EXTH-48. NOVEL COMBINATION THERAPY IN PRECLINICAL GLIOMA MODELS USING THE CELL CYCLE STABILIZING COMPOUND ARGYRIN F AND CHECKPOINT INHIBITION

Glioblastoma are incurable aggressive tumors and remain a therapeutic challenge. Glioblastoma frequently harbor alterations in the retinoblastoma pathway with subsequent cell cycle abnormalities. Here, we aimed to investigate the anti-glioma activity of the cell cycle-stabilizing compound Argyrin F and its potential treatment-induced vulnerabilities to exploit possibilities for novel combination therapies. We investigated cell viability, clonogenic survival, cell cycle status and immunoblots of human and murine glioma cells treated with Argyrin F. Moreover, we established an ex vivo glioma model using residual freshly resected tissue from patients, i.e. patient-derived microtumors (PDMs). Additionally, we extracted autologous tumor infiltrating lymphocytes (TILs) to perform co-culturing experiments. We performed mass spectrometry-based immunopeptidomics and used the orthotopic syngeneic SMA560/VM/Dk glioma mouse model. Argyrin F displayed anti-glioma efficacy in glioma cell lines in vitro and in PDM models ex vivo. Moreover, Argyrin F treatment induced cell cycle arrest, reduced clonogenic survival in vitro and prolonged survival in vivo. Argyrin F-treated SMA560 glioma displayed 4.6-fold more glioma-infiltrating CD8+ T cells. We discovered a distinctive treatment-induced immunopeptidome. Combination of Argyrin F plus PD-1 antibody increased cellular toxicity in PDM/TILs co-cultures ex vivo and prolonged overall survival compared with monotherapies in vivo. We conclude that our experimental data suggest a novel combination of Argyrin F plus PD-1 blockade and its clinical translation.

CTIM-22. NIVOLUMAB AND BEVACIZUMAB FOR RECURRENT GLIOBLASTOMA; T-CELL REACTIVITY AGAINST AUTOLOGOUS TUMOR CELLS

INTRODUCTION Glioblastoma is an aggressive brain tumor with a median survival of 14.6 months. We have no standard treatment for relapse and known options have limited effect. Novel treatments are necessary to improve survival and quality of life. METHODS We present our trial; phase II open label, two-armed translational study of Nivolumab and Bevacizumab for recurrent GBM, who have failed Stupp’s regimen. Patients are included in two arms depending on the possibility of salvage neurosurgical resection. Both arms receive Nivolumab and Bevacizumab administrated every second weekend, and the surgical arm also receive Nivolumab 7 days prior surgery. Forty-four patients were included by January 2021; 20 in each arm (four screen-failures). In the surgical arm, 20 fresh tumor samples as well as paired tissue from primary tumor were available. Tumor infiltrating lymphocytes (TILs) and tumor digest were produced in vitro from recurrent settings. Young TILs were expanded from fresh tumor fragments after minimal-culture, whereas rapidly expanded TILs (REP TILs) were obtained after massive expansion. By intracellular cytokine staining, we investigated the TIL reactivity after exposure to autologous tumor digest in order to evaluate whether the TILs were tumor-reactive, non-reactive or bystanders. RNA and whole exome sequencing were available before and after treatment. RESULTS Material from 19 patients was analyzed (one out of the 20 collected biopsies was limited in size, therefore no tumor digest could be produced). Four out of 19 TIL samples showed tumor reactivity after exposure to the autologous tumor digest. Tumor reactivity was ranged between 1,2 to 13,6 tox% in CD8+ TILs and between 2,8 to 10,9 tox% in CD4+ TILs. By flowcytometry we found, IgG4+ CD3+ TILS from tumor biopsies, meaning that Nivolumab were found in the brain. Currently controls are included to evaluate these results. CONCLUSIONS Updated results will be presented at SNO.

CTIM-33. PHASE II TRIAL OF VACCINE IMMUNOTHERAPY IN PRIMARY GLIOBLASTOMA: ADJUNCTIVE AUTOLOGOUS DENDRITIC CELLS PULSED WITH LYSATE FROM IRRADIATED SELF-RENEWING AUTOLOGOUS TUMOR CELLS (AV-GBM-1)

In primary glioblastoma (GBM), overall survival (OS) is poor despite standard aggressive therapy. Adjunctive AV-GBM-1 vaccine immunotherapy may improve OS. In this multi-institutional phase II trial, key eligibility criteria for intent-to-treat (ITT) enrollment were: (1) primary GBM, (2) age < 70 years when GBM was resected, (3) successful GBM cell culture, (4) successful monocyte collection by leukapheresis, (5) KPS > 70 post-surgery, and (6) plan to treat with concurrent RT/TMZ. Dendritic cells (DC) were differentiated from monocytes by culturing in IL-4 and granulocyte-macrophage colony stimulating factor (GM-CSF). AV-GBM-1 consisted of autologous DC incubated with autologous tumor antigens contained in the lysate of irradiated cultured GBM cells. After recovery from RT/TMZ, doses were admixed with 500 mcg GM-CSF; up to 8 doses were injected subcutaneously over 6 months. Patients were not excluded by apparent progression or pseudo-progression post RT/TMZ. OS and progression-free-survival (PFS) were calculated from ITT enrollment. The success rate was 97% for both GBM cell cultures and collection of monocytes; 60/60 vaccines were successfully manufactured. Median age was 59 years. 57 patients received 392 injections. After two weekly injections there were significant increases in plasma lipocalin-2 and angiopoietin-1, and decreases in thrombospondin-5, angiotensinogen, and beta-fibroblast growth factor. The most common adverse events attributed to AV-GBM-1 were local injection site reactions (16%) and flu-like symptoms (10%). With follow up from 15.2 to 32 months, median PFS and OS were 10.3 (8.5,11.6 95% CI) and 16.0 (13.0,21.3 95% CI) months respectively. OS was better in the 25 patients who had methylguanine-methyltransferase (MGMT) methylation and/or isocitrate dehydrogenase (IDH) mutation. Age was not independently correlated with survival. From date of first injection, OS was not increased in 14 patients who were treated with alternating electrical tumor-treating fields. CONCLUSION: feasibility, safety, and PFS were encouraging. A phase III trial is in development. Clinicaltrials.gov NCT03400917.

IMMU-07. REPROGRAMMING OF MICROGLIA/MACROPHAGES IN GLIOBLASTOMA IMPROVES ANTI-TUMOR T CELL RESPONSES

Glioblastoma (GBM) still remains incurable. In search for new treatment modalities immunotherapy might be attractive but highly depends on a sufficient infiltration and function of effector T cells in an immunosuppressive microenvironment. In this study, we analyzed if blocking or reprogramming M2polarized glioma-associated microglia/macrophages (GAMs) could improve spontaneous effector T cell responses and thus enhance the effectiveness of immunotherapy. This was tested by sorting patient-derived CD11b+ cells from GBM tissues and treating these GAMs with small molecule inhibitors (SMI) targeting the colony stimulating factor1 receptor (CSF1R). Especially CD11b+ cells treated with the SMI GW2580 presented with a reduced expression of the M2 marker CD163 (p < 0.01) and an increased expression of HLA-DR (p < 0.05). Conditioned media of SMI-treated GAMs also contained significantly higher levels of nitrite (p < 0.001) and a reduced concentration of the immunosuppressive cytokine IL-6 (p < 0.05). Moreover, gene expression profiles of GW2580-treated GAMs showed a shift from an immunosuppressive towards a pro-inflammatory phenotype. Most importantly, the addition of conditioned media of GW2580-treated GAMs to a co-culture of autologous tumor and T cells significantly reduced the number of live tumor cells as compared to the use of conditioned media of untreated GAMs (p < 0.05). Interestingly, in some cases the ability of T cells to transmigrate through a dense barrier of autologous tumor-derived endothelial cells could also be increased. In summary, we showed that CSF-1R blockade with the SMI GW2580 can reprogram GAM phenotype and thereby improve T cell activation. This strongly suggests further studies on the use of GW2580 in combination with immunotherapeutic approaches for the treatment of GBM.

TAMI-34. TARGETING CSF1R AND PD1 IN EXPERIMENTAL GLIOMA

The treatment of glioblastoma remains a challenge. Novel therapeutic strategies are urgently needed. Targeting the immunosuppressive glioblastoma-associated microenvironment is an interesting approach in this regard. Tumor-associated macrophages represent a distinct population of tumor-infiltrating immune cells with tumor-promoting features. The colony stimulating factor-1/ colony stimulating factor-1 receptor (CSF-1/CSF1R) axis plays an important role for macrophage differentiation and survival inside the tumor microenvironment. We thus aimed at investigating the antiglioma activity of CSF1R inhibition alone or in combination with blockade of programmed death (PD) 1. We detected CSF1R expression in paired tissue samples of primary and corresponding progressive glioblastoma. We investigated anti-CSF1R treatment alone or in combination with anti-PD1 antibodies in the orthotopic syngeneic VM/Dk SMA560 glioma mouse model, evaluated post-treatment effects and assessed treatment-induced cytotoxicity in a coculture model of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) ex vivo. Anti-CSF1R monotherapy increased the latency until the onset of neurological symptoms. Combinations of anti-CSF1R and anti-PD1 antibodies prolonged the latency until the onset of neurological symptoms and led to long-term survivors in vivo. Immunohistochemical analysis of post-treatment SMA-560 glioma revealed a modulation of the microenvironment, including increased T cell infiltration and reduced numbers of tumor-associated macrophages. Furthermore, we observed treatment-induced cytotoxicity of combined anti-CSF1R and anti-PD1 treatment in the PDM/TILs cocultures ex vivo. Taken together, our data indicates that CSF1R is a promising therapeutic target for glioblastoma, potentially in combination with PD1 inhibition.

64 A cloning and expression system of the neoantigen-specific TCRs from tumor-infiltrating lymphocytes by single-cell sequencing of paired TCRα and TCRβ chains

BackgroundT-cells that target tumor neoantigens arising from cancer mutations are the primary mediators of cancer immunotherapies. Identifying neoantigens and T-cells that recognize them is essential for T-cell-based immunotherapy. However, neoantigen-reactive Tumor-infiltrating lymphocytes (TILs) are highly differentiated or exhausted with a limited proliferative capacity; it is challenging to expand them for a sufficient number to probe their specificity. Therefore, we developed a novel cloning and expression system to examine TCRs discovered by single-cell sequencing of TILs for their neoantigen-specificity.MethodsTILs of lung cancer and sarcoma were analyzed. Surgically removed tumors were divided into several pieces. They were enzymatically digested to prepare fresh tumor digest (FTD) and cryopreserved. They were used to generate TIL cultures and perform WES and RNA-Seq to identify tumor-specific mutations. MHCflurry was used to predict the binding affinity of potential epitopes arising from these mutations to HLA class I. Peptides that were predicted to bind to patients‘ own MHC class I molecules strongly were then synthesized. Single TILs isolated with the ICELL8® cx system (Takara Bio) were dispensed into a nanowell TCR chip containing preprinted barcodes. Barcoded cDNAs were PCR-amplified in-chip, pooled off-chip, and used as a template in the TCR-specific PCR or for the whole transcriptome library generation of 5’ ends of all transcripts. Based on single-cell transcriptome data and TCR profiles of TILs, we predict and prioritize neoantigen-specific TCRs and cloned them into siTCR® retrovirus vectors. These TCRs were transduced into SUP-T1-based reporter cells in which ZsGreen fluorescent protein expression is controlled by AP-1 and NFAT binding sites. TCR-expressing reporter cells were cocultured with patient autologous APCs pulsed with a pool of candidate neoantigen peptides. ZsGreen expression indicates that TCRs match their cognate neoantigens.ResultsIn a lung cancer patient, we set up 18 TIL cultures and obtained 12 TILs. TILs were cocultured with FTD; IFN-γ production was measured by ELISA to evaluate their reactivity to the autologous tumor. NGS identified 197 somatic mutations, 4 fusion genes, and 8 highly expressed cancer-testis antigens. Among them, 339 candidate peptides were synthesized and screened. In addition, we cloned 3 pairs of TCRαβ chains from most expanded TIL cultures and 4 TCRs from ex vivo TILs with exhausted phenotype. Two reporter cells that express TCRs from exhausted TILs responded to the same neoantigen peptide.ConclusionsGenerating TCR expressing cell lines facilitated the identifying neoantigens and their cognate TCR sequences from patients.Ethics ApprovalG3545

179 CD8+CD69+ Expanded Tumor Infiltrating Lymphocytes from Soft Tissue Sarcoma Have Increased Tumor-Specific Functional Capacity

BackgroundAdoptive cell therapy (ACT) utilizing tumor infiltrating lymphocytes (TIL) has demonstrated durable responses in patients with metastatic melanoma and offers potential for other solid tumors. Preclinical experience with expanded TIL from soft tissue sarcoma (STS) demonstrates less frequent tumor-specific reactivity compared to melanoma samples, limiting the potential for efficacy.1 We hypothesized that CD69+ TIL have increased tumor-specific reactivity, which can be manipulated in culture, thereby offering an opportunity to enhance the antitumor effect of this cellular immunotherapy product.MethodsPatients were enrolled on an IRB-approved protocol and TIL were expanded from fresh surgical specimens. After enzymatic digestion, tumor single cell suspensions were cultured in media containing 10% human serum and IL-2 (6000IU/mL). Expanded TIL were then enriched for CD8+ using magnetic bead isolation and CD69+ by flow cytometry cell sorting (FACS). After co-culture with autologous tumor digest, functional capacity was compared between bulk TIL and enriched TIL by evaluation of IFN-gamma (IFNg) and Granzyme B (GzB) secretion. Capacity for direct tumor cytotoxicity was assessed by Cr51 assay after co-culture of autologous immortalized cell lines with expanded TIL subpopulations after enrichment.ResultsFollowing co-culture with autologous tumor digest, CD69+ TIL demonstrated increased IFNg secretion compared to CD69- TIL in 6 samples (1.4–4.2x, p<0.05). CD8+ enriched TIL (75% compared to bulk) had higher relative IFNg secretion in both CD69+ and CD69- subsets (4.2 and 5.8x, respectively, p<0.001). Maximal IFNg secretion was seen from TIL that were both CD69+ sorted and CD8+ enriched, demonstrating an synergistic effect (16.3x vs Bulk CD69-, 4.2x vs Bulk CD69+, 2.8x vs CD8 enriched CD69- ; p<0.001). Functional capacity was also assessed by GzB secretion with similar results. CD69+ TIL had increased relative secretion (1.8–2.2x) compared to CD69- TIL (p< 0.01). CD8+ enriched TIL had increased relative GzB secretion in both CD69- and CD69+ sorted fractions (1.4x, 1.2x, respectively, p<.05). CD69+ sorted and CD8+ enriched TIL demonstrated an additive effect (2.6x vs Bulk CD69-, p<0.01; 1.2x vs Bulk CD69+, p<0.05; 1.8x vs CD8 enriched CD69-, p<0.01). CD8+ enriched CD69+ sorted TIL had greater relative cytotoxicity (3x, p<0.05) at 40:1 E:T ratio against autologous tumor cell lines compared to bulk expanded TIL (figure 1).Abstract 179 Figure 1Functional capacity of CD69+ TIL is demonstrated by increased secretion of GzB (A) and IFNg (B) after co-culture with autologous tumor digest. CD69+ TIL have greater cytotoxicity against autologous immortalized cell lines compared to bulk TIL at 40:1 E:T ratio (C).ConclusionsTIL expanded from STS demonstrate greater tumor-specific functional capacity and cytotoxicity after CD8 enrichment and CD69+ FACS compared to bulk expanded TIL. These data validate the strategy to enhance CD8+CD69+ TIL during culture to yield a more efficacious cellular immunotherapy product.AcknowledgementsThis work was funded by NIH K08CA252642Trial Registration n/aReference1. Mullinax JE, Hall M, Beatty M, Weber AM, Sannasardo Z, Svrdlin T, Hensel J, Bui M, Richards A, Gonzalez RJ, Cox CA, Kelley L, Mulé JJ, Sarnaik AA, Pilon-Thomas S. Expanded Tumor-infiltrating Lymphocytes From Soft Tissue Sarcoma Have Tumor-specific Function. J Immunother 2021 Feb-Mar 01;44(2):63–70.Ethics ApprovalAbstract cites IRB-approved protocol in methods section.Consent n/a