Immunotherapy Bridge 2018
SITC Session—evolving topics in cancer immunotherapy: tumor microenvironment—oral communications
O1 Cancer immunosuppression induced by albumin derived neo-structures
Canimguide Therapeutics AB, Scheelevägen 2, 223 81 Lund, Sweden
Journal of Translational Medicine 2019, 17(Supp 1):1
Background: Better understanding of immunosuppressor mechanisms is a prerequisite in order to enhance the efficacy of cancer immunotherapy. As the serum concentration of interleukin-6 (IL-6) is increased in the majority of advanced cancer patients, the occurrence of unknown factors inducing this cytokine was further explored.
Materials and methods: Immunoregulatory albumin neo-structures were identified by adsorption to immune cell receptors, further analysed by 2-D gel electrophoresis, MALDI-TOF and sequencing according to Edman. Binding of this neo-structure to LFA-1 was investigated using immunohistochemistry. Production of IL-6 was determined by ELISA.
Results: Proteolytic fragmentation or denaturation of normal serum albumin was found to generate conformational changes, neo-structures, with the capacity to induce IL-6 production by normal mononuclear blood cells (PBMC). Analysis of albumin sequences identified a sequence inducing IL-6. Further analysis of immune cell binding albumin neo-structures also identified a potent immunosuppressor, P3028, binding to LFA-1 and CD25 on immune cells and thereby inhibiting lymphocyte proliferation and migration and NK-cell cytotoxicity. The immunosuppressor 3028 is a physiological blocker of LFA-1, a β2-integrin of fundamental importance for multiple activities of the immune system, crucial for immune mediated cancer control. Blockade of LFA-1 inhibits: Initiation of an immune response, lymphocyte proliferation, lymphocyte recruitment to tumours, migration of these cells within tumours and their cytolytic activity. Blocking of 3028 either by antibodies directed to this structure or by a complementary binding peptide (P28R) reverse cancer related immunosuppression in an in vitro lymphocyte proliferation assay. 3028-structures are frequently found to block LFA-1 in animal as well as human tumours and ex vivo treatment of tumour sections with P28R efficiently unblock this receptor. The 3028-structure is expressed in all types of tumours studied so far, e.g. breast, colon, prostate cancer, squamous cell cancer of the oral cavity, renal cell carcinoma and melanoma. Treatment of spontaneous tumours in dogs by injecting P28R subcutaneously, blocking the immunosuppressor 3028, results in a significantly enhanced inflammatory infiltrate, eradication of tumour cells and an almost complete histopathological regression after a single injection within 5 days.
Conclusions: This investigation identified and characterized a physiological inhibitor of LFA-1, of fundamental importance for multiple activities of the immune system, crucial for immune mediated cancer control. Blocking of the immunosuppressor 3028 is a new therapeutic strategy for reversal of cancer related immunosuppression.
O2 Targeted next generation sequencing for the evaluation of tumor mutation burden
Francesca Fenizia1, Raffaella Pasquale1, Cristin Roma1, Francesca Bergantino1, Nicoletta Chicchinelli1, Paolo Graziano2, Gerardo Botti3, Fabiana Tatangelo3, Giosuè Scognamiglio3, Matilde Lambiase1, Alessia Iannaccone1, Nicola Normanno1
1Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, Italy; 2Unit of Pathology, IRCCS “ Casa Sollievo della Sofferenza” Hospital, San Giovanni Rotondo, Foggia, Italy; 3Pathology Unit, Istituto Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, Italy
Journal of Translational Medicine 2019, 17(Supp 1):2
Background: Tumour mutation burden (TMB) is defined as the total number of mutations per coding area of a tumour genome and it has been associated with clinical response to Immune checkpoint inhibitors (IOs) in different tumor types, including colorectal cancer (CRC) with mismatch repair deficiency . Targeted next generation sequencing (TS) can be an alternative to whole exome sequencing (WES) to determine TMB in clinical practice . We performed TMB analysis on tumor cell lines and CRC samples and compared the results with microsatellite instability (MSI) status to evaluate the clinical robustness of a TS approach.
Materials and methods: Genomic DNA (gDNA) was extracted from CRC cell lines using the QIAgen DNeasy Blood&Tissue kit. To obtain gDNA from FFPE CRC samples, we optimized an extraction method that overcomes fixation issues, which cause an increase of deamination mutations, thus altering TMB values. The extraction workflow includes enzymatic removal of deamination artifacts, which result in sequencing errors. For both samples, TMB libraries were prepared with the Oncomine Tumor Mutation Load Assay (Thermofisher) and sequenced on the Ion S5 platform. TMB was defined as the total number of somatic SNVs divided by number of bases with sufficient coverage. MSI status was evaluated by means of the Bethesda panel and the Idylla MSI assay (Biocartis) .
Results: We first evaluated the TMB in 8 cell lines and the results were correlated with the massively parallel sequencing data from > 1600 genes performed on the same cell lines available on cBioPortal , demonstrating the ability of the panel to infer WES-TMB (R2 = 0.979). In our cell lines, a strong correlation between TMB and MSI status was also observed. The optimized extraction workflow allowed to overcome DNA damage due to fixation and obtain successful sequencing in all the FFPE CRC samples analyzed. TMB evaluation performed on 32 CRC tumor tissues from the Pascale Institute biobank was compared to the MSI results. Significant differences were found in TMB values of MSI-High (MSI-H, n = 9, median value: 40.25) versus microsatellite stable (MSS) cases, as expected (n = 23, median value: 10.97) (Fig. 1; Mann–Whitney test: P < 0.0001).
Conclusions: These data suggest that the developed workflow for TMB testing provides results in line with the expected tumor mutational load. Targeted sequencing can represent a valid approach that might be translated into clinical practice to ensure that patients receive an appropriate TMB test.
Yarchoan M, Hopkins A, Jaffee EM. Tumor Mutational Burden and Response Rate to PD-1 Inhibition. N Engl J Med. 2017;377(25):2500–1.
Chalmers ZR, Connelly CF, Fabrizio D, Gay L, Ali SM, Ennis R, et al. Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden. Genome Med. 2017;9(1):34.
Umar A, Boland CR, Terdiman JP, Syngal S, de la Chapelle A, Ruschoff J, et al. Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst. 2004;96(4):261–8.
Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012;483(7391):603–7.
O3 Activation of immune response in refractory patients to standard treatment
Marcella Occelli1, Andrea Abbona2, Dario Sangiolo3,4, Cristiana Lo Nigro1, Ornella Garrone1, Antonella Falletta2, Merlotti Anna1, Chiara Varamo3,4, Massimo Aglietta3,4, Loretta Gammaitoni4, Martino Monteverde2, Marco Merlano1,2
1Ospedale Santa Croce e Carle Cuneo, Cuneo, Italy; 2Laboratorio di Oncologia Traslazionale, Fondazione Arco Cuneo, Cuneo, Italy; 3Department of Oncology, University of Torino, Torino, Italy; 4Candiolo Cancer Institute FPO-IRCCS, Torino, Italy
Journal of Translational Medicine 2019, 17(Supp 1):3
Background: The TRANSLATE project started in 2016 to test the immune effects of metronomic cyclophosphamide, daily low-dose IL-2 every other week, and a single flash of radiotherapy (RT) in peripheral blood.
The rationale is based on self vaccination induced by radiotherapy (RT) 8 Gy single fraction on one metastatic lesion, T cells expansion by IL-2 treatment and selective Tregs down regulation by lowdose cyclophosphamide administration.
Materials/methods: We enrolled patients with end-stage breast, colon, kidney and prostate cancer. Analysis was performed at baseline, the day after RT, after 28 days from treatment start and at disease progression. Assay focused on Tregs, CD8+, NK, MDSC, CD3-PD1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17a, TNFα, IFNγ, TGFβ. We divided patients into two groups depending on the time of disease progression (A > 3 months; B, < 3 months). We report preliminary data with the aim to show the changes observed post-RT in 20 pts.
Results: At baseline, group B had higher rates of CD3-PD1, higher IL-2, IL-13, TNFα, and TGFβ; group A had higher IFN γ, IL-4, and IL-12. After RT, we observed a difference between the two groups in the rates of CD3-PD1 (lower in group B) and Tregs (higher in group A). Among cytokines, only TNFα reached a statistical significance (higher in group B). We also observed that TGFβ and IL-6 were higher in group B and IFNγ was higher in group A. The longitudinal analyses showed that CD3-PD1 remained stable between basal and post-RT in group A but decreased in group B. Tregs marginally increased in group A. TNFα, TGFβ, IL-4, IL-6, and IL-12 increased in group B. IL-4 decreased in group A and IL-6 and IL-12 also marginally decreased in the same group. IFNγ slightly increases in group A.
Conclusions: The limited number of patients reduced the interpretation of the study. However, following RT a positive trend of Th2 cytokines is observed in patients with early progressing disease, without the expected surge of IFNγ that was instead observed in patients with better outcome. Additional analyses are in progress and will be presented.
O4 Dysregulation of immune modulating molecules and signaling pathways in dendritic cells/DC-based vaccines generated from advanced-stage melanoma patients
Deena M Maurer1, Patricia Santos2, John M Kirkwood2, Ahmad A. Tarhini7, Hussein A. Tawbi5, David Stroncek6, Ping Jin6, and Lisa H Butterfield1,2,3,4
1Department of Immunology, University of Pittsburgh, Pittsburgh PA; 2Department of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh PA; 3Department of Surgery, University of Pittsburgh Cancer Institute, Pittsburgh PA; 4Department of Clinical and Translational Science, University of Pittsburgh, Pittsburgh PA; 5MD Anderson Cancer Center, University of Texas, Houston TX; 6Department of Transfusion Medicine, National Institutes of Health, Bethesda MD; 7Lerner School of Medicine, Case Western Reserve University, Cleveland OH
Journal of Translational Medicine 2019, 17(Supp 1):4
Background: Stage IV melanoma has a 5-year survival rate of only 15–20%. Immune checkpoint blockade has shown therapeutic efficacy in a subset of melanoma patients, often those with pre-existing antitumor immunity. Therapeutic vaccines targeting melanoma-associated antigens are commonly immunogenic, but only rarely effective in promoting clinical responses, suggesting a clear need for further improvement.
Materials and methods: To promote strong anti-tumor immune responses in melanoma patients, we created a vaccine consisting of autologous dendritic cells (DC) transduced ex vivo with a recombinant adenovirus encoding three shared melanoma antigens: Tyrosinase, MART-1, and MAGE-A6 (TMM2). Monocyte-derived DC were first matured with IFN-γ + LPS, and then transduced with recombinant adenovirus encoding TMM2 and administered to patients (n = 35) 3× via bi-weekly intradermal injections in a Phase I trial. Human microarrays were used to analyze gene expression profiles of patient immature, mature, and adenovirus-transduced DC.
Results: Genomic analyses revealed that melanoma patient (but not healthy donor ) DC exhibit a significant increase in expression of transcripts encoding immunosuppressive molecules, such as IDO1 and TXN after ex vivo maturation and viral transduction, when compared to individual-matched immature DC. DC-associated transcripts correlating significantly with clinical outcome include LAMP1, DDO, CLEC4A and NLRP2 linked to antigen-presentation, aspartate degradation, plasmacytoid DC and an inhibitor of TBK1/Type-1 IFN signaling, respectively.
Protein-based analyses indicate ICOSL downregulation on the surface of immature, mature, and transduced DC in melanoma patients versus HD. NFkB signaling, known to regulate ICOSL expression in DC, appears selectively dysregulated in mDC generated from melanoma patients versus HD.
Conclusions: Our profiling data suggest deficiencies in NFkB and Type-1 IFN signaling and costimulatory molecule expression (ICOS-L), as well as elevated expression of immunosuppressive gene products (IDO1, TXN), may serve to limit the immunostimulatory capacity of melanoma patient-derived DC and derivative DC-based vaccines. These data provide clues for targeted manipulation of patient DC to develop improved vaccines implementing DC for the treatment of advanced-stage melanoma patients.
Trial Registration Identifier NCT01622933.
Jin, P., T. H. Han, J. Ren, S. Saunders, E. Wang, F. M. Marincola, and D. F. Stroncek. 2010. Molecular signatures of maturing dendritic cells: implications for testing the quality of dendritic cell therapies. J. Transl. Med. 8: 4.
O5 Class I HDAC inhibitor domatinostat beneficially affects phenotype and functionality of T cells in the tumor microenvironment, and synergizes with PD-1/LAG3 checkpoint blockade
Svetlana Hamm, Ulrike Parnitzke, René Bartz, Frank Hermann
SC AG, Planegg-Martinsried, Germany
Journal of Translational Medicine 2019, 17(Supp 1):5
Background: Anti-PD-1 plus anti-LAG3 therapy was well tolerated in melanoma patients and has shown clinical activity in patients progressive on PD-1 therapy. However, a significant proportion of patients still exhibit a priori resistance or suffer from disease progression. Domatinostat, a selective HDAC class I inhibitor, has been preclinically reported to upregulate MHC molecules, tumor-associated antigen expression, and increase inflammatory signature and T cell infiltration into tumors. Here, we provide preclinical data on the triple combination of domatinostat, anti-PD-1 plus anti-LAG3 antibodies.
Materials and methods: Anti-tumoral efficacy and the impact on tumor microenvironment were analyzed in a syngeneic C38 model with a low response rate to anti-PD-1 and no response to anti-LAG3 treatment, reflecting refractory clinical situation. Clinical equivalent dose of domatinostat was used to ensure translational relevance.
Results: Domatinostat and anti-PD1 in monotherapy reduced C38 tumor growth resulting in 10% and 25% regressions, respectively. The combination of domatinostat plus anti-PD-1 antibody resulted in a stronger tumor growth control with 60% regressing tumors. Anti-LAG3 treatment alone or in combination with either domatinostat or anti-PD-1 did not show added anti-tumoral efficacy. However, addition of domatinostat to anti-PD-1+ anti-LAG3 double combination resulted in a higher anti-tumor activity with a tumor regression rate of 80%. FACS analysis revealed that domatinostat increased expression of MHC class I molecules on tumor cells, and MHC class II molecules on tumor cells, Ly6G+ myeloid derived suppressor cells and M1 macrophages. Domatinostat-mediated upregulation of MHC class II and co-stimulatory molecules [Bretz et al. AACR 2017] allows to hypothesize that the addition of an anti-LAG3 antibody, which blocks detrimental effect of MHC class II/LAG3 engagement on T cell activity, may beneficially affect T cell response by supporting T helper cell activation and function. Consistently, in combination with both checkpoint inhibitors domatinostat strongly enhanced the proportion of proliferating cytotoxic T cells (CTLs) in the tumors and reduced exhaustion phenotype of CTLs and T helper cells.
Conclusions: Domatinostat has previously demonstrated beneficial immunomodulatory effects in syngeneic preclinical tumor models in combination with immunostimulating agents as well as with immune checkpoint blockade. Currently, domatinostat is in clinical evaluation in combination with pembrolizumab in advanced melanoma patients refractory or non-responding to anti-PD-1 (“SENSITIZE” study; NCT03278665). The triple combination of domatinostat, anti-PD-1 and anti-LAG3 in the C38 syngeneic mouse model resulted in a high rate of complete responses, superior to any double combination, suggesting a favorable synergy justifying and warranting clinical investigation.