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Purpose: Intratumoral oncolytic virotherapy may overcome anti–PD(L)-1 resistance by triggering pro-inflammatory remodeling of the tumor microenvironment. This pilot study investigated ONCOS-102 (oncolytic adenovirus expressing GM-CSF) plus anti–programmed cell death protein 1 (PD)-1 therapy in anti–PD-1–resistant melanoma. Patients and Methods: Patients with advanced melanoma progressing after prior PD-1 blockade received intratumoral ONCOS-102 either as priming with 3 doses (3 × 1011 viral particles) during Week 1 [Part 1 (sequential treatment)] or as 4-dose priming and 8 booster doses every 3 weeks [Part 2 (combination treatment)]. From Week 3, all patients received pembrolizumab every 3 weeks (≤8 doses). The primary endpoint was safety. Objective response rate (ORR), progression-free survival, and immunologic activation in repeat biopsies were also investigated. Results: In 21 patients (Part 1, n = 9; Part 2, n = 12) ONCOS-102 plus pembrolizumab was well tolerated: most adverse events (AE) were mild/moderate in severity. Pyrexia (43%), chills (43%), and nausea (28%) were the most common ONCOS-102–related AEs. There were no dose-limiting toxicities. ORR was 35% [response evaluation in solid tumors (RECIST) 1.1, irRECIST]. Reduction in size of ≥1 non-injected lesions observed in 53% patients indicated a systemic effect. In injected tumors, persistent immune-related gene expression and T-cell infiltration were associated with clinical benefit. Viral persistence and efficacy in injected and non-injected lesions without additional toxicity supported Part 2 dosing regimen in future studies. Conclusions: ONCOS-102 plus pembrolizumab was well tolerated and led to objective responses in patients with anti–PD-1–resistant advanced melanoma. ONCOS-102 promoted T-cell infiltration, particularly cytotoxic CD8+ T cells, which persisted at Week 9, driving clinical benefit. Further investigation of ONCOS-102 plus PD-1 blockade is warranted. See related commentary by Levi and Boland, p. 3

The CD40 receptor is an attractive target for cancer immunotherapy. Although a modest pharmacodynamic effect is seen in patients following administration of CD40-targeting monoclonal antibodies (mAb), the doses that could be safely administered do not result in a meaningful clinical response, most likely due to the limited therapeutic window associated with systemic CD40 activation. To overcome this issue, we developed a multispecific DARPin construct, α-FAPxCD40, which has conditional activity at the site of disease. α-FAPxCD40 activation of CD40 depends on binding to fibroblast activation protein (FAP), a cell-surface protease overexpressed in the stroma of solid tumors. In vitro studies demonstrated that α-FAPxCD40 potently activates human antigen-presenting cells in the presence, but not in the absence, of FAP-positive cells. After intravenous injection, a murine surrogate construct (α-mFAPxCD40) accumulated in FAP-positive tumors, elicited rejection of 88% of these tumors, and induced memory antitumor immunity. Importantly, in contrast to the mouse anti-CD40 tested in parallel, the in vivo antitumor activity of α-mFAPxCD40 was associated neither with elevated blood cytokines nor with hepatotoxicity, both of which contribute to the clinical dose-limiting toxicities of several CD40 mAb. This study demonstrates that α-(m)FAPxCD40 engages CD40 in an FAP-restricted manner, leading to tumor eradication without signs of peripheral toxicity. This distinct preclinical profile suggests that a favorable therapeutic index may be achieved in humans. It further supports the development of α-FAPxCD40, currently tested in a first-in-human clinical study in patients with solid tumors (NCT05098405).

Supplementary Figure from A Multispecific Anti-CD40 DARPin Construct Induces Tumor-Selective CD40 Activation and Tumor Regression

Dysfunctional T cells present in malignant lesions are characterized by a sustained and highly diverse expression of inhibitory receptors, also referred to as immune checkpoints. Yet, their relative functional significance in different cancer types remains incompletely understood. In this study, we provide a comprehensive characterization of the diversity and expression patterns of inhibitory receptors on tumor-infiltrating T cells from patients with non–small cell lung cancer. In spite of the large heterogeneity observed in the amount of PD-1, Tim-3, CTLA-4, LAG-3, and BTLA expressed on intratumoral CD8+ T cells from 32 patients, a clear correlation was established between increased expression of these inhibitory coreceptors and progression of the disease. Notably, the latter was accompanied by a progressively impaired capacity of T cells to respond to polyclonal activation. Coexpression of several inhibitory receptors was gradually acquired, with early PD-1 and late LAG-3/BTLA expression. PD-1 blockade was able to restore T-cell function only in a subset of patients. A high percentage of PD-1hi T cells was correlated with poor restoration of T-cell function upon PD-1 blockade. Of note, PD-1hi expression marked a particularly dysfunctional T-cell subset characterized by coexpression of multiple inhibitory receptors and thus may assist in identifying patients likely to respond to inhibitory receptor–specific antibodies. Overall, these data may provide a framework for future personalized T-cell–based therapies aiming at restoration of tumor-infiltrating lymphocyte effector functions. Cancer Immunol Res; 3(12); 1344–55. ©2015 AACR.

Time-lapse fluorescence microscopy of CMV-pp65-specific T cell mediated killing of MCSP+ tumor cells (Colo38) incubated with 15nM of EBV-pMHCI-IgG as control. Constructs are indicated in the respective panel. T cells are stained in red (PKH-26, Sigma), tumor cells in green (CMFDA, LifeTechnologies). Effector to target ratio was 3:1. Real-time is indicated in the lower left corner in h:m:s:ms (h=hours, m=minutes, s=seconds, ms=milliseconds), scale bar is shown in the lower right corner 0-50 µm (µm=micrometre).

Protein Purification, Flow cytometry, Time lapse live cell imaging by confocal microscopy, Stimulation of PBMCs with homo-and heterodimeric CMV-pMHCI-IgGs, Pharmacokinetic measurement.

Tumor cells escape immune eradication through multiple mechanisms, including loss of antigenicity and local suppression of effector lymphocytes. To counteract these obstacles, we aimed to direct the unique cytomegalovirus (CMV)-specific immune surveillance against tumor cells. We developed a novel generation of fusion proteins composed of a tumor antigen–specific full immunoglobulin connected to a single major histocompatibility class I complex bearing a covalently linked virus-derived peptide (pMHCI–IgG). Here, we show that tumor antigen–expressing cancer cells, which are decorated with pMHCI–IgGs containing a HLA-A*0201 molecule associated with a CMV-derived peptide, are specifically eliminated through engagement of antigen-specific CD8+ T cells isolated from peripheral blood mononuclear cell preparations of CMV-infected humans. These CD8+ T cells act without additional expansion, preactivation, or provision of costimulatory signals. Elimination of tumor cells is induced at similar concentrations and with similar time kinetics as those seen with bispecific T-cell engagers (BiTE). However, while BiTE-like reagents indiscriminately activate T cells through binding to the T-cell receptor complex, pMHCI–IgGs selectively engage antigen-specific, constantly renewable, differentiated effector cytotoxic T lymphocytes to tumor cells, thereby representing a novel class of anticancer immunotherapeutics with potentially improved safety and efficacy profiles. Cancer Immunol Res; 3(7); 764–76. ©2015 AACR.

Time-lapse fluorescence microscopy of CMV-pp65-specific T cell mediated killing of MCSP+ tumor cells (Colo38) incubated with 15nM of CMV-pMHCI-IgG. Constructs are indicated in the respective panel. T cells are stained in red (PKH-26, Sigma), tumor cells in green (CMFDA, LifeTechnologies). Effector to target ratio was 3:1. Real-time is indicated in the lower left corner in h:m:s:ms (h=hours, m=minutes, s=seconds, ms=milliseconds), scale bar is shown in the lower right corner 0-50 µm (µm=micrometre).

Supplementary Table 1. Clinical data of NSCLC patients. Supplementary Figure 1. Calculation of the iR score. Supp. Figure 2. Activation of PBMC from healthy donors by polyclonal stimulation.

Immunologic pathway genes

Purpose:Intratumoral oncolytic virotherapy may overcome anti–PD(L)-1 resistance by triggering pro-inflammatory remodeling of the tumor microenvironment. This pilot study investigated ONCOS-102 (oncolytic adenovirus expressing GM-CSF) plus anti–programmed cell death protein 1 (PD)-1 therapy in anti–PD-1–resistant melanoma.Patients and Methods:Patients with advanced melanoma progressing after prior PD-1 blockade received intratumoral ONCOS-102 either as priming with 3 doses (3 × 1011 viral particles) during Week 1 [Part 1 (sequential treatment)] or as 4-dose priming and 8 booster doses every 3 weeks [Part 2 (combination treatment)]. From Week 3, all patients received pembrolizumab every 3 weeks (≤8 doses). The primary endpoint was safety. Objective response rate (ORR), progression-free survival, and immunologic activation in repeat biopsies were also investigated.Results:In 21 patients (Part 1, n = 9; Part 2, n = 12) ONCOS-102 plus pembrolizumab was well tolerated: most adverse events (AE) were mild/moderate in severity. Pyrexia (43%), chills (43%), and nausea (28%) were the most common ONCOS-102–related AEs. There were no dose-limiting toxicities. ORR was 35% [response evaluation in solid tumors (RECIST) 1.1, irRECIST]. Reduction in size of ≥1 non-injected lesions observed in 53% patients indicated a systemic effect. In injected tumors, persistent immune-related gene expression and T-cell infiltration were associated with clinical benefit. Viral persistence and efficacy in injected and non-injected lesions without additional toxicity supported Part 2 dosing regimen in future studies.Conclusions:ONCOS-102 plus pembrolizumab was well tolerated and led to objective responses in patients with anti–PD-1–resistant advanced melanoma. ONCOS-102 promoted T-cell infiltration, particularly cytotoxic CD8+ T cells, which persisted at Week 9, driving clinical benefit. Further investigation of ONCOS-102 plus PD-1 blockade is warranted.See related commentary by Levi and Boland, p. 3

Supplementary methods

Supplementary figures

The CD40 receptor is an attractive target for cancer immunotherapy. Although a modest pharmacodynamic effect is seen in patients following administration of CD40-targeting monoclonal antibodies (mAb), the doses that could be safely administered do not result in a meaningful clinical response, most likely due to the limited therapeutic window associated with systemic CD40 activation. To overcome this issue, we developed a multispecific DARPin construct, α-FAPxCD40, which has conditional activity at the site of disease. α-FAPxCD40 activation of CD40 depends on binding to fibroblast activation protein (FAP), a cell-surface protease overexpressed in the stroma of solid tumors. In vitro studies demonstrated that α-FAPxCD40 potently activates human antigen-presenting cells in the presence, but not in the absence, of FAP-positive cells. After intravenous injection, a murine surrogate construct (α-mFAPxCD40) accumulated in FAP-positive tumors, elicited rejection of 88% of these tumors, and induced memory antitumor immunity. Importantly, in contrast to the mouse anti-CD40 tested in parallel, the in vivo antitumor activity of α-mFAPxCD40 was associated neither with elevated blood cytokines nor with hepatotoxicity, both of which contribute to the clinical dose-limiting toxicities of several CD40 mAb. This study demonstrates that α-(m)FAPxCD40 engages CD40 in an FAP-restricted manner, leading to tumor eradication without signs of peripheral toxicity. This distinct preclinical profile suggests that a favorable therapeutic index may be achieved in humans. It further supports the development of α-FAPxCD40, currently tested in a first-in-human clinical study in patients with solid tumors (NCT05098405).

In recently performed phase I/II clinical studies, we have demonstrated successful delivery of vectorized transgene-encoding mRNA payloads to solids tumors with substantial clinical benefit. In patients, persistent and prolonged transgene expression correlated with positive clinical outcome, and thus temporal expansion of the transgene expression profile is expected to improve the therapeutic benefit even further. Recently, circular RNA (circRNA) has been discovered as a novel class of endogenously expressed RNA. CircRNAs, in contrast to mRNAs, are resistant to exo-nucleolytic decay which results in high intra-cellular stability and persistent expression within cells. Therefore, circRNA-based systems serve as an interesting new technology towards prolonged effects of treatment. Here, we show that our proprietary vector system, circAde, allows high expression of circRNAs through spliceosome-dependent biogenesis. By comparing the protein output from circAde and mRNA-based vectors, we show that the circAde-system out-performs conventional mRNA-based design, both in terms of transgene yield and temporal sustainability of expression. Moreover, using a panel of proof-of-concept-reporters, we show that certain design-features greatly influence the circRNA production and subsequent protein expression from the vector. Without the 5’cap, circRNAs are dependent on IRESs (internal ribosome entry sequences) for effective translation. By conducting two orthogonal IRES screens on 1000+ putative IRES elements, we have identified the most effective IRES sequence in melanoma and lung-cancer cells supporting effective circRNA production and high-yield protein expression, thereby improving our circAde vector system even further. Our results open a new platform towards development of novel, more effective strategies for the treatment of cancer and other settings in which durable transgene expression is desirable. Citation Format: Eoghan T. O'leary, Sara Vikberg, Emilie Foord, Erik D. Wiklund, Lone Ottesen, Victor Levitsky, Thomas B. Hansen. circAde: a circRNA-based system for prolonged and more effective treatment of cancer. [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 5335.

BackgroundMalignant pleural mesothelioma (MPM) is an aggressive malignancy without curative treatment. Standard of care (SOC) include pemetrexed/cisplatin and nivolumab/ipilimumab with median overall survival in unresectable disease of 12.1 months and 18.1 months respectively.1 2 ONCOS-102 is a granulocyte-macrophage colony stimulating factor (GM-CSF) expressing oncolytic adenovirus (Ad5/3-D24-GMCSF) with a unique ability to both prime and boost immune responses. The aim of the study was to assess efficacy and safety of ONCOS-102 in combination with SOC chemotherapy in 1st and 2nd line unresectable MPM.MethodsTwenty patients (experimental arm) were allocated to receive ONCOS-102 given intratumorally under CT or US guidance at a dose of 3 x 1011 VP on Day 1, 4, 8, 36, 78 and 120 plus six cycles of SOC starting on Day 22. Eleven patients (control group) received SOC. Imaging was done at baseline, Day 43–64 and 127–148 with regular monitoring of blood and biopsy based immune markers. Primary objective was safety and tolerability. Secondary objectives were ORR, PFS and OS as well as immunological activation. An analysis of 24 month survival data compared randomised only patients excluding six patients in the single-arm safety lead-in.Results24-month survival rate for 1st line pts was 50% in the experimental group and 0% in the control group with mOS of 25.0 months and 13.5 months respectively (N.S). Based on censoring, mOS in the experimental group will be within 21.9 – 25.0 months range. mOS across both 1st and 2nd line was 19.3 and 18.3 months for experimental and control patients (N.S). mPFS was 9.8 months in the experimental group and 7.6 in the control group (N.S.).ConclusionsThe survival rate of patients receiving ONCOS-102 in combination with SOC was seen to be numerically higher than previously reported for SOC or nivolumab/ipilimumab. Improved survival was associated with ONCOS-102 induced immune activation with a favourable TME modulation providing scientific rationale for combination with check point inhibition.Trial RegistrationClinicalTrials.gov NCT02879669ReferencesVogelzang, et al, Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 2003;21: 2636–44.Baas P, et al, First-line nivolumab plus ipilimumab in unresectable pleural mesothelioma (CheckMate 743): a multicentre, randomised, open-label, phase 3 trial. The Lancet 2021; 397: 375–386.Ethics ApprovalThis study was approved by the IRBs of all the participating sites in Madrid, Barcelona, Rennes and Poitiers.

BackgroundSolid tumors exhibit highly variable compositions of immune infiltrates. Therapeutic compounds driving uniform remodeling of tumor microenvironment (TME) across tumor types may improve the efficacy of cancer immunotherapy. ONCOS-102, a granulocyte-macrophage colony stimulating factor (GM-CSF)-expressing oncolytic adenovirus (Ad5/3-D24-GMCSF), was tested for its safety, therapeutic efficacy and capacity to remodel TME in recently completed phase I/II clinical studies in anti-PD-1 refractory melanoma (NCT03003676) and malignant pleural mesothelioma (MPM) (NCT02879669).MethodsBiopsies were obtained from tumor lesions of patients treated with intra-tumoral injections of ONCOS-102 in combination with chemotherapy or pembrolizumab for MPM and melanoma, respectively. Tumor immune infiltrates were analyzed by immunohistology using several antibody panels. On-treatment biopsies were compared to paired baseline samples as wells as to samples from control patients treated with chemotherapy alone in the case of MPM. Gene expression data obtained by next generation RNA sequencing were used to complement the immunohistology analysis and all results were correlated to clinical outcomes.ResultsComparative TME analysis of anti-PD-1 refractory melanoma and MPM tumors revealed noticeably lower baseline T-cell infiltration in mesothelioma. Thus, fractions of CD8+ T-cells were significantly below 10% in 80% of MPM biopsies while approaching or exceeding this level in 60% of melanoma baseline samples. Comparison of tumor biopsies obtained at baseline or on-treatment, demonstrated increased infiltration by both CD4+ and CD8+ T-cells in large proportions of melanoma (CD4+: 13/20 (65%); CD8+: 16/19 (84%) and MPM (CD4+: 10/15 (67%); CD8+: 9/15 (60%) tumor lesions in response to ONCOS-102. Frequencies of cytotoxic T-cells with high granzyme-B expression also increased in response to the treatment in both tumor types, in particular when assessed as percentage of total CD8+ T-cells. Other observed changes induced by ONCOS-102 in samples taken from CR, PR and SD patients with MPM or melanoma included increased CD8/Treg ratio and modulation of PD-L1 expression. Biological and clinical importance of these findings was further supported by correlation between modulation of several subsets of genes related to the process of T-cell activation, such as cytotoxic granule components and co-stimulatory molecules, and clinical response to ONCOS-102 in melanoma and both tumor response and overall survival in MPM patients.ConclusionsONCOS-102 drives pro-inflammatory modulation of immune TME across tumor types of different origins, anatomical locations and immunological baseline characteristics. Our data support potential of ONCOS-102 to serve as a potent immune sensitizing agent in combination therapies with various classes of immunomodulatory compounds and chemotherapy.

Background: Defining molecular parameters of clinical response to immune modulators may lead to development of novel biomarkers, new therapeutic targets and drug combinations in cancer treatment. We recently completed phase I/II testing of ONCOS-102, a GM-CSF-encoding oncolytic adenovirus (Ad5/3-D24-GMCSF), for therapeutic efficacy and capacity to remodel tumor micro-environment (TME) in combination with pembrolizumab (pem) in patients (pts) with non-resectable, stage III-IV, anti-PD-1 resistant/refractory (R/R) melanoma (NCT03003676). Here we report comparative, longitudinal gene expression analysis of tumor samples from pts with complete/partial response (CR/PR), stable (SD) or progressive (PD) disease by RECIST1.0. Methods: In study part 1, n=9 pts received three intra-tumoral (i.t.) injections of ONCOS-102 every 3 days during the first week of treatment. Pem was administered i.v. at 10 mg/kg from day 22 every 3 weeks for 6 months. In study part 2, n=12 pts received additional injections of ONCOS-102 at day 15 and then every 3 weeks along with pem. On-treatment tumor biopsies were collected at baseline, day 22 and 64. Total RNA sequencing followed by differential gene expression analysis using DESeq1 was conducted on n=17 pts to disclose dynamic transcriptome changes associated with ONCOS-102 administration and clinical response. Results: Baseline biopsies of CR/PR pts showed 1,5-2 fold higher levels of expression of innate and adaptive immune response-related genes compared to PD pts. Treatment with ONCOS-102 upregulated immune related genes at day 22 across most tumors with no clear PR/PD differentiation. However, at day 64 a striking distinction between pts with CR/PR compared to PD emerged, showing 4-5 fold increase in expression levels for several immune-related gene categories including check-points, co-stimulatory and cellular cytotoxicity genes. TME remodeling in CR/PR pts was sustained at least until day 64. On individual genes,higher and more sustained upregulation of several co-stimulatory molecules (4-1BB, OX40, CD28) and checkpoints (LAG-3, TIGID, TIM-3) was seen in responders. Expression of adenovirus genes was more prevalent in CR/PR (2 of 3 pts) than PD (0 of 2 pts) on day 64, suggesting that tumor susceptibility to virus entry and replication drives clinical benefit of ONCOS-102. Our findings were supported by analysis of other gene subsets and comparison of different schedules of ONCOS-102 administration. Conclusions: ONCOS-102 drives pro-inflammatory modulation of the TME in PD-1 R/R melanoma tumors. Clinical response ONCOS-102 is associated with efficient viral gene expression and strong, sustained activation of immune-related genes, which may be targets for future combinations of ONCOS-102 and immune-modulators beyond PD-1/PD-L1 inhibitors. Citation Format: Erik Digman Wiklund, Lukasz Kuryk, Sandeep Kumar, Magnus Jaderberg, Victor levitsky, Thomas Birkballe Hansen, Anne-Sophie Moller, Alexander Shoushtari. Modulation of immune gene expression by intra-tumoral oncolytic adenovirus ONCOS-102 is associated with clinical response in anti-PD-1 refractory/resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1983.

8561 Background: MPM is an aggressive malignancy without curative treatment. ONCOS-102 is an oncolytic adenovirus expressing GM-CSF (Ad5/3-D24-GMCSF) with a clinically documented ability to stimulate local and systemic immune responses and re-modulate the tumor microenvironment, both as a monotherapy and in combination with anti-PD1 blockade. The study objectives included determining safety and tolerability, efficacy and immunological activation in repeat tumor biopsies, and correlations with clinical outcomes. Methods: Following a safety run-in of n = 6 pts, 25 patients were randomized to receive ONCOS-102 intratumorally under CT or US guidance at a dose of 3 x 1011 Virus Particles on Day 1, 4, 8, 36, 78 and 120, plus six cycles of P/C starting on Day 22, or control comprising six cycles of P/C only. Both treatment-naïve (1L) and previously treated patients (2L) were enrolled. Imaging was done at baseline, Day 43-64 and 127-148 and tumor biopsy were collected at baseline and at Day 36. Final survival analysis (n = 25) was performed after 30 mo follow up. Multiplex immunofluorescence for immune cell subsets, RNASeq and qPCR was performed on repeat tumor samples. Results: The most frequent adverse events were anaemia, neutropenia and asthenia reported by > 50% in both groups with more frequent reports of pyrexia and nausea in the experimental (exp) group. No difference in the rate of severe events (Gr 3/4 acc to NCI CTCAE vs 4.0) were observed. 30-month survival rate (n = 25) was 34.3 % vs 18.2 % (NS) with mOS of 19.3 mo (95% CI: 4.6, NA) vs 18.3 mo (95% CI: 3.1, 28.9) in the exp group vs control. For patients treated in the 1L setting, 30 mo survival was 33.3 % in the exp group (n = 8) and 0% in the control group (n = 6) with mOS of 25.0 months and 13.5 months, respectively (NS). mPFS was unchanged from prior cut-offs; 9.8 months in the exp group and 7.6 in the control (NS). ONCOS-102 + P/C induced a pronounced increase in tumor infiltration by CD4+, CD8+ and granzyme B expressing CD8+ T-cells as well as M1:M2 macrophage polarization in patients with disease control (n = 13) vs progressing patients (n = 3). The data from RNAseq and qPCR analysis will be presented. Conclusions: The addition of ONCOS-102 to P/C was well tolerated by MPM patients and resulted in numerically improved 30-month survival rate in the overall population, and improved mOS in chemotherapy-naïve patients, albeit not statistically significant. Substantial immunological activation in tumor associated with ONCOS-102 was demonstrated, correlating with clinical benefit. Further exploration of ONCOS-102 as a treatment option in MPM is warranted Clinical trial information: NCT02879669.

Background Malignant pleural mesothelioma (MPM) is a rare, aggressive malignancy without curative treatment. Majority of patients receive pemetrexed/cisplatin as standard of care (SoC). Median overall survival in unresectable disease is 12 months. ONCOS-102 is a granulocyte-macrophage colony stimulating factor (GM-CSF) expressing oncolytic adenovirus (Ad5/3-D24-GMCSF) with a unique ability to both prime and boost immune responses. The aim of the study was to assess immune and clinical responses as well as safety in patients with 1st and 2nd line unresectable MPM. Methods Eligible patients (experimental arm, n=20) received ONCOS-102 given intratumorally under CT or US guidance at a dose of 3 × 10 × 11 on Day 1, 4, 8, 36, 78 and 120 plus six cycles of SoC starting on Day 22. The control group (n=11) received only SoC. Imaging was done at baseline, Day 43–64 and 127–148. Patients were monitored regularly for immunological assessment including lesional biopsies (baseline and Day 36). Primary objective was safety and tolerability. Secondary objectives were immunological activation, ORR, PFS and OS as well as correlation between immunological activation and clinical outcome. Results There were no safety concerns nor DLTs. In 1st line patients ORR/DCR was 30%/90% in the experimental group and 33%/83% in the control group. 2nd line patients had ORR/DCR of 11%/67% in the experimental group and 60%/80% in the control group. 12-month survival rate for the 1st line pts was 64% in the experimental group and 50% in the control group. PFS and OS are still to be reported. The treatment with ONCOS-102 induced strong upregulation of multiple genes associated with immune activation in tumor lesions. Profound innate and adaptive immune activation was observed in the experimental vs control group that was associated with better clinical outcome. In addition to an increase in intra-tumoral cytotoxic T-cells (10/15 pts), the treatment with ONCOS-102 resulted in polarization from M2 to M1 macrophages. An upregulation of PD-L1 was reported in 9/15 pts in the experimental group vs 2/5 pts in the control arm, highlighting the potential of ONCOS-102 as an immunosensitizing agent for combinatory therapies with checkpoint inhibitors. Conclusions ONCOS-102 treated patients benefited from superior immune activation compared to patients receiving SoC with preliminary signals of clinical efficacy. Upregulation of adaptive immunity and cytotoxicity related gene expression, PD-L1 level and M2 to M1 macrophage polarization indicate that ONCOS-102 can induce a favourable TME modulation thus providing a scientific rationale for combination with check point inhibition. Trial Registration ClinicalTrials. gov NCT02879669 Ethics Approval This study was approved by the IRBs of all the sites in Madrid, Barcelona, Rennes and Poitiers.

Development of plant vascular tissues involves tissue identity specification, growth, pattern formation and cell-type differentiation. Although later developmental steps are understood in some detail, it is still largely unknown how the tissue is initially specified. We used the early

Development of biologics or cell therapeutics which target MHC class I:peptide (pMHC) complexes for recognition and elimination of tumor cells is hindered by low affinity, cross-reactivity or challenging biochemical properties of antibody- and T-cell receptor-based binders. We hypothesized that DARPin® proteins may be particularly effective in solving this problem due to structural characteristics of their antigen binding surface and excellent biophysical properties. Panels of binders highly specific for a given pMHC complex were isolated from DARPin® libraries through several rounds of selection and counter selection on the relevant or irrelevant but structurally similar pMHC complexes using ribosome display. DARPin® binders were successfully isolated against pMHC complexes composed of different MHC class I alleles with various peptides derived from either tumor associated antigens or non-self viral proteins. A selected panel of DARPin® binders specific to HLA-A2 molecule in association with SLLMWITQC (SLL peptide), a peptide derived from NY-ESO-1, was used to create bi-specific T-cell engagers containing another moiety binding to the epsilon component of the CD3 complex, thus allowing highly sensitive analysis of pMHC specificity and potential cross-reactivity. Using a number of cellular assays, including peptide pulsing of TAP-deficient T2 cells, we confirmed high specificity of selected DARPin® proteins to the HLA-A2:SLL complex. Architectural fine tuning and sequence engineering allowed us to further increase the potency of the selected candidates without compromising the specificity. This was manifested as effective T cell mediated activation by relevant DARPin® constructs in the presence of HLA-A2+/NY-ESO-1+ tumor cells. Furthermore, HLA-A2+/NY-ESO-1+ cells but not HLA-A2+/NY-ESO-1- cells were effectively killed in the presence of engineered HLA-A2:SLL-specific DARPin® T-cell engagers. The versatility of the DARPin® platform and the engineering experience gained with NY-ESO-1 did allow us to identify highly potent and specific DARPins® proteins for additional clinically attractive pMHC targets.Alanine and X-scan mutagenesis demonstrated that, in many cases, interactions with several peptide residues located across the entire peptide sequence are critical for DARPin® protein binding to the pMHC complex. These data suggest that peptide residues exposed outside of the MHC peptide binding grove create the focal point of MHC:peptide:DARPin® proteins interactions. Further molecular and cellular analysis of DARPin® protein specificity will be evaluated in order to de-risk for potential clinically relevant toxicity.In conclusion, we show that the DARPin® technology platform may be highly instrumental in developing a new class of anti-cancer therapeutics based on specific targeting of pMHC complexes presented selectively by cancer cells. Citation Format: Natalia Venetz, Sandra Müller, Tim Schulte, Stefanie Fischer, Maria Paladino, Nicole Pina, Nadir Kadri, Sandra Bruckmaier, Andreas Cornelius, Tanja Hospodarsch, Tatyana Sandalova, Victor Levitsky, Adnane Achour, Marcel Walser. Application of the DARPin® technology for specific targeting of tumor-associated MHC class I:peptide complexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1349.

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti–4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor–mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP–4-1BBL (RG7826) and CD19–4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen–mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP–4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer–bearing rhesus monkey. Combination of FAP–4-1BBL with tumor antigen–targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP– or CD19–4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8(+) T cells. FAP– and CD19–4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

// Melissa M. Norstrom 1, * , Emelie Radestad 2, * , Berit Sundberg 2 , Jonas Mattsson 2, 3 , Lars Henningsohn 4 , Victor Levitsky 5, 8 , Michael Uhlin 2, 3, 6, 7 1 Pharmaceutical Sciences (PS), Roche Pharmaceutical Research and Early Development (pRED), Roche Innovation Center, Basel, Switzerland 2 Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden 3 Centre for Allogeneic Stem Cell Transplantation (CAST), Karolinska University Hospital, Huddinge, Sweden 4 Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institute, Stockholm, Sweden 5 Roche Pharmaceutical Research and Early Development (pRED), Roche Innovation Center, Zurich, Switzerland 6 Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden 7 Department of Applied Physics, Royal Institute of Technology, Stockholm, Sweden 8 Current address: Oncology Research, Molecular Partners AG, Zurich, Switzerland * These authors have contributed equally to this work Correspondence to: Emelie Radestad, e-mail: emelie.radestad@ki.se Keywords: benign prostatic hyperplasia, prostate-infiltrating lymphocytes, immunophenotyping, cytokine/chemokine profiling, inflammation Received: December 03, 2015 Accepted: February 28, 2016 Published: March 14, 2016 ABSTRACT Benign prostatic hyperplasia (BPH) is a common chronic non-malignant condition whose prevalence substantially increases with age. Immune cell infiltration and pro-inflammatory mediators have been implicated in the pathogenesis. Here, we characterized 21 extracellular markers on prostate-infiltrating lymphocytes (PILs) and analyzed expression of 26 soluble proteins in prostate tissue obtained from BPH patients ( n = 31). These data were correlated with clinical parameters and compared with peripheral blood mononuclear cells (PBMCs) ( n = 10). Increased frequencies of T cells expressing co-inhibitory receptors LAG-3, PD-1, TIM-3 or CTLA-4, and co-stimulatory receptors CD28, OX40 or 4-1BB were observed in BPH tissue compared to PBMCs. These findings are consistent with chronic activation and possible functional exhaustion of PILs that may be further augmented by several identified pro-inflammatory factors, such as IL-8 and MCP-1, promoting inflammation and chemotaxis of immune cells to the prostate. Prostate size and plasma prostate-specific antigen levels positively correlated with IL-8 and MCP-1 concentrations, and frequencies of T cells expressing CTLA-4 and TIM-3. It remains to be established whether the link between inflammation and BPH progression supported by our findings reflects a progressive failure of the immune system leading to decreased immune surveillance and development of prostate cancer.

CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily which can activate both innate and adaptive tumor immunity, making it an attractive target for cancer immunotherapy. Systemic administration of agonistic anti-CD40 antibodies has shown signs of activity in cancer patients, but dose-limiting toxicity has prevented the exploration of the full dose range and possibly has impaired clinical efficacy. To overcome this issue, we developed a multispecific DARPin® therapeutic candidate, MP0317, intended to activate CD40 receptor locally, rather than systemically, in the tumor microenvironment through cross-linking with fibroblast activation protein (FAP) that is abundantly present in the stroma of many solid tumors, but has limited expression in healthy adult tissues. Using primary human 1) B cells and 2) dendritic cells, MP0317 activated the CD40 pathway in vitro. Upregulation of CD80, CD86, CD69 and IL12 occurred exclusively in the presence of FAP-positive, but not with FAP-negative cells, confirming a mechanism of action strictly dependent on FAP-mediated cross-linking. A surrogate mouse-specific FAP x CD40 DARPin® molecule (mFAP x mCD40) was generated and tested in murine cell-based in vitro assays showing comparable results as MP0317 with a strict FAP-dependent activation of CD40. The mFAP x mCD40 was also active in vivo and inhibited substantially the progression of FAP-positive tumors. Moreover, in contrast to an anti-mouse CD40 antibody (clone FGK45), the antitumor activity of mFAP x mCD40 was neither associated with elevated blood cytokine levels nor with hepatotoxicity, both of which manifest as dose limiting toxicities of some of the clinical CD40 activating antibodies. These data support a mode of action, both in vitro and in vivo, that is dependent on FAP-mediated crosslinking of CD40 receptor resulting in a tumor-localized CD40 activation without peripheral or off-tumor organ toxicity. In conclusion, we have designed a tumor-targeted CD40 agonistic DARPin® therapeutic candidate that is able to activate the CD40 receptor locally in FAP-positive tumors producing impressive antitumor activity in the absence of systemic toxicity. Citation Format: Nicolo Rigamonti, Sarah Jetzer, Niels Van der Velden, Omar Abdelmotaleb, Sophie Barsin, Jonas Schwestermann, Susanne Mangold, Yvonne Kaufmann, Ralph Bessey, Andrea Valeri, Heidi Poulet, Christof Zitt, Pamela A. Trail, Victor Levitsky, Niina Veitonmäki, Clara Metz. A tumor-targeted CD40 agonistic DARPin® molecule leading to antitumor activity with limited systemic toxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1073.

Following the clinical success of checkpoint inhibitors, cancer immunotherapy is rapidly expanding into combination treatments to enhance response rates and duration. Engagement of co-stimulatory molecules from the tumor necrosis factor receptor (TNFR) superfamily, including 4-1BB, may be a promising approach to enhance the benefits of cancer immunotherapy. Agonistic antibodies against the costimulatory receptor 4-1BB (CD137) have been shown to effectively enhance the anti-tumor activity of checkpoint inhibitors and other agents in preclinical animal models. However, the clinical development of 4-1BB agonistic antibodies has met with limited success thus far. Anti-4-1BB monoclonal antibodies have either been reported to cause significant dose-limiting hepatotoxicity or demonstrated limited efficacy as single agent therapeutics. We generated a DARPin® therapeutic candidate, MP0310 (AMG 506), which comprises domains binding to 4-1BB and fibroblast activation protein (FAP). MP0310 triggers 4-1BB activation only if bound and clustered via FAP which is abundantly expressed by cancer associated fibroblasts present in many solid tumors. In vitro functional assays indicate that MP0310 is a potent T cell co-stimulator in the presence of FAP-expressing cells. In vivo activity of tumor-targeted 4-1BB agonism was assessed in the HT-29 colon carcinoma xenograft model in PBMC humanized mice in combination with a T cell engager. Consistent with tumor targeting, MP0310 enhanced intra-tumoral CD8 T cell expansion while showing only limited systemic activity. We used a translational pharmacokinetic-pharmacodynamic (PK-PD) modeling approach to integrate in vitro and in vivo data to support the estimation of a minimal anticipated biological effect level (MABEL) and the relevant dose range for first in human (FIH) studies. In addition to a PK model describing the MP0310 concentrations over time in mouse and monkey, direct and indirect response models were used to describe receptor occupancy (RO), intra-tumoral CD8 T cell infiltration and peripheral CD8 count kinetics. Predictions from the combined PK and PD models using average intra-tumoral and peripheral drug concentrations (Cav) provide a MABEL dose with minimal expected systemic PD effects at 20% RO as well as the anticipated therapeutic dose range in humans. In conclusion, our PK/PD-modeling approach provides support for the selection of a safe starting dose for MP0310 (AMG506) in patients. It provides a rationale for selecting the maximum tested dose, and may help reduce the number of cancer patients receiving sub-therapeutic doses. MP0310 (AMG 506) is currently being evaluated in a Ph1 clinical study. Citation Format: Alexander Link, Laurent Juglair, Heïdi Poulet, Guy Lemaillet, Christian Reichen, Patricia Schildknecht, Ivana Tosevski, Joanna Robinson, Niina Veitonmäki, Jörg Herbst, Keith Dawson, Christof Zitt, Camila de Almeida, Rik de Greef, Victor Levitsky, Michael T. Stumpp, Hong Ji, Elmar Vom Baur. Selection of first-in-human clinical dose range for the tumor-targeted 4-1BB agonist MP0310 (AMG 506) using a pharmacokinetic/pharmacodynamics modeling approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2273.

Based on the unprecedented clinical efficacy of PD-1/PD-L1 pathway checkpoint inhibitors (CPI), non-redundant immune checkpoints like TIM-3, LAG-3, TIGIT or BTLA are currently being targeted, by combinatorial approaches using monospecific or bispecific antibodies. Up-regulation of TIM-3 has been described as an adaptive CPI resistance mechanism, and internal prevalence data on archival samples of CPI-naïve and -experienced patients showed co-expression of PD-1 and TIM-3 in various tumor types, consistent with literature reports. Here, we describe RG7769 (PD1-TIM3), a novel avidity driven heterodimeric PD-1/TIM-3 1+1 bispecific CrossMabVH-VL intentionally designed as high affinity PD-1 (KD 250 pM, 37°C) and low affinity TIM-3 (KD 130 nM, 37°C) Fab-moieties to specifically target PD-1+ and PD-1+ TIM-3+ T cells through avidity gain, while bypassing PD-1- TIM3+ myeloid and NK cells. In contrast to IgG4-based PD-1 antibodies and conventional IgG1-based TIM-3 Fc-effector function competent antibodies, RG7769 harbors a PG LALA containing heterodimeric KiH IgG1 Fc-region rendering the BsAb refractory to drug shaving by FcgR-expressing macrophages in the TME, while retaining IgG-pharmacokinetics. RG7769 binds to PD-1 with higher affinity than pembrolizumab and nivolumab. X-ray crystallography demonstrated that the humanized PD-1 binding Fab recognizes a unique glycosylated epitope on PD-1, and potently blocks the PD-1/PD-L1 and PD-1/PD-L2 interactions in both biochemical and reporter cell line assays. The humanized TIM-3 binding arm was identified for maximal functional activity using mixed lymphocyte reaction (MLR) assays. Compared with bivalent TIM-3 antibodies, RG7769 shows reduced binding to TIM-3+ myeloid and NK cells, but binds preferentially to dysfunctional T cells expressing PD-1 or both PD-1 and TIM-3, like tumor infiltrating lymphocytes (TILs) in the tumor microenvironment. By virtue of its monovalency, RG7769 induced low antibody internalization on activated T cells when compared with bivalent TIM-3 antibodies, overcoming a major cellular sink for TIM-3 antibodies. In functional assays, RG7769 showed increased IFN-γ secretion by in vitro generated tumor-specific T-cells, increased ex vivo tumor-specific effector functions of T cells from PBMCs of melanoma patients, and enhanced the anti-tumor-activity of TILs from melanoma patients when compared to the monospecific parental PD-1 antibody. Finally, RG7769 showed superior efficacy in controlling s.c. MC38 tumor growth in huPD-1/huTIM-3 transgenic C57/BL6 mice compared to the parental PD-1 antibody. In summary, these preclinical data support the use of RG7769 as a monotherapy and as combination partner for the treatment of patients with solid/hematological tumors. A phase I study is currently ongoing in patients with advanced metastatic solid tumors (NCT03708328). Citation Format: Laura Laura Codarri Deak, Stefan Seeber, Mario Perro, Patrick Weber, Laura Lauener, Standford Chen, Sonja Offner, Stefan Dengl, Friederike Hesse, Adrian Zwick, Marco Boettger, Alexander Bujotzek, Jörg Benz, Guy Georges, Georg Fertig, Valeria Lifke, Jens Fischer, Stephane Leclair, Victor Levitsky, Marta Canamero, Juha Lindner, Sara Colombetti, Stefanie Bendels, Christophe Boetsch, Matthias Fueth, Merlind Muecke, Henry Kao, Pablo Umana, Christian Klein. RG7769 (PD1-TIM3), a novel heterodimeric avidity-driven T cell specific PD-1/TIM-3 bispecific antibody lacking Fc-mediated effector functions for dual checkpoint inhibition to reactivate dysfunctional T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2270.

Development of biologics or cell therapeutics which target MHC class I:peptide (pMHC) complexes for recognition and elimination of tumor cells is hindered by low affinity, cross-reactivity or challenging biochemical properties of antibody- and T-cell receptor-based binders. We hypothesized that DARPin® proteins may be particularly effective in solving this problem due to structural characteristics of their antigen binding surface and excellent biophysical properties. Panels of binders highly specific for a given pMHC complex were isolated from DARPin® libraries through several rounds of selection and counter selection on the relevant or irrelevant but structurally similar pMHC complexes using ribosome display. DARPin® binders were successfully isolated against pMHC complexes composed of different MHC class I alleles with various peptides derived from either tumor associated antigens or non-self viral proteins. A selected panel of DARPin® binders specific to HLA-A2 molecule in association with SLLMWITQC (SLL peptide), a peptide derived from NY-ESO-1, was used to create bi-specific T-cell engagers containing another moiety binding to the epsilon component of the CD3 complex, thus allowing highly sensitive analysis of pMHC specificity and potential cross-reactivity. Using a number of cellular assays, including peptide pulsing of TAP-deficient T2 cells, we confirmed high specificity of selected DARPin® proteins to the HLA-A2:SLL complex. This was manifested as effective activation of T-cells in the presence of relevant DARPin® constructs tested at a range of concentrations and HLA A2 positive cells pulsed with the SLL or irrelevant HLA A2 binding peptides. Furthermore, HLA-A2+/NY-ESO-1+ cells but not HLA-A2+/NY-ESO-1- cells were effectively killed in the presence of HLA-A2:SLL-specific T-cell engagers. Alanine scanning mutagenesis demonstrated that, in many cases, interactions with several peptide residues located across the entire peptide sequence are critical for DARPin® protein binding to the pMHC complex. These data suggest that peptide residues exposed outside of the MHC peptide binding grove create the focal point of MHC:peptide:DARPin interactions. Further molecular and cellular analysis of DARPin® protein specificity using additional molecular and cellular approaches will be presented in order to de-risk for potential clinically relevant toxicity which may result from off-target interactions with other pMHC complexes structurally resembling the tumor-associated pMHC complex. In conclusion, we show that the DARPin® technology platform may be highly instrumental in developing a new class of anti-cancer therapeutics based on specific targeting of pMHC complexes presented selectively by cancer cells. Citation Format: Marcel Walcer, Natalia Venetz, Tim Schulte, Stefanie Fischer, Nicole Bassler, Maria Paladino, Nicole Pina, Denis Villemagne, Sandra Bruckmaier, Andreas Cornelius, Tanja Hospodarsch, Loic Duffet, Tatjana Sandalova, Adnane Achour, Victor Levitsky. Application of the DARPin® technology for specific targeting of tumor-associated MHC class I:peptide complexes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 690.

BACKGROUND Benign prostatic hyperplasia (BPH) is the most common benign adenoma and prostate cancer is the most frequent malignancy in men over 50 years of age in the Western world, where it remains a significant health problem. Prostate lesions are known to contain immune cells, which may contribute to the immune control of tumor progression. However, due to their low numbers and restricted access to necessary material it is difficult to isolate immune cells from prostate tissue to characterize their immunological features. METHODS An efficient and robust method was developed to process prostate tissue and isolate immune cells for phenotypic analysis by multicolor flow cytometry as downstream application. Fresh prostate tissue from 11 patients undergoing surgery for bladder outlet obstruction due to BPH was processed to evaluate the number, viability, yield, and frequency of various immune cell types. RESULTS The presented method does not include enzymatic digestion nor incubation steps at 37°C, increasing cellular viability and avoiding possible phenotypic modification. Various immune cell populations were detected in all patient samples and the median cellular viability was 90%. The number of detected events of individual cell populations varied between patients. The median frequency of different immune cell populations also varied, being 87% for the CD3- and 15% for the CD3+ cell population. CONCLUSIONS This novel method will allow the phenotypic characterization of immune cell populations present in tumor tissue of prostate cancer patients and promote development of novel approaches to immunotherapy of the disease. Prostate 74:1391–1399, 2014. © 2014 Wiley Periodicals, Inc.

CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor receptor superfamily which can activate both innate and adaptive immune system, making it an interesting target for tumor immunotherapy. Systemic administration of agonistic CD40 antibodies (Ab) has shown signs of activity in cancer patients, but dose-limiting toxicity impaired the clinical efficacy. New approaches are therefore needed to increase the therapeutic index of CD40-targeting molecules and achieve better clinical outcomes. Here, we report an alternative approach designed to activate CD40 specifically in the tumor microenvironment (TME), and not systemically, in order to increase efficacy and reduce systemic toxicity. This novel approach is based on a bispecific DARPin® molecule, targeting CD40 and fibroblast activation protein (FAP) alpha, intended to induce immune activation only when clustered by binding to FAP-expressing cells in the TME. The bispecific FAP x CD40 DARPin®molecule was tested in a reporter assay and in additional cell assays using primary human 1) B cells, 2) macrophages and 3) dendritic cells. These studies demonstrated CD40 activation only in the presence of FAP-positive, but not with FAP-negative cells, confirming a mechanism of action strictly dependent on FAP-mediated cross-linking. A surrogate mouse-specific FAP x CD40 DARPin® molecule (mFAP x CD40) was generated and tested in similar in vitro assays and showed FAP-dependent activation of CD40 and comparable results as the human construct. In vivo experiments, performed in tumor-free mice, showed a comparable half-life between mFAP x CD40 and an anti-mouse CD40 Ab (clone FGK45). However, mFAP x CD40, in contrast to the FGK45 Ab, did not increase the serum level of IL-6, supporting a mode of action that is dependent on FAP-mediated crosslinking of CD40 receptor. In additional studies mFAP x CD40 was active and inhibited the growth of FAP+ tumors. There were no signs of toxicity with mFAP x CD40 in contrast to the FGK45 Ab which resulted in body weight loss. In conclusion, we have generated bispecific agonist FAP x CD40 DARPin® molecules able to activate the CD40 pathway with a targeting (FAP)-dependent mechanism of action. Citation Format: Nicolo Rigamonti, Anja Schlegel, Sophie Barsin, Jonas Schwestermann, Susanne Mangold, Yvonne Kaufmann, Christof Zitt, Niina Veitonmäki, Victor Levitsky, Clara Metz. Fibroblast activation protein (FAP)-selective delivery of CD40 agonistic DARPin®molecule for tumor-localized immune activation [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 3251.

Background: Antibodies against several agonistic immunomodulatory receptors proved to be highly efficacious in preclinical tumor models especially in combination with other immunomodulatory compounds and treatment modalities. However, their clinical application met with limited success and was associated with systemic toxicities which are believed to be driven by Fc-receptor-mediated cross-linking of antibody molecules. We developed a new class of biologics based on the DARPin® technology platform, which allow tumor-localized engagement of immunomodulatory agonists in the absence of Fc-receptors. Preclinical in vitro and in vivo results indicate that DARPin®-based compounds are at least comparable to conventional antibodies in agonistic potency. The localization of their activity to the tumor site relative to the systemic activity of agonistic MAbs should improve the safety and therefore the therapeutic index for drug candidates which exploit this mode of action. Methods: We generated multi-specific DARPin® therapeutics, which bind to human fibroblast activation protein (FAP) and either to 4-1BB, OX40 or CD40 receptors. Each of these receptors can effectively enhance immune responses through a set of distinct and non-redundant mechanisms. Functional properties of these molecules were characterized using appropriate sets of biochemical methods and cell-based assays utilizing reporter cells lines or various subsets of primary immune cells. The FAP-targeted 4-1BB agonist referred to as MP0310, which represents our most advanced drug candidate, was also characterized in vivo in terms of its pharmacokinetics and pharmacodynamics in mice as well as cynomolgus monkeys. The in vivo efficacy of MP0310 was analyzed both in monotherapy and in combination with tumor-specific T-cell engagers using a xenotransplantation human tumor model of immunodeficient mice engrafted with human PBMCs. Results and Conclusions: All analyzed FAP-targeted DARPin®-based agonistic immunomodulators efficiently induced activation of the NFkB pathway in reporter cell lines in a dose dependent manner but only upon crosslinking through binding to FAP. The same pattern of functional activity was exhibited by the molecules in assays with appropriately selected human primary immune cell subsets. We show that MP0310 promotes tumor regression in a dose dependent manner when co-administered with a tumor-specific T-cell engaging bi-specific antibody into tumor-bearing immunodeficient NGS mice engrafted with human PBMCs. The observed effect correlated well with 4-1BB receptor occupancy in blood and an increase of CD8 T lymphocytes in the tumors. Urelumab, a clinically tested 4-1BB agonistic antibody associated with liver toxicity, led to signs of severe graft-versus-host disease (GVHD) and weight loss in the humanized NOG mice. In contrast, mice treated with several doses of MP0310 showed only minor signs of GVHD and marginal weight loss. Thus, tumor targeted DARPin® agonists of co-stimulatory molecules can enhance T-cell activity in a manner independent of Fc-crosslinking and localized primarily to the tumor site, thereby avoiding the systemic toxicity seen with antibodies against the same immunologic targets. This approach with DARPin® therapeutics can be broadly applied to a range of tumor targets and immune receptors. Citation Format: Victor Levitsky, Alexand Link, Michael T. Stump, Elmar vom Baur. Development of tumor-targeted agonistic Immunomodulators using the DARPin® technology platform [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B026.

EBV is associated with a broad range of malignancies. Adoptive immunotherapy of these tumors with EBV-specific CTL proved useful. We generated a panel of primary human T cells specific to various EBV antigens (i.e. Epstein-Barr nuclear antigen 3A, 3B and BamHI-M leftward reading frame) via transfer of modified TCR genes that are either coupled to CD3zeta or Fc(epsilon)RIgamma. TCR-transduced T cells from 20-60% of donors (total number of 25) demonstrated specific lysis of EBV peptide-loaded target cells, whereas lymphoblastoid cell lines expressing native EBV antigens were not killed by any of the EBV-specific T cell populations. This non-responsiveness, confirmed at the level of nuclear factor of activated T cells activation, is not due to receptor configuration since identical receptor formats specific for melanoma antigens successfully re-targeted T cells to native melanoma cells. In an effort to generate a more potent receptor, we introduced a CD28 domain into one of the EBV-specific TCR. This TCR did not affect the cytotoxic response of re-targeted T cells, but dramatically enhanced antigen-specific IFNgamma production. We therefore conclude that these novel CD28-containing EBV-specific TCRs provide a basis for further development of TCR gene transfer to treat EBV-induced diseases.

The syntaxin 11 (STX11) gene is mutated in a proportion of patients with familial haemophagocytic lymphohistiocytosis (FHL) and exocytosis of cytotoxic granules is impaired in STX11-deficient NK cells. However, the subcellular localization, regulation of expression and molecular function of STX11 in NK cells and other cytotoxic lymphocytes remain unknown. Here we demonstrate that STX11 expression is strictly controlled by several mechanisms in a cell-type-specific manner and that the enzymatic activity of the proteasome is required for STX11 expression in NK cells. In resting NKL cells, STX11 was localized in the cation-dependent mannose-6-phosphate receptor (CD-M6PR)-containing compartment, which was clearly distinct from cytotoxic granules or Rab27a-expressing vesicles. These subcellular structures appeared to fuse at the contact area with NK-sensitive target cells as demonstrated by partial colocalization of STX11 with perforin and Rab27a. Although STX11-deficent allo-specific cytotoxic T-lymphocytes efficiently lysed target cells and released cytotoxic granules, they exhibited a significantly lower extent of spontaneous association of perforin with Rab27a as compared with STX11-expressing T cells. Thus, our results suggest that STX11 promotes the fusion of Rab27a-expressing vesicles with cytotoxic granules and reveal an additional level of complexity in the spatial/temporal segregation of subcellular structures participating in the process of granule-mediated cytotoxicity.

BCR-triggering shown as the first known B-cell-specific signal enhancing cellular sensitivity to TRAIL. TRAIL is known to cause death in tumor cells, but physiological regulation of its activity remains poorly characterized. We demonstrate that BCR triggering sensitizes transformed centroblast-like BL cells and peripheral blood memory B cells to TRAIL-mediated apoptosis. The sensitization correlated with surface down-regulation and intracellular retention of TRAIL-R4, along with changes in the expression of several Bcl-2 protein family members. Although enhancing FAS-mediated cell death, CD40 activation protected B cells from TRAIL-induced apoptosis. Combination of Ig cross-linking with CD40 ligation did not prevent TRAIL-R4 down-regulation but induced changes in the mitochondria-regulated pathway of apoptosis that are known to be associated with resistance to TRAIL. Human CD5+ B cells, presumably stimulated by reactivity to self without immunological help, exhibited very high ex vivo sensitivity to TRAIL. Our results define the first B-lymphocyte-specific physiological signal that increases cellular sensitivity to TRAIL. This may be important for our understanding of TRAIL involvement in the control of B cell responses and aid in designing TRAIL-based therapies for B cell lymphomas.

We have previously shown that factors secreted by activated CTLs induce apoptosis in a panel of glioblastoma lines. In this study, we analyzed the expression of death receptors, activation of caspases and mRNA expression of 96 apoptotic genes in glioblastoma lines either sensitive or resistant to supernatant of activated CTLs. Our results indicate that exposure to supernatant triggers several pathways of caspase activation in glioblastoma lines involved in the initiation of both extrinsic and intrinsic apoptosis. High steady-state levels of Bcl-2 were identified as potentially accounting for the resistance of a proportion of glioblastoma lines to factors secreted by activated CTLs.

Immunomodulating agents have revolutionized anti-cancer therapy. However, monotherapy is often not sufficient, and development of combination treatments is hampered by cumulative toxicity. In an attempt to overcome this challenge, a tumor-restricted agonistic 4-1BB/FAP DARPin drug candidate, which induces T-cell co-stimulation only when clustered by binding to fibroblast activation protein alpha (FAP) expressing cells, has been developed. FAP is a type II membrane-bound glycoprotein abundantly expressed in the stroma of many solid tumors by cancer-associated fibroblasts. As shown previously using in vitro and in vivo models (HT-29), co-stimulation induced by a FAP-targeted 4-1BB agonistic DARPin molecule leads to enhanced activation and expansion of CD8+ T-cells. To support clinical development of the drug candidate, tumor localization and accumulation were studied by whole-body SPECT/CT imaging and quantitative biodistribution using Indium-111 labeled DARPin molecules in a human colorectal adenocarcinoma (HT-29) xenograft model in CD1 nude mice. Immunohistochemical staining of tumor stroma confirmed local expression of FAP. Labeled 4-1BB/FAP DARPin molecules specifically accumulated in FAP-expressing tumor in vivo. SPECT/CT imaging and biodistribution revealed a maximum tumor accumulation of around 15% of the injected dose per gram of tissue around 72 h post injection. High tumor/blood ratios were observed one week post injection because the activity in the blood decreased according to the expected serum half-life of 26 h, determined in separate pharmacokinetic studies in BALB/c mice following single dose intravenous bolus injections. Based on the decrease of radioactivity in the tumor, a tumor residence half-life of approximately 4 days was calculated, indicating an extended tumor retention potentially due to FAP binding. No accumulation was observed in the muscle tissue that was choosen as a rather weakly-perfused control tissue. Taken together, FAP-targeting of a 4-1BB agonist DARPin molecule resulted in expected high tumor accumulation and retention compared to an untargeted version of the molecule, both relevant observations for further preclinical and clinical studies. These findings suggest that tumor-targeting via FAP has the potential to induce T-cell activation restricted to the tumor site, and thereby reducing toxicities caused by systemic 4-1BB activation. In conclusion, immunostimulatory drugs with tumor-targeted activity may have the potential to circumvent current limitations of immunotherapy and allow safe and effective use, in particular in combination therapy. Citation Format: Christian Reichen, Ralph Bessey, Christine DePasquale, Stefan Imobersteg, Martin Behe, Alain Blanc, Roger Schibli, Alexander Link, Laurent Juglair, Joanna Taylor, Patricia Schildknecht, Julia Hepp, Elmar vom Baur, Hong Ji, Christof Zitt, Victor Levitsky, Keith M. Dawson, Michael T. Stumpp, Dan Snell. FAP-mediated tumor accumulation of a T-cell agonistic FAP/4-1BB DARPin drug candidate analyzed by SPECT/CT and quantitative biodistribution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3029.

Agonistic antibodies against the T cell costimulatory receptor 4-1BB (CD137) have proved to be very efficacious anti-tumor agents in preclinical animal models. However, clinical development of 4-1BB agonistic antibodies has met with limited success thus far. Anti-4-1BB monoclonal antibodies have either been reported to cause significant dose-limiting hepatotoxicity or demonstrated limited efficacy as single agent therapeutics. Here we describe the generation of a tumor-targeted 4-1BB agonist aimed at inducing more effective triggering of 4-1BB without associated systemic toxicity. Tumor targeting is achieved via fibroblast activation protein (FAP) which is abundantly expressed by cancer associated fibroblasts present in many solid tumors. Drug candidate MP0310 comprises DARPin domains binding to 4-1BB and FAP and is devoid of an antibody Fc domain. Compared to first generation monoclonal antibodies targeting 4-1BB, MP0310 shows high potency in vitro and less systemic activation in vivo. In vitro reporter and T cell assays indicate that MP0310 is a potent T cell co-stimulator whose activity is restricted to the presence of FAP-expressing cells. In humanized mouse xenograft studies, FAP-targeted 4-1BB activation induced potent co-stimulation of CD8 T cells leading to tumor growth inhibition. On the other hand, the DARPin molecule did not induce effects associated with strong systemic activation such as hepatotoxicity or exacerbation of graft versus host disease observed in such models, unlike the first generation FcγR-dependent 4-1BB antibodies. In addition, no systemic activation of T cell proliferation was observed in the absence of FAP-positive tumors. In healthy cynomolgus monkeys, administration of MP0310 did not induce systemic stimulation of memory T cell proliferation in contrast to an anti-4-1BB antibody despite MP0310 being fully cross-reactive to cyno 4-1BB and binding effectively to cyno FAP. Therefore, we conclude that the tumor-restricted co-stimulation of 4-1BB may prevent toxicities caused by systemic 4-1BB activation and provide a safe and effective way to boost anti-tumor T cell responses. This could allow more effective dosing and better combination therapies with checkpoint inhibitors and other immune stimulating drugs. MP0310 is in preparation to enter clinical development. Citation Format: Alexander Link, Julia Hepp, Christian Reichen, Patricia Schildknecht, Ivana Tosevski, Joanna Taylor, Laurent Juglair, Alexander Titz, Mirela Matzner, Ralph Bessey, Christof Zitt, Guy Lemaillet, Joerg Herbst, Keith M. Dawson, Hong Ji, Victor Levitsky, Dan Snell, Michael T. Stumpp, Andreas Harstrick, Elmar vom Baur. Preclinical pharmacology of MP0310: A 4-1BB/FAP bispecific DARPin drug candidate promoting tumor-restricted T-cell costimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3752.