will receive up to 8 injections into a single tumor lesion over a 12 weeks period. The primary objective is to determine safety and tolerability, secondary/explorative objectives are to evaluate tumor response and changes in tumor tissue and blood-based biomarkers. The study has been initiated and enrollment is ongoing. Clinical trial identification: NCT03291002. Legal entity responsible for the study: CureVac AG, Paul-Ehrlich-Str. 15, 72076 Tübingen, Germany. Funding: Has not received any funding. Disclosure: P. Terheyden: Honoraria, Travel expenses: Novartis, Merck, Roche, BMS, Biofrontera; Advisory board: Roche, BMS, Sanofi Novartis, Merck. L. Heinzerling: Consultant: CureVac AG PI in clinical study at site Erlangen. P. Mohr: Consultant or advisory role: Merck, Roche, GSK, BMS, Novartis, SciBase, LEO; Honoraria: GSK, Roche, Merck, BMS, Novartis, LEO, SciBase; Research funding: Merck. F. Kiecker: Payments, Travel grants for symposia, Advisory boards, Meetings: Amgen, BMS, Merck-Serono, MSD, Novartis, Pierre-Fabre, Regeneron, Roche. J.C. Becker: Speaker honoraria: Amgen, MerckSerono and Pfizer; Advisory board honoraria: Amgen, CureVac, eTheRNA, Lytix, MerckSerono, Novartis, Rigontec, Takeda; Research funding: Boehringer Ingelheim, BMS, MerckSerono. U. Gnad-Vogt: Stock, Other ownership interests: Bristol-Myers Squibb. All other authors have declared no conflicts of interest.

We recently completed a phase I/IIa trial of RNActive® CV9201, a novel mRNA-based therapeutic vaccine targeting five tumor-associated antigens in non-small cell lung cancer (NSCLC) patients. The aim of the study presented here was to comprehensively analyze changes in peripheral blood during the vaccination period and to generate hypotheses facilitating the identification of potential biomarkers correlating with differential clinical outcomes post RNActive® immunotherapy. We performed whole-genome expression profiling in a subgroup of 22 stage IV NSCLC patients before and after initiation of treatment with CV9201. Utilizing an analytic approach based on blood transcriptional modules (BTMs), a previously described, sensitive tool for blood transcriptome data analysis, patients segregated into two major clusters based on transcriptional changes post RNActive® treatment. The first group of patients was characterized by the upregulation of an expression signature associated with myeloid cells and inflammation, whereas the other group exhibited an expression signature associated with T and NK cells. Patients with an enrichment of T and NK cell modules after treatment compared to baseline exhibited significantly longer progression-free and overall survival compared to patients with an upregulation of myeloid cell and inflammatory modules. Notably, these gene expression signatures were mutually exclusive and inversely correlated. Furthermore, our findings correlated with phenotypic data derived by flow cytometry as well as the neutrophil-to-lymphocyte ratio. Our study thus demonstrates non-overlapping, distinct transcriptional profiles correlating with survival warranting further validation for the development of biomarker candidates for mRNA-based immunotherapy.

### P189 Rational combinations of intratumoral T cell and myeloid agonists mobilize abscopal responses in prostate cancer #### Casey Ager1, Matthew Reilley2, Courtney Nicholas1, Todd Bartkowiak1, Ashvin Jaiswal1, Michael Curran1 ##### 1Department of Immunology, University of Texas MD Anderson

Abstract Background: Preclinical studies demonstrated that local radiotherapy (RT) acts synergistically with RNActive mRNA vaccines to enhance anti-tumor effects and increase tumor-infiltrating lymphocytes. BI 1361849 is a therapeutic vaccine comprising optimized mRNA constituents encoding six NSCLC-associated antigens. Interim data of a phase Ib study, employing local RT to increase the immune mediated tumor control by BI 1361849, have been previously published (J Clin Oncol 34, 2016, suppl; abstr e20627). Here we report results of immune response analyses as well as updated safety and efficacy data. Methods: Patients (pts) with stage IV NSCLC were enrolled in three cohorts based on histological and molecular NSCLC subtypes (squamous and non-squamous cell with/without activating epidermal growth factor receptor (EGFR) mutations). Pts received two vaccinations with BI 1361849 before local RT to a single tumor lesion (thoracic, bone, lymph node, skin/subcutaneous) was administered in four consecutive daily fractions of 5 GY. Vaccination was continued until start of subsequent anti-cancer therapy. Maintenance pemetrexed (mP) and epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) were given in parallel according to the label where indicated. The primary endpoint was safety; secondary endpoints included objective response, progression-free survival (PFS) and overall survival (OS). Cellular and humoral immune responses were measured ex vivo by multifunctional intracellular cytokine staining, IFN-γ ELISpot, and ELISA in pre- and post-treatment blood samples. Results: 26 pts were enrolled. 15 pts received mP, two received EGFR TKIs. Most frequent AEs were mild to moderate injection-site reactions and flu-like symptoms. No BI 1361849-related SAEs were reported. Based on preliminary data following up to 110 weeks of exposure, one confirmed PR was observed in a pt on mP, 13 pts (52%) experienced SD (8 pts on mP, 2 pts on EGFR-TKI and 3 pts without concomitant maintenance treatment, associated with 15% tumor shrinkage outside the radiation field in one pt without maintenance). 25 pts were available for immune response analysis. Preliminary data indicate that BI 1361849 was capable of eliciting antigen-specific immune responses in the majority of the patients including cellular and humoral immune responses. Conclusion: BI 1361849 can be safely combined with local RT and mP treatment. Shrinkage of non-irradiated lesions and prolonged disease stablization was observed in a subset of pts, mainly in combination with mP. Data indicate immunogenicity of BI 1361849. Analyses of cellular and humoral immune responses will be presented, as well as updated clinical data. Citation Format: M. M. Hipp, M. Sebastian, C. Weiss, M. Früh, M. Pless, R. Cathomas, W. Hilbe, G. Pall, T. Wehler, J. Alt, H. Bischoff, M. Geißler, F. Griesinger, J. Kollmeier, A. Papachristofilou, F. Doener, M. Fotin-Mleczek, H. S. Hong, K. J. Kallen, U. Klinkhardt, S. D. Koch, E. Niehus, B. Scheel, A. Schröder, T. Seibel, U. Gnad-Vogt, A. Zippelius. Phase Ib trial of the RNActive cancer vaccine BI 1361849 (CV9202) and local radiotherapy in patients with stage IV non-small cell lung cancer (NSCLC) with disease control after first-line chemotherapy or during therapy with an epidermal growth factor receptor tyrosine kinase inhibitor: Updated clinical results and immune responses [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 B072.

CV9103 is a prostate-cancer vaccine containing self-adjuvanted mRNA (RNActive®) encoding the antigens PSA, PSCA, PSMA, and STEAP1. This phase I/IIa study evaluated safety and immunogenicity of CV9103 in patients with advanced castration-resistant prostate-cancer.

Protein‐ and peptide‐based tumor vaccines depend on strong adjuvants to induce potent immune responses. Here, we demonstrated that a recently developed novel adjuvant based on a non‐coding, long‐chain RNA molecule, termed RNAdjuvant®, profoundly increased immunogenicity of both antigen formats. RNAdjuvant® induced balanced, long‐lasting immune responses that resulted in a strong anti‐tumor activity. A direct comparison to Poly(I:C) showed superior efficacy of our adjuvant to enhance antigen‐specific multifunctional CD8+ T‐cell responses and mediate anti‐tumor responses induced by peptide derived from HPV‐16 E7 protein in the syngeneic TC‐1 tumor, a murine model of human HPV‐induced cervical cancer. Moreover, the adjuvant was able to induce functional memory responses that mediated complete tumor remission. Despite its remarkable immunostimulatory activity, our RNA‐based adjuvant exhibited an excellent pre‐clinical safety profile. It acted only locally at the injection site where it elicited a transient but strong up‐regulation of pro‐inflammatory and anti‐viral cytokines as well as cytoplasmic RNA sensors without systemic cytokine release. This was followed by the activation of immune cells in the draining lymph nodes. Our data indicate that our RNA‐based adjuvant is a safe and potent immunostimulator that may profoundly improve the efficacy of a variety of cancer vaccines.

BACKGROUND:Advanced non-small cell lung cancer (NSCLC) represents a significant unmet medical need. Despite advances with targeted therapies in a small subset of patients, fewer than 20% of patients survive for more than two years after diagnosis. Cancer vaccines are a promising therapeutic approach that offers the potential for durable responses through the engagement of the patient's own immune system. CV9202 is a self-adjuvanting mRNA vaccine that targets six antigens commonly expressed in NSCLC (NY-ESO-1, MAGEC1, MAGEC2, 5 T4, survivin, and MUC1).METHODS/DESIGN:The trial will assess the safety and tolerability of CV9202 vaccination combined with local radiation designed to enhance immune responses and will include patients with stage IV NSCLC and a response or stable disease after first-line chemotherapy or therapy with an EGFR tyrosine kinase inhibitor. Three histological and molecular subtypes of NSCLC will be investigated (squamous and non-squamous cell with/without EGFR mutations). All patients will receive two initial vaccinations with CV9202 prior to local radiotherapy (5 GY per day for four successive days) followed by further vaccinations until disease progression. The primary endpoint of the study is the number of patients experiencing Grade >3 treatment-related adverse events. Pharmacodynamic analyses include the assessment of immune responses to the antigens encoded by CV9202 and others not included in the panel (antigen spreading) and standard efficacy assessments.DISCUSSION:RNActive self-adjuvanted mRNA vaccines offer the potential for simultaneously inducing immune responses to a wide panel of antigens commonly expressed in tumors. This trial will assess the feasibility of this approach in combination with local radiotherapy in NSCLC patients.TRIAL REGISTRATION:Clinicaltrials.gov: NCT01915524/EudraCT No.: 2012-004230-41.

Two-component mRNA-based vaccine (RNActive®) combines high antigen expression with strong immune stimulation. We have previously shown that intradermal administration of RNActive® vaccines induced balanced, potent and long-lasting immune responses in both mice and humans. Here, we characterized in detail early events after vaccine injection to better understand the self-adjuvant capacity of mRNA-based vaccines. Shortly after intradermal administration, mRNA was present in both non-leukocytic and leukocytic cells, the latter being mostly represented by MHC class II-expressing cells. Further studies revealed that 24 h after intradermal administration RNActive® was detectable in dendritic cells (DCs) within the draining lymph nodes (dLNs). Full genome microarray analyses of the skin tissues showed that RNActive® vaccine transiently altered the gene expression profile at the injection site. Various chemokines such as CXCR3 ligands (including CXCL9, CXCL10 and CXCL11) as well as CCL2, CCL4, CCL5 and CCL12, whose pleiotropic functions include recruitment and activation of macrophages, NK cells and DCs, were up-regulated early after RNActive® treatment. Additionally, gene expression of multiple proinflammatory cytokines such as IL-6 or TNF-α was increased. In corroboration with the microarray data, after injection of mRNA vaccine we observed a strong production of chemokines including CXCL9, CXCL10, CCL3, CCL4, CCL5 as well as proinflamatory cytokines (IL-6, TNF-a) at the site of injection. No changes in the cytokine serum level were detected suggesting a local immunostimulation with no signs of cytokine release syndrome. The innate immune responses in the skin were followed by the increased cellularity as well as an enhanced activation of a wide range of the immune cells in the dLNs including CD8+ T-cells, B cells, γδ T-cells, NK and NKT cells. In summary, our data indicate that the self-adjuvanted RNActive® vaccine induces strong innate immune response at the site of administration followed by the activation of the immune cells in the dLNs. These results provide a possible mechanistic explanation of a potent and balanced adaptive immunity induced by mRNA-based vaccine which is a novel vaccination platform for an efficient cancer immunotherapy.

Meeting abstracts CV9104 is a novel mRNA-based therapeutic vaccine for prostate cancer encoding the prostate cancer-associated antigens PSA, PSMA, PSCA, STEAP, PAP and MUC1 by engineered mRNA molecules optimized for protein expression and immunostimulation (RNActive®). CV9103, an mRNA-based

Meeting abstracts Recently, the RNActive technology has been developed to generate highly active cancer vaccines. The antigen of choice is encoded by a sequence engineered mRNA that increases antigen expression up to 5 orders of magnitude. Immunogenicity of the RNActive vaccines is ensured by a