Intratumoral T-cell Research Articles

Changes in Tumor Immune Micro-Environment in Diffuse Large B-Cell Lymphoma (DLBCL): A Comparative Study of Relapsed Versus Diagnostic DLBCL

Introduction The tumor immune microenvironment (TiME) of DLBCL at diagnosis bears many biomarkers known to predict sensitivity to checkpoint blockade therapy (CBT). Approximately 25% of DLBCL, harboring PDL1 gene alteration, exhibit an immune inflamed phenotype. However, CBT studies in relapsed DLBCL show very low response rates. The immune landscape of DLBCL in the setting of relapse has not yet been characterized. We hypothesized that the TiME can be influenced by the therapy, explaining the CBT lack of efficacy. We explored the TiME evolution by comparing the tumor infiltrating lymphocytes (TIL) and the immune evasion signature by protein and gene expression (GE) in paired biopsies of DLBCL at diagnosis and at relapse. Method This study included patients with DLBCL treated with first-line R-CHOP-like regimen, in which we had access to nodal biopsies obtained pre-treatment and at relapse. Assessment of TIL and PDL1+ cell content was carried out by CD3, CD4, CD8 immunostaining and with PDL1 (Ventana, SP263) and PAX5 double staining. These were performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections cut at 4µm. The proportion of each cell-type was expressed in relation to total nucleated cells. PDL1+ cell proportion was expressed for both total cells and tumor cells. The paired non-parametric Wilcoxon test, double-sided with p=0.05, was used for comparison. GE using a panel of 760 cancer immune genes was performed with the digital hybridization NanoString platform on paired FFPE specimens. NSolver software was used for normalization on housekeeping genes, differential expression (DE) and cell-type profiling. The correlation between IHC and GE variation were computed using the Spearman test. Analysis of enriched pathways on combined patient data were performed with GSEA software, using two gene sets for T-cell activation and immune evasion validated in B-cell lymphoma, and Ingenuity Pathway Analysis (IPA) for the 339 highest changed genes. The pathway enrichment analysis was reported for false discovery rate (FDR) <0.05. Results Twenty DLBCL patients with paired diagnostic and relapse biopsies were studied. IHC was performed on 19 patients (necrotic tissue for one patient) and GE by NanoString on 6 patients. The median number of treatments received prior to obtaining the relapsed biopsy was 3 (1-6). Three patients had prior exposure to CBT, none from the patients in whom GE was performed. Our population was composed of 2/3 germinal center B-cell (GCB) DLBCL. Six patients had transformed DLBCL. At relapse, CD4 and CD8 cells density was significantly reduced compared to diagnosis. The median percentages of CD3, CD4 and CD8 were 15%, 7.5% and 5% at diagnosis and 7.5%, 2.5% and 2.5% at relapse, respectively (Figure 1A). The median proportion of PDL1+ cells (n=15) did not differ at relapse compared to pre-treatment (5.25% and 5%, respectively) (Figure 1B). At diagnosis, 87% of biopsies were found to be positive for PDL1 (≥1% of positive cells) and 73% at relapse. The PDL1 positive cells were mostly immune cells; only 3 samples had tumor cells expressing PDL1 at pre-treatment and 4 at relapse with <1% of positive tumor cells in all cases. The GE profile detected 63 genes that were significantly different in relapse vs pre-treatment samples (p<0.005), including 8 up-regulated and 55 down-regulated. The correlation for T-cell abundance variation between IHC and GE was high (r=0.94, p=0.017). For the subset of 6 patients in whom GE was performed, the T-cells were not significantly reduced as measured by both IHC and GE. However, the GSEA for immune escape genes and T-cell activation signatures were both down-regulated (FDR=0.033 and 0.010, respectively) (Figure 2). IPA predicted decreased activation of leukocytes (z-score=-6.317) and Th2 activation pathway (z-score=-3.182). Conclusion At relapse, a proportion of DLBCL have a significant reduction in T cell infiltration. As well, they acquire a non-T cell-inflamed TiME. The T-cell inhibition seems to be independent of local T-cell immunoregulatory mechanisms as the immune escape signature is also reduced. This non immunogenic, "cold" condition could explain the lack of efficacy of CBT in the relapsed setting. Prior exposure to steroid containing therapy might be a trigger for intra-tumor T-cell desertion and downregulation. This observation must be confirmed on a larger population. Further analyses will be presented. Disclosures Johnson: BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Seattle Genetics: Honoraria; Merck: Consultancy, Honoraria; Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; BMS: Consultancy, Honoraria; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Assouline:Janssen: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria.

Histone deacetylase inhibition promotes intratumoral CD8+ T-cell responses, sensitizing murine breast tumors to anti-PD1

Histone deacetylase (HDAC) inhibitors impair tumor cell proliferation and alter gene expression. However, the impact of these changes on anti-tumor immunity is poorly understood. Here, we showed that the class I HDAC inhibitor, entinostat (ENT), promoted the expression of immune-modulatory molecules, including MHCII, costimulatory ligands, and chemokines on murine breast tumor cells in vitro and in vivo. ENT also impaired tumor growth in vivo-an effect that was dependent on both CD8+ T cells and IFNγ. Moreover, ENT promoted intratumoral T-cell clonal expansion and enhanced their functional activity. Importantly, ENT sensitized normally unresponsive tumors to the effects of PD1 blockade, predominantly through increases in T-cell proliferation. Our findings suggest that class I HDAC inhibitors impair tumor growth by enhancing the proliferative and functional capacity of CD8+ T cells and by sensitizing tumor cells to T-cell recognition.

TMIC-23. A SEQUENTIAL IMAGING STRATEGY TO STUDY ONCOLYTIC VIRUS INFILTRATION, REPLICATION AND TUMOR MICROENVIRONMENTAL PERTURBATIONS, EX VIVO

Abstract The use of oncolytic viruses (OV) as therapeutic agents for glioblastoma multiforme (GBM) is an emerging clinical focus. OV have the potential to reinvigorate the immune contexture in the tumor microenvironment (TME). However, current pre-clinical models are insufficient to study the immune effects of OV. Here, we describe an imaging approach using a human ex-vivo platform to study the effects of OV on tumor-associated immune cells in GBM. Using the rQNestin34.5v.1 herpes simplex virus (rQNestin) we treated human GBM ex-vivo, incorporating autologous patient-derived peripheral-blood mononucleated cells and whole tissue fragments. We treated GBM (n=10) with rQNestin, capable of expressing GFP, and profiled GFP expression as a proxy for replication. We analyzed serial sections of H&E, HSV-1, and multiplex immunohistochemistry (mIHC) to investigate the impact of rQNestin on the spatial context of the immune compartment. Cell Signaling Technology antibodies, CD3e (D7A6E™,ID:85061), CD68 (D4B9C,ID:76437), CD11c (D3V1E,49420), MHC-Class II ((HLA-DRB)LGII-612.14,NA) and Pan-keratin (C11,4545) were optimized for identifying intratumoral T-cells (CD3e+), macrophages (CD68) and dendritic cells (CD11c+MHCII+) in relation to the surrounding tissue architecture (pan cytokeratin). Using the serial-section imaging approach, we overlaid infiltration and replication of rQNestin. Additionally, strategic sectioning identified dynamic spatial alterations in the immune compartment, via visualization of the tumor-immune interface with indications of infiltration and replication. Quantitative pathology showed dynamic re-arrangement of immune cells - in spatial orientation, quantity and localization, relative to the tumor, under rQnestin pressure, ex-vivo. Here, we report that our ex-vivo human tumor model can be used to evaluate the effects of OV in the TME – not only viral replication but also the direct effect on the immune compartment. In particular, we can assess changes in immune subsets post-OV treatment in a 3-D manner, therefore providing greater insight into their spatial arrangement and distribution. These findings could develop our understanding of anti-tumor OV mechanisms.

Differences in Tumor Microenvironment Dictate T Helper Lineage Polarization and Response to Immune Checkpoint Therapy

Immune checkpoint therapy (ICT) shows encouraging results in a subset of patients with metastatic castration-resistant prostate cancer (mCRPC) but still elicits a sub-optimal response among those with bone metastases. Analysis of patients' bone marrow samples revealed increased Th17 instead ofTh1 subsets after ICT. To further evaluate the different tumor microenvironments, we injected mice with prostate tumor cells either subcutaneously or intraosseously. ICT in the subcutaneous CRPC model significantly increases intra-tumoral Th1 subsets and improves survival. However, ICT fails to elicit an anti-tumor response in the bone CRPC model despite an increase in the intra-tumoral CD4 Tcells, which are polarized to Th17 rather than Th1 lineage. Mechanistically, tumors in the bone promote osteoclast-mediated bone resorption that releases TGF-β, which restrains Th1 lineage development. Blocking TGF-β along with ICT increases Th1 subsets and promotes clonal expansion of CD8 Tcells and subsequent regression of bone CRPC and improves survival.

90. Fecal Microbiota Transplantation in Metastatic Melanoma Patients Resistant to Anti-PD-1 Treatment

BackgroundMost metastatic melanoma patients treated with Programed cell Death (PD)-1 blockers fail to achieve a durable response. The gut microbiota profoundly affects host immunity, and fecal microbiota transplantations (FMT) have been shown to enhance anti-PD-1 effectiveness in murine models. We report initial safety and efficacy results from the first patients treated in a Phase I study of FMT and re-induction anti-PD-1 therapy in anti-PD-1 refractory metastatic melanoma.MethodsFMT donors were two metastatic melanoma patients who achieved a durable complete response to treatment. FMT recipients were metastatic melanoma patients who failed at least one anti-PD-1 line of treatment. FMT was conducted by both colonoscopic and oral administration, followed by anti-PD-1 re-treatment. Each recipient underwent pre- and post-treatment stool sampling, tissue biopsy of both gut and tumor, and total body imaging.ResultsFive patients with treatment-resistant metastatic melanoma were recruited. No FMT-related or immunotherapy-related adverse events were observed. To assess engraftment of the new microbiota, recipients were paired with their respective donors and stool 16S rDNA gene sequence analysis was performed. Sequencing results demonstrated post-FMT compositional dissimilarity (Unweighted UniFrac, P = 0.04, FDR q = 0.22) between the two recipient–donor groups. Specific taxonomic dynamics included post-FMT increased abundance of Paraprevotellaceae, previously associated in descriptive studies with responsiveness to treatment, and significant reductions in abundance of β-proteobacteria, previously associated with reduced response to treatment. Immunohistochemical stains of biopsies demonstrated an increased post-FMT infiltration of antigen presenting cells (CD68+) in the gut (paired T-test, P = 0.008) and in the tumor (P = 0.0076). Post-treatment intra-tumoral CD8+ T-cell infiltration was also increased. Three patients had a partial or complete response to treatment post-FMT.ConclusionFMT in metastatic melanoma patients seems to be safe and may alter recipient gut microbiota to resemble that of a responder donor. This alteration may result in intra-tumoral T-cell activity, and conferred clinical and radiological benefit in several recipients previously unresponsive to treatment.Disclosures All Authors: No reported Disclosures.

1900P - Activated effector T cells co-expressing multiple inhibitory receptors (IRs) are enriched in the tumor immune microenvironment in high grade serous ovarian cancer (HGSOC)

BackgroundIn HGSOC, the positive association between tumor-infiltrating lymphocytes (TILs) and survival suggests that immunotherapy might be beneficial. Despite this, initial attempts had limited efficacy. The pattern of IRs expression displayed by HGSOC TILs has not been fully determined. We characterized the phenotype of TILs in HGSOC to identify immunosuppressive pathways limiting anti-tumor T-cell activity. MethodsPeripheral blood (PB), primary tumor and ascites were retrieved from HGSOC patients admitted for primary surgery in our Institute. Tumor immune infiltrate was characterized on 34 formalin-fixed paraffin-embedded specimens by immunohistochemistry (IHC) for lineage markers (CD20, CD3, CD8, CD4, CD163) and inhibitory molecules (PD-1, PD-L1, LAG-3, TIM-3, VISTA).T cells isolated from tumor, ascites and PB of 14 patients were also characterized by flow cytometry for the expression of lineage and memory markers (CD3, CD4, CD8, CD45RA, CD62L, CD95), inhibitory molecules (PD1, CTLA-4, LAG-3, TIGIT, TIM-3, 2B4, GITR, KLRG1, CD39, CD160) and the activation marker CD137. ResultsThe entity of immune infiltration was heterogeneous: 2/34 of cases (6%) were immune desert; all other cases displayed a wide range of immune cell density, dominated by T lymphocytes and macrophages. B cells were scanty. Intratumoral T cells (ITTCs) were present in 30/34 cases (88.2%), almost constantly CD8. PD-1, LAG-3 and TIM-3 were expressed by 61.9%, 61.7% and 61.7% of ITTCs respectively. VISTA was rarely expressed. PD-L1 was expressed by neoplastic cells and immune cells (mainly macrophages) in 40% and 82.3% of samples, respectively. Flow cytometry confirmed the expression by TILs of several IRs (mainly PD-1, CD39, TIM-3 and LAG-3), with variable percentages of positive cells. Compared with corresponding PB and ascites, TILs were enriched in effector memory lymphocytes and in cells co-expressing PD-1, CD39 and CD137. ConclusionsThese data suggest that HGSOC is infiltrated by antigen-experienced T lymphocytes displaying features of both activation and partial exhaustion. The anti-tumor activity of such cells still needs to be determined. Legal entity responsible for the studyThe authors. FundingAIRC Associazione Italiana Ricerca sul Cancro. DisclosureAll authors have declared no conflicts of interest.

New Clones on the Block

Although immune checkpoint blockade (ICB) has yielded striking clinical responses in subsets of cancer patients, the mechanism of action is still unclear. In a recent issue of Nature Medicine, Yost etal., 2019 report that the Tcell clones that dominate the intra-tumoral Tcell landscape after ICB are distinct from those prior to treatment, a phenomenon referred to by the authors as "clonal replacement."

CXCR1- or CXCR2-modified CAR T cells co-opt IL-8 for maximal antitumor efficacy in solid tumors

Chimeric antigen receptor (CAR) T-cell therapy targeting solid tumors has stagnated as a result of tumor heterogeneity, immunosuppressive microenvironments, and inadequate intratumoral T cell trafficking and persistence. Early (≤3 days) intratumoral presentation of CAR T cells post-treatment is a superior predictor of survival than peripheral persistence. Therefore, we have co-opted IL-8 release from tumors to enhance intratumoral T-cell trafficking through a CAR design for maximal antitumor activity in solid tumors. Here, we demonstrate that IL-8 receptor, CXCR1 or CXCR2, modified CARs markedly enhance migration and persistence of T cells in the tumor, which induce complete tumor regression and long-lasting immunologic memory in pre-clinical models of aggressive tumors such as glioblastoma, ovarian and pancreatic cancer.

Tumor-reprogrammed resident T cells resist radiation to control tumors

Successful combinations of radiotherapy and immunotherapy depend on the presence of live T cells within the tumor; however, radiotherapy is believed to damage T cells. Here, based on longitudinal in vivo imaging and functional analysis, we report that a large proportion of T cells survive clinically relevant doses of radiation and show increased motility, and higher production of interferon gamma, compared with T cells from unirradiated tumors. Irradiated intratumoral T cells can mediate tumor control without newly-infiltrating T cells. Transcriptomic analysis suggests T cell reprogramming in the tumor microenvironment and similarities with tissue-resident memory T cells, which are more radio-resistant than circulating/lymphoid tissue T cells. TGFβ is a key upstream regulator of T cell reprogramming and contributes to intratumoral Tcell radio-resistance. These findings have implications for the design of radio-immunotherapy trials in that local irradiation is not inherently immunosuppressive, and irradiation of multiple tumors might optimize systemic effects of radiotherapy.

The Effect of Dexamethasone on the Αpd-1/L1 and Radiotherapy Stimulated Anti-Tumor Response

Dexamethasone is a potent immunosuppressant. In current practice, if a cancer patient requires dexamethasone, αPD-1/L1 therapy is either stopped or not initiated due to a presumed counteraction of αPD-1/L1’s immune-stimulation. This delay or cessation in αPD-1/L1 treatment likely worsens outcomes. Additionally, many cancer patients require radiotherapy (RT) which can also stimulate an anti-tumor T-cell response. There is a known synergy between αPD-1/L1 and RT leading to improved local and distant tumor control (abscopal effect). Therefore, determining whether dexamethasone impairs the αPD-1/L1 response alone and in combination with RT is an important clinical question. B16F10 tumors were established subcutaneously on the bilateral flanks of wt C57BL/6 mice. Following tumor engraftment, dexamethasone was initiated at a low (0.25-0.5 mg/kg) or high (2.5 mg/kg) dose. Once tumors were palpable, αPD-1 or αPD-L1 was administered with or without tumor RT. Tumor RT (10 Gy in one fraction) was directed at one flank tumor while tumors on both flanks were monitored for growth. Tumor specific T-cells were tracked using a tetramer specific for an LCMV viral glycoprotein inserted into the B16F10 tumor cell line. Neither low (LD) nor high dose (HD) dexamethasone impaired tumor control with αPD-L1 (LD + αPD-L1vs αPD-L1 alone (p=0.40); HD + αPD-L1vs αPD-L1 alone (p=.14); each vs untreated control (p<0.05)). LD led to a mild reduction in intra-tumoral tumor-specific CD8 T-cell numbers. However, concurrent dexamethasone and αPD-L1 treatment showed a robust increase in intra-tumoral tumor-specific CD8 T-cells relative to untreated controls (5.5 fold). Combined RT and αPD-1 improved local tumor control and stimulated an abscopal effect that was not impaired by LD. T-cell analysis did show a reduction in the numbers of tumor specific CD8 T-cells in the RT + αPD-1 mice which received dexamethasone compared to the RT + αPD-1 alone group. Our work suggests that LD should not be an absolute contraindication to checkpoint blockade administration, and a clinical benefit persists for the immune-stimulatory effects of both αPD-1/L1 and RT even in the presence of supra-physiologic doses of glucocorticoids. More study is needed for HD.

Anti-CTLA-4 Activates Intratumoral NK Cells and Combined with IL15/IL15Rα Complexes Enhances Tumor Control.

Antibodies targeting CTLA-4 induce durable responses in some patients with melanoma and are being tested in a variety of human cancers. However, these therapies are ineffective for a majority of patients across tumor types. Further understanding the immune alterations induced by these therapies may enable the development of novel strategies to enhance tumor control and biomarkers to identify patients most likely to respond. In several murine models, including colon26, MC38, CT26, and B16 tumors cotreated with GVAX, anti-CTLA-4 efficacy depends on interactions between the Fc region of CTLA-4 antibodies and Fc receptors (FcR). Anti-CTLA-4 binding to FcRs has been linked to depletion of intratumoral T regulatory cells (Treg). In agreement with previous studies, we found that Tregs infiltrating CT26, B16-F1, and autochthonous Braf V600E Pten -/- melanoma tumors had higher expression of surface CTLA-4 (sCTLA-4) than other T-cell subsets, and anti-CTLA-4 treatment led to FcR-dependent depletion of Tregs infiltrating CT26 tumors. This Treg depletion coincided with activation and degranulation of intratumoral natural killer cells. Similarly, in non-small cell lung cancer (NSCLC) and melanoma patient-derived tumor tissue, Tregs had higher sCTLA-4 expression than other intratumoral T-cell subsets, and Tregs infiltrating NSCLC expressed more sCTLA-4 than circulating Tregs. Patients with cutaneous melanoma who benefited from ipilimumab, a mAb targeting CTLA-4, had higher intratumoral CD56 expression, compared with patients who received little to no benefit from this therapy. Furthermore, using the murine CT26 model we found that combination therapy with anti-CTLA-4 plus IL15/IL15Rα complexes enhanced tumor control compared with either monotherapy.

Immune Desertic Landscapes in Hepatocellular Carcinoma Shaped by β-Catenin Activation.

About one third of cases of hepatocellular carcinoma (HCC) show gain-of-function mutations of CTNNB1 (β-catenin) that correlate with sparse intratumoral T-cell content, as observed previously in an ample spectrum of malignancies, and there is mounting preliminary evidence that such HCC cases are refractory to treatment with PD-1 checkpoint inhibitors. Elegant hepatocarcinogenesis experiments by in vivo gene transfer to mouse hepatocytes show that coexpression of active forms of β-catenin result in poor T-cell infiltrates, faster progression in immunocompetent hosts, and unresponsiveness to immunotherapy with checkpoint inhibitors.See related article by Ruiz de Galarreta et al., p. 1124.

Abstract 4967: Neoadjuvant chemotherapy promotes a transient increase of intratumoral T-cell density in colorectal liver metastases

Abstract Background: In metastatic colorectal cancer, only the microsatellite instable (MSI) subgroup of cases has so far demonstrated major responses to immune checkpoint inhibition. Increased infiltration of T lymphocytes in presumed non-responsive tumors has been shown to facilitate response to immune checkpoint inhibition. Cytotoxic chemotherapy might induce immunogenic cell death and cause infiltration of immune cells, turning “cold” tumors into “hot”. In this work we investigated whether standard-of-care neoadjuvant chemotherapy (NACT) would influence T-cell infiltration in the tumor microenvironment of colorectal liver metastases (CLM) in a substudy of patients included in the OSLO-COMET trial (n=280; NCT01516710). Materials and methods: Patients (n=92; bearing 144 metastatic tumors) had resectable CLM, and of these, 45 patients received NACT, in most cases fluoropyrimidine-based, with the addition of oxaliplatin (n=36) or irinotecan (n=8). Quantification of total T-cells (Ttot), cytotoxic T-cells, T-helper cells, and regulatory T-cells was performed by immunohistochemical staining of serial sections with antibodies against CD3, CD8, CD4, and FOXP3, respectively. Hotspots from the invasive margin (IM), intratumoral region (IT), and adjacent liver tissue (NLi) were analyzed. T-cell density was reported as number of T-cells/mm2. Results: The density of Ttot in the IM region (median 2360 cells/mm2) was 7 times greater than in the IT region (319 cells/mm2) and 13 times greater than in the NLi region (183 cells/mm2), with similar findings for T-cell subtypes. Comparing patients that had received NACT to patients that had not, there was no difference in T-cell infiltration in the IM and NLi regions, but a non-significant trend towards increased T-cell density was detected in the IT region after NACT, exemplified by Ttot 353 vs 292 cells/mm2 (p=0.2). Exploring the influence of the time interval between NACT completion and surgery by receiver operating characteristic curve analysis, 9.5 weeks was identified as the time point that separated the cases most clearly by T-cell density. Interestingly, cases with a shorter than 9.5-week interval had a significantly higher intratumoral T-cell count than cases with the longer interval; with median Ttot 491 vs 236 cells cells/mm2; p&amp;lt;0.0001. Similar findings were detected for all T-cell subsets in the IT region. Conclusion: The main finding of this work was that NACT administration was associated with a transient increase of intratumoral T-cell density in CLM, potentially promoting an MSI-like tumor immune phenotype. The results suggest that when exploring combinations of cytotoxic therapy and immune checkpoint inhibition, the timing of individual treatment components may be essential. To further optimize treatment schedules, biomarkers reflecting T-cell infiltration may be a useful tool for tailoring personalized therapy. Citation Format: Kjersti Flatmark, audun E. Berstad, Vegar Dagenborg, Bjørn Edwin, Åsmund A. Fretland, Krzysztof Grzyb, Eirik Høye, Marius Lund-Iversen, Serena E. Marshall, Anne H. Ree, Sheraz Yaqub. Neoadjuvant chemotherapy promotes a transient increase of intratumoral T-cell density in colorectal liver metastases [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 4967.

Abstract CT042: Fecal microbiota transplantation (FMT) and re-induction of anti-PD-1 therapy in refractory metastatic melanoma patients - preliminary results from a phase I clinical trial (NCT03353402)

Abstract Background: The majority of metastatic melanoma patients treated with Programed cell Death (PD)-1 blockade fail to achieve durable response. The gut microbiota profoundly affects host immunity, and fecal microbiota transplantations (FMT), which transfers the entire gut microbiota from one host to another, has been shown to enhance anti-PD-1 effectiveness in murine models. We report initial safety and efficacy results from the first three patients treated on a Phase I study of FMT and re-induction anti-PD-1 therapy in anti-PD-1 refractory metastatic melanoma. Methods: FMT donors were two metastatic melanoma patients who achieved a durable complete response. FMT Recipients were metastatic melanoma patients who failed at least one anti-PD-1 line of treatment. FMT was conducted by both colonoscopy and oral ingestion of stool capsules, followed by anti-PD-1 re-treatment (Nivolumab, BMS). Each recipient underwent pre- and post-treatment stool sampling, tissue biopsy of both gut and tumor, and total body imaging. Results: Recipients #1 and #3 received fecal implants from Donor #1, while Recipient #2 receive implants from Donor #2. No FMT-related or immunotherapy-related adverse events were observed. To assess engraftment of the new microbiota, recipients were paired with their respective donors and stool 16S rDNA gene sequence analysis performed. Ideal engraftment from a single donor would result in identical microbiota composition between that donor and recipients. In the case of two donors, ideal engraftment would result in two distinctive recipient-donor compositions. Sequencing results demonstrated post-FMT compositional dissimilarity (Unweighted UniFrac, p=0.04, FDR q=0.22) between the two recipient-donor groups. Immunohistochemical stains of biopsies demonstrated an increased post-FMT infiltration of antigen presenting cells (CD68+) in the gut (paired T test, p=0.008) and in the tumor (p=0.076). Post-treatment intra-tumoral CD8+ T-cells infiltration was also increased (p=0.096), especially in recipients #1, #3. Recipient #1 and Recipient #3 demonstrated clinical and radiological benefit from treatment. Conclusion: FMT in metastatic melanoma patients seemed to be safe and may alter recipient gut microbiota to resemble that of a responder donor. This alteration may result in intra-tumoral T-cell activity, which was translated to a clinical and radiological benefit in two recipients. Citation Format: Erez N. Baruch, Ilan Youngster, Rona Ortenberg, Guy Ben-Betzalel, Lior H. Katz, Adi Lahat, Iris Barshack, Daniela Dick-Necula, Ronac Mamtani, Naamah Bloch, Bella Ungar, Daniel Rotin, Camila Avivi, Liat Anafi, Yael Steinberg-Silman, Nethanel Asher, Ronnie Shapira-Frommer, Tal Brosh-Nissimov, Yael Eshet, Stephen Raskin, Hagit Harati, Jenny Melnichenko, Jacob Schachter, Omry Koren, Gal Markel, Ben Boursi. Fecal microbiota transplantation (FMT) and re-induction of anti-PD-1 therapy in refractory metastatic melanoma patients - preliminary results from a phase I clinical trial (NCT03353402) [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 CT042.

Abstract 4979: Ofranergene Obadenovec (VB-111), an anti-cancer gene therapy, induces immunologic responses in solid tumors transforming cold tumors to hot tumors

Abstract Introduction: Ofranergene Obadenovec (VB-111) is a non-replicating adenovirus 5 (Ad-5, El-deleted) carrying a proapoptotic human Fas-chimera transgene that targets angiogenic blood vessels and leads to vascular disruption. Improved responses seen among patients experiencing post treatment fever suggest that VB-111’s mode of action involves induction of a tumor directed immune response. The following experiments were conducted to assess if VB-111 may alter the immunogenic profile of the tumor. Methods: Biopsies were obtained from 3 patients with recurrent platinum-resistant ovarian cancer treated with intravenous VB-111 1x1013 viral particles (VPs) every 2 months in combination with weekly paclitaxel. H&amp;E and Immunohistochemistry (IHC) were performed for CD8 and CD4 intratumoral T-cells, and results were compared to pre-treatment specimens and to untreated controls. In parallel, in the Lewis Lung Carcinoma (LLC) model, mice were randomly treated with intravenous saline or VB-111 at doses of 1x109 or 1x1011 VPs. Upon sacrifice lungs were harvested and weighted. Tumor burden was assessed, and Immunohistochemistry with anti-CD8 antibody for the presence of infiltrating cytotoxic T-cells was performed. Results: Specimens from Ovarian Cancer taken before treatment with VB-111 showed no or minimal T-cell infiltration. One month after VB-111 treatment, metastatic lesions demonstrated increase in CD8 (up to 74 CD8+ cells/HPF) and CD4 tumor infiltrating T-cells. At 4.5 months following first drug administration (post 3rd dose) a liver lesion showed necrotic and fibrotic tissue with no viable tumor, lymphocytic aggregate, intensive staining for CD-8 and CD-4 T-cells and pigmented macrophages. In mice induced with LLC tumor and treated with saline, large tumor masses were observed in the lungs, and the calculated average tumor burden was 0.883g. Administration of VB-111 at 1x109 and 1x1011 VPs reduced tumor burden by 42% and 72% respectively. Concurrently, the number of tumor infiltrating CD8 T-cells was increased in correlation with VB-111 treatment dose. Conclusion: Pre clinical and clinical data suggest that VB-111 induces an Immunotherapeutic effect manifested locally with tumor infiltration with CD-8 T-cells, and evidence of tumor necrosis. The viral vector may promote transformation of the tumor microenvironment from immunologically "cold" to "hot", enhancing immune cell recognition and immune activation. Citation Format: Ronnie Shapira-Frommer, Tamar Rachmilewitz Minei, Iris Barshack, Itzhak Mendel, Niva Yakov, Yael C. Cohen, Eyal Breitbart, Dror Harats, Richard T. Penson. Ofranergene Obadenovec (VB-111), an anti-cancer gene therapy, induces immunologic responses in solid tumors transforming cold tumors to hot tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4979.

Abstract 2331: Intratumor adoptive transfer of IL-12 mRNA transiently engineered anti-tumor CD8+ T cells

Abstract Adoptive T-cell therapy with TILs, CAR-T cells, and TCR-engineered T-cells is achieving remarkable clinical efficacy. Retroviral gene transfer of IL-12 into the T cell cultures markedly enhances efficacy but has shown unacceptable severe side effects in the clinic. To ensure transient gene transfer, tumor specific CD8 T cells were engineered by electroporation of mRNA encoding single chain IL-12 into OT1 and Pmel-1 TCR transgenic lymphocytes. Engineered T cells were injected intratumorally and achieved complete rejections not only of the injected lesion but also of distant concomitant tumors. Efficacy was further enhanced if a small dose of agonist anti-CD137 mAb was co-injected or if CD137-ligand mRNA was co-electroporated. Importantly, intratumor T-cell administration was more efficacious that the intravenous or subcutaneous routes. Therapeutic effects were dependent on IFNγ, cDC1 dendritic cells and endogenous T cells. Interestingly, treatment induced epitope spreading of the immune response to antigens not recognized by the adoptively transferred lymphocytes. Efficacy was also achieved when mouse TILs cultures were used and human TIL cultures can also be transiently IL-12 mRNA engineered. Intratumor release, transient IL-12 mRNA-engineering and repeated administration jointly constitute a safe, feasible and powerful cancer immunotherapy strategy. Note: This abstract was not presented at the meeting. Citation Format: Inaki Etxeberria, Elixabet Bolaños, Alvaro Teijeira, Arantza Azpilicueta, Jose Ignacio Quetglas, Alfonso Rodriguez Sanchez-Paulete, Itziar Otano, Uxua Mancheño, Sandra Hervas-Stubbs, Susana Inoges, Saray Garasa, Maite Alvarez, Pedro Berraondo, Ignacio Melero. Intratumor adoptive transfer of IL-12 mRNA transiently engineered anti-tumor CD8+ T cells [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 2331.

Abstract 4753: Attenuating colorectal cancer cell growth and enhancing immunogenicity through rational combination of MEK and HDAC inhibition

Abstract Background: Studies have shown that MEK inhibitors (MEKi) can reduce tumorigenic growth signaling in colorectal cancer (CRC) through inhibition of the RAS pathway which is upregulated in over 40% of CRC tumors, while HDAC inhibitors (HDACi) lead to cell cycle arrest, inhibit angiogenesis, and induce apoptosis in neoplastic cells. In addition to a direct effect on neoplastic cells, MEKi have also demonstrated the potential to increase immune response to the tumor through upregulation of MHC I on tumor cells, induction of intra-tumoral T-cell infiltration, and enhancement of anti-PDL1 activity. HDAC inhibitors lead to epigenetic alterations which have similarly shown an ability to increase tumor immunogenicity, leading to the hypothesis that these agents may be combined with immunotherapy treatments for enhanced anti-tumor effect. In this study we first analyzed the effect of MEKi and HDACi combination treatment in both resistant and sensitive CRC cell lines to assess for synergy in the attenuation of cancer cell proliferation. We then demonstrated the effect of combination MEKi and HDACi therapy on tumor immunogenicity through increased expression of tumor immune markers. Methods: We assessed the efficacy of a novel MEKi agent, HL-085, in comparison with trametinib in six different CRC cell lines. Treatment with each agent was separately administered with a maximum concentration of 10 uM. Similarly, five different HDAC inhibitors were tested in each of these cell lines, with a maximum concentration of 1 uM. Optimal combination of MEK inhibitor HL085 and HDAC inhibitor class I/II (OKI-005) were then chosen based on this data. Cellular proliferation was assessed after 72 hours with Cell Titer Glo. Flow cytometry of a panel of immunogenic markers was then performed alongside a viability stain to assess for expression level changes of MCH class I in CRC cells. Results: Combinatorial effects of dual treatment with MEK inhibitor HL085 and HDAC inhibitor OKI-005 are observed in both resistant and sensitive cell lines with synergistic effect produced in the SW620 and Lovo cell lines (MEKi sensitive) as well as the GP2D (MEKi resistant) cell line. No synergistic effect was observed in the HCT15, DLD1 and LS513 cell lines. Flow cytometry showed an increase in MHC class I expression in neoplastic cells with combined MEKi and HDACi treatment compared to individual agent treatment and control, demonstrating increased immunogenicity of CRC cells with combination treatment. Conclusions: Combination treatment with a novel MEK inhibitor HL085 and the HDAC inhibitor OKI-005 demonstrates synergy in three out of six colorectal cancer cell lines. We further show increased immunogenicity of CRC cells through enhanced expression of MCH I on tumor cell surface, indicating rationale for further investigation of combination MEKi and HDACi with immunotherapy in the treatment of metastatic colorectal cancer. Citation Format: Mihailo Miljanic, Nisha Holay, Anna Capasso, Todd Triplett, Milad Soleimani, Uma Giri, Carla Van Den Berg, Gail Eckhardt. Attenuating colorectal cancer cell growth and enhancing immunogenicity through rational combination of MEK and HDAC inhibition [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 4753.

CXCR4 antagonism in combination with IDO1 inhibition weakens immune suppression and inhibits tumor growth in mouse breast cancer bone metastases.

PurposeTo investigate whether inhibition of the CXCL12/CXCR4 axis or IDO1 could produce antitumor effects in a metastasized breast cancer immunocompetent animal model.MethodsBreast cancer metastasis models were established in mice. CXCR4 inhibitor and IDO1 inhibitor were used to evaluate the anticancer effects.ResultsCombination treatment using the CXCR4 antagonist AMD3465 and the IDO1 inhibitor D1MT successfully delayed the progression of breast cancer bone metastases. AMD3465 reduced the number of intratumoral regulatory T-cells (Tregs) and myeloid-derived suppressor cells (MDSCs), while D1MT facilitated the antitumor effects of intratumoral CD8+ T-cells. IDO1 inhibition elevated the expression of perforin, granzyme-B, and IFN-γ in CD8+ T-cells, and AMD3465 treatment weakened the potential immune suppressive effects of Tregs and MDSCs. As a result, combination treatment significantly prolonged tumor-bearing mouse survival in two metastasis models, and these antitumor effects relied on overexpression of indoleamine 2, 3-dioxygenase 1 (IDO1), an enzyme that modulates the immune response and impairs immune attack in ovarian cancers CXCR3+ CD8+ cytotoxic T-cell function.ConclusionThe current study provides preclinical evidence that AMD3465 treatment in combination with IDO1 inhibition may be a promising therapeutic regimen for refractory metastasized breast cancers.

Intratumoral CD3+ and CD8+ T-cell densities in patients with deficient DNA mismatch repair (dMMR) metastatic colorectal cancer (mCRC) receiving programmed death-1 (PD-1) blockade.

3532 Background: Colorectal cancer with dMMR display heterogeneity in the extent of intratumoral T-cell infiltration which may explain their variable responsiveness to PD-1 blockade. We examined the association of intratumoral CD3+ and CD8+ T-cell densities (TCD) with objective response rate (ORR) and response duration in patients with dMMR mCRCs receiving pembrolizumab (PEM). Methods: Record review was performed on 12 patients with dMMR mCRC treated with PEM (200 mg intravenously every 3 weeks) after failure of prior chemotherapy [median no. of regimens was 1 (range 1-4)] between 01/2015 and 12/2017. CD3+ and CD8+ TCDs were analyzed in the primary tumor core (CT) and at the invasive margin (IM) by immunohistochemistry and automated image analysis to determine density score (0 to 100) for each T-cell subtype and compartment (Ventana Medical Systems, Inc.). Patients were categorized as 1) responders [CR (complete response) + PR (partial response)] vs. non responders [SD (stable disease) + PD (progressive disease)] per RECIST version 1.1, and 2) by duration of response (&lt; or &gt; 12 months). Results: Median follow-up post PEM was 19.5 (9-41) months. Responders included 2 CR and 5 PR; non-responders included 4 SD and 1 PD. The ORR and median time to response were 58.3% (7/12) and 12 weeks (range 9-40), respectively. CD3+ and CD8+ TCD scores were higher in responders vs. in non-responders as well as in patients who had disease control for &gt; 12 months; differences were greatest for CD8+ CT (Table). Conclusions: Among patients treated with PEM, data suggest higher intratumoral CD3+ and CD8+ TCDs in responders versus non-responders and in those with a longer duration of disease control. If confirmed, TCDs may potentially predict responsiveness to PD-1 blockade in dMMR mCRCs. [Table: see text]

Mandated Cool Housing Temperature and Adrenergic Stress reduce the efficacy of radiation and mask the “Abscopal Effect” in mouse models of cancer

Abstract Mice used for modeling cancer and anti-tumor immunity experience increased adrenergic stress due to IACUC-mandated cool housing temperatures. We wondered if this source of stress could be impacting the efficacy of ionizing radiation and the frequency of an “abscopal” effect (in which tumors outside the field of irradiation exhibit a response, an effect linked to enhanced systemic anti-tumor immunity). When mice bearing two separate CT26 tumors were housed at a thermoneutral temperature (TT; ~30°C) and 6 Gy of radiation was delivered to one tumor, control of both the irradiated and non-irradiated tumors was much greater than that seen in both tumors of mice housed at standard cool temperature (ST; ~22°C). Moreover, when tumor bearing mice housed at ST were treated with the pan β-blocker, propranolol and radiation, we observed a significantly improved growth control of irradiated and non-irradiated tumors. These effects were lost in SCID mice, and in mice depleted of CD8+T-cells. Experiments using knockout mice specifically identified a role for the β2 adrenergic receptor in mediating these effects. Moreover, we found that intra-tumoral effector T-cells, including IFN-γ+, GzmB+, TNFα+ and T-bet+CD8+T-cells, as well as the ratio of IFNγ+CD8+T-cells to Tregs, increased while numbers of MDSC and Tregs decreased, in mice treated with β-blocker and radiation. Finally, cured mice treated with propranolol and radiation rejected local and distant rechallenges in a tumor-specific manner. In conclusion, these data suggest that adrenergic stress plays a major role in regulating the efficacy of radiation therapy. Blockade of β2 adrenergic signaling could be a useful new strategy in the radiation oncology clinic.