PD-1 Ab Research Articles

Axitinib in combination with anti–PD-1 ab (pucotenlimab) plus intra-hepatic injection of oncolytic virus (OH2), in patients with mucosal melanoma and liver metastasis: An open-label phase I trial.

e21524 Background: The combination of anti-PD-1 plus axitinib as systemic therapy had promising antitumor activity in mucosal melanoma, but with limit effect in mucosal melanoma with liver metastasis, which might due to immunosuppression in liver metastasis. Oncolytic virus can heighten the immune response and turn over the immunosuppressive state, which has shown its efficacy in liver metastaic as Intra-hepatic injection. So we initiated this phase I study to evaluate axitinib combination with anti–PD-1 ab plus oncolytic virus in liver metastasis mucosal melanoma. Methods: Eligible pts included those over 18 years old, mucosal melanoma with injectable liver metastasis confirmed by biopsy with or without extra-hepatic metastasis. Pts received intravenous pucotenlimab Q3W combined plus axitinib 5 mg orally twice a day, combined with ultrasound guided intratumoral injection of OH2 Q2W (107CCID50/mL, 8ml per injection). Liver biopsy would be performed at first tumor evaluation (8-12weeks). The primary endpoint was ORR; secondary endpoints included toxicity, disease control rate (DCR), PFS and OS. Results: From Dec 2021 to Jan 2024, 20 pts were eligible and enrolled. Baseline characteristics: median age 56 yrs; 70% got previous treatment, (35% PD-1 resistant). 90% got extra-hepatic metastasis: regional or distant lymph node 45%, lung 40%,; LDH>ULN 50%; median size of target lesions: 61.5mm(12-155mm); median number of liver metastasis: 3(1-18) with median liver lesions 42.5mm(13-230mm). Till the last flow up, 15 pts were evaluable with a median follow-up of 9.39 months. The global ORR was 26.7% (4/15) with 42.9% lesionshrink, injection lesions decrease (43.8%) and non-injection lesion 23.1%. The DCR was 46.7% (7/15). There are still 8 pts on treatment at last follow up. The median PFS was 4.5m, and no pts who had response showed progression till last follow up, the mean DOR was 11.0m. Median OS is not reached. Biopsies of 13 pts for injected lesions at 8 to 12 weeks after first injection were examined to determine the situation of tumor regression and TIL infiltration. Among them, 3 pts (2 PD and 1 PR) had no melanoma cells residual by immunohistochemistry with impressive TIL infiltration. The biopsy showed better pathological response than radiological response which might explain some pts still got survival benefit on treatment after radiological PD. Most adverse events (AE) were grade 1-2 and manageable. Grade 3-4 treatment-related AE showed 30% pts, with diarrhea and oral ulceration as the most common. No treatment-related deaths occurred. Conclusions: Systemic anti-PD-1 plus axitinib combined intralesional injection of Oncolytic virus has shown preliminary efficacy in mucosal melanoma pts with liver metastases with manageable toxicity. Clinical trial information: NCT04206358 , NCT05070221 .

Abstract LB257: Possible involvement of PDL1 intrinsic signaling to acquired resistance to immunotherapy for renal cell carcinoma (RCC)

Abstract Immune checkpoint inhibitor (ICI) therapy has shown unprecedented results in RCC treatment, but durable responses have been hampered by acquired resistance (AR, initially respond but eventually develop cancer progression). Mechanisms for AR remain ambiguous. Previously, we reported elevated blood GPNMB to strongly associate with AR development. To study molecular mechanisms for AR-linked GPNMB elevation, we created an ICI-resistant RenCa (R-RenCa) mouse model by repeated selection of largest tumors in mice treated with PD1 Ab ICI. Parental cells (Gpnmblow) respond to ICI (S-RenCa), while R-RenCa is completely unresponsive to ICI and acquired Gpnmb overexpression (but PDL1 remained unchanged), recapitulating strong association of GPNMB with AR development. R-RenCa tumors displayed more immunosuppressive and angiogenic phenotypes and enhanced response to anti-Gpnmb Ab. In probing for genetic alterations unique to R-RenCa, whole exome seq analysis showed R-RenCa to bear a greater tumor mutation burden (184.9 and 174.6 mut/Mb for R- and S-RenCa, respectively) but no somatic mutations in beta-2 microglobulin and IFN-signal molecule genes known to be involved in AR. RNA-seq analysis showed; more transcriptionally active genes in R-RenCa tumors (heatmap analysis); more responsive genes to ICI (Volcano analysis); and dysregulation in microphthalmia (Mitf) family gene expression. Mitf RNA was upregulated 3-fold, but Tfe3 and other members downregulated 2-3-fold. RNA start sites of Mitf transcripts are highly heterogenous in R-RenCa compared to S-RenCa (Sashimi plot), indicating generation of more isoforms. Since Gpnmb promoter has E-boxes (DNA binding sites) for Mitf factor, we examined whether Mitf is responsible for Gpnmb expression using promoter-Luc reporter assays and found 4-fold higher in R-RenCa, which was lost completely in E-box-mutated promoter. In seeking transcriptional activators for Mitf gene, we found Sox10 factor to be upregulated in R-RenCa. The molecular link of Sox10-Mitf-Gpnmb was shown by the data of gene-knock down experiments that Sox10-siRNA diminished Mitf and Gpnmb expression completely, and Mitf-siRNA had no effect on Sox10 expression but diminished Gpnmb expression. We then studied mechanisms for Sox10 activation and found PDL1 signaling (triggered by Ab-crosslinking) to induce Sox10 RNA expression in tumor cells, which was 80% abrogated by deleting the RMLDVEKC cytoplasmic motif known to protect tumor cells from IFN cytotoxicity. For human relevance of Sox10-Mitf axis activation, we employed nested qRT-PCR of cell-free RNA (cfRNA) isolated from plasma of RCC patients (n=3) immediately before and after AR. SOX10 and MITF cfRNAs were increased by 2.3 ± 0.85 and 1.77 ± 0.05-fold, respectively, between these two time points. Thus, PDL1 signaling is involved in AR by dysregulating the SOX10-MITF-GPNMB cascade, indicating the potential of the cascade-targeting to break AR hindrance for ICI therapy. Citation Format: Jin-Sung Chung, Lei Guo, Vinita Popat, Ponciano D. Cruz, Lin Xu, Hans Hammers, Kiyoshi Ariizumi. Possible involvement of PDL1 intrinsic signaling to acquired resistance to immunotherapy for renal cell carcinoma (RCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB257.

Abstract 5260: Combination activity of eribulin liposomal formulation and anti-PD-1 antibody after tumor regrowth during anti-PD-1 antibody treatment in mice

Abstract Eribulin (ERI) has been reported a microtubule dynamics inhibitor with unique tumor microenvironment modulations such as vascular remodeling activity. In the previous meeting (AACR2022, 2023), we reported that ERI and liposomal formulation of ERI (ERI-LF) also have immunomodulatory activity that induces CD8+ T cells via its vascular remodeling activity and ERI-LF showed more potent immune modulation and combination activities with anti-PD-1 antibody (PD-1 Ab) than ERI in syngeneic mice models and humanized mice models. Although immune-checkpoint inhibitors (ICI) are using as a key drug for various types of cancers in clinical setting, several clinical questions regarding suitable therapies after ICI therapy and efficacy of ICI rechallenge are remaining. In this study, we evaluated anti-tumor activities of combination of ERI-LF and PD-1 Ab in two syngeneic transplantation models using mouse non-small lung cancer cells, P-glycoprotein-knockout (Pgp-KO) KLN205 cells and Pgp-KO LL2 cells. As a result, combination of 1mg/kg ERI-LF (Q7D×2) with PD-1 Ab (200 ug/head, twice a week ×2 weeks) showed significant stronger antitumor activity compared with each monotherapy in only Pgp-KO KLN205 model, in which ERI-LF increased intratumoral microvascular density as a vascular remodeling activity, not in Pgp-KO LL2 model. We next conducted continuous treatment of PD-1 Ab in Pgp-KO KLN205 model to investigate tumor regrowth during treatment of PD-1 Ab. In this model, tumor growth was inhibited by PD-1 Ab until about two weeks after initiation of the treatment and then this inhibitory activity disappeared after two weeks even by continuous treatment of PD-1 Ab. We performed flow cytometry analysis (FCM) for tumor-infiltrating immune cells and RNAseq analysis using regrowing tumors compared with non-treatment tumors. Expressions of mRNA of several immune inhibitory receptors were upregulated in tumors with regrowth, although CD4+ cells and CD8+ cells were also increased in FCM using tumor samples. Furthermore, we investigated anti-tumor activity of combination of ERI-LF and PD-1 Ab against tumors which changed from inhibitory phase to regrowth phase during PD-1 Ab treatment in Pgp-KO KLN205 model. The combination of ERI-LF at 1mg/kg (Q7D×3) and PD-1 Ab (200 ug/head, twice a week ×3 weeks) also showed potent anti-tumor activity against tumors with regrowth by prior PD-1 Ab treatment as well as PD-1 Ab-naïve tumor, suggesting that this combination therapy might be effective after prior ICI therapy. Currently, Phase 1b/2 clinical trial of ERI-LF plus nivolumab in patients with selected solid cancers (NCT04078295) is underway. Patients with or without prior ICI therapy were enrolled in a small cell lung cancer cohort of this clinical study. Our preclinical results might provide a scientific rationale for a uniqueness of ERI-LF as a post ICI therapy. Citation Format: Moe Tamura, Yuki Niwa, Taro Semba. Combination activity of eribulin liposomal formulation and anti-PD-1 antibody after tumor regrowth during anti-PD-1 antibody treatment in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5260.

Alum-tuned hyaluronic acid-based hydrogel with immune checkpoint inhibition for immunophoto therapy of cancer

Alum-crosslinked hyaluronic acid-dopamine (HD) hydrogel containing indocyanine green (ICG) with anti-programmed cell death-1 (PD-1) antibody (Ab) administration was developed for immunophoto therapy of cancer. Alum modulates the rheological characteristics of hydrogel for enabling syringe injection, shear-thinning feature, and slower biodegradation. In addition, alum in HD-based hydrogel provided CD8+ T cell-mediated immune responses for cancer therapy. ICG in the hydrogel under near-infrared (NIR) light exposure may induce hyperthermia and generate singlet oxygen for selective cancer cell killing. HD/alum/ICG hydrogel injection with NIR laser irradiation elevated PD-1 level in CD8+ T cells. Administration of PD-1 Ab aiming at highly expressed PD-1 in T cells may amplify the anticancer efficacies of HD/alum/ICG hydrogel along with NIR laser. HD/alum/ICG hydrogel with NIR light may have both CD8+ T cell-linked immune responses and ICG-related photodynamic/photothermal effects. Additional injection of immune checkpoint inhibitor can ultimately suppress primary and distant tumor growth by combination with those therapeutic actions.

A phase II trial of paclitaxel plus biweekly cetuximab for patients with recurrent or metastatic head and neck cancer previously treated with both platinum-based chemotherapy and anti-PD-1 antibody.

6032 Background: An important unmet need in treatment options remains for patients with recurrent or metastatic head and neck squamous cell carcinoma (R/M-HNSCC) previously treated with both platinum-based chemotherapy and anti-PD-1 antibody (PD-1 Ab). Recent retrospective studies suggest that previous treatment with Immune checkpoint inhibitor might augment the efficacy of subsequent chemotherapy. This phase II trial aimed to evaluate the efficacy and safety of paclitaxel plus biweekly cetuximab (PTX+bwCmab) for patients with R/M-HNSCC previously treated with both platinum and PD-1 Ab. Methods: This is a single-arm, multicenter phase II trial. Key eligibility criteria were R/M-HNSCC; previous treatment with both platinum-based chemotherapy and PD-1 Ab; ECOG performance status 0 or 1; measurable disease per RECIST v1.1; and adequate organ function. PTX+bwCmab consisted of weekly paclitaxel 100 mg/m2 (days 1, 8, 15) and biweekly cetuximab 500 mg/m2 (days 1, 15) with a cycle of 28 days. Primary endpoint was objective response rate (ORR). We set a null hypothesis of 10% and alternative of 30% with a one-sided alfa of 0.05 and power of 90%. Secondary endpoints were progression-free survival (PFS), overall survival (OS), disease control rate (DCR), and adverse events (AEs) (CTCAE v5.0). Binominal confidence intervals (CIs) for ORR and DCR were estimated by the exact method. Time-to-event analyses were calculated by the Kaplan-Meier method with 95% CIs. Results: Between August 2020 and August 2022, 35 patients were enrolled, of whom 32 were evaluable for response. ORR was 68.7% (95% CI: 49.9-83.8), which met the prespecified criteria for the primary endpoint of ORR. With a median follow-up period for survivors of 16.6 months, median PFS and OS were 5.7 and 13.4 months, respectively. DCR was 93.7% (95% CI: 79.1-99.2). The most common AEs of any grade were skin rash (65%), peripheral nerve neuropathy (45%), neutropenia (40%), fatigue (40%) and paronychia (37%). Grade 3 and 4 adverse events included neutropenia (34%), mucositis (8%), pneumonitis (8%) and febrile neutropenia (5%). These AEs were manageable and no treatment-related death within 30 days was observed. Conclusions: PTX+bwCmab showed highly encouraging efficacy and tolerability in RM-HNSCC patients previously treated with both platinum and PD-1 Ab. Although preliminary, these results suggest that this is a promising treatment option for this hitherto untreatable patient group. Further evaluation is warranted. Clinical trial information: jRCTs051200040 .

Analysis of overall survival (OS) and progression-free survival (PFS) in the phase 1b clinical trial of anti–PD-1 ab (toripalimab) plus intrahepatic injection of orienX010 in stage IV melanoma with liver metastases.

9564 Background: Advanced melanoma with liver metastasis has a lower response rate and survival even in immunotherapy era which might because of its immunosuppressive tumor microenvironment. CPI combined with TVEC had showed a remarkable in cutaneous melanoma. The initial results of the phase 1b trial, systemic toripalimab (anti-programmed cell death-1 antibody) combined with Intratumoral injection of liver with OrienX010 - a HSV-1-derived oncolytic virotherapy with expression of GM-CSF in liver metastasis patients without extra-hepatic injectable lesions had shown its efficacy. Here we present the PFS and overall survival (OS) outcomes. Methods: Eligible pts included those over 18 with injectable liver metastasis confirmed by biopsy with or without extra-hepatic metastasis; the ocular melanoma and brain metastasis were excluded. Pts received intravenous toripalimab Q2W combined with ultrasound guided intratumoral injection of OrienX010 Q2W (8×107 pfu/ml, 10ml per injection) until intolerance or disease progression per iRECIST criteria. Liver biopsy would be performed at baseline and first tumor evaluation (8-12weeks). The primary endpoint was toxicity; secondary endpoints included ORR, disease control rate (DCR), PFS and OS. NCT04206358. Results: From Jul 2019 to Feb 2023, 30 pts enrolled. 60.0% pts primary from mucosal,; 73.3% got extra-hepatic metastasis; median size of injected lesions: 24mm(10-94mm); median number of liver metastasis: 7(1-10); median number of injection: 10 (3-36). Among these pts, 29 pts could be evaluated for efficacy. The median PFS was 7.0months (95%CI: 4.7-9.3 months) and the median OS was not reached. The 3-year OS rates 51.5%. The global ORR by investigator was 20.7% (6/29), DCR 48.3% (14/29); the response rate was 31.0%(9/29) for injected lesions, 30.0%(6/20) for non-injected lesions in liver, and 27.8% (5/18) for extra-hepatic metastases. For pts（21.7%( 2 PR and 3 SD)）with no melanoma cells residual by immunohistochemistry in biopsies the median PFS was 14.0 months (95%CI: 3.7-24.4 months), and it was much longer than that of other pts which was 4.1 months (95%CI: 1.4-6.8 months). The median OS of the pts with no melanoma cells was 19.7 months (95%CI: 7.5-31.9 months). Most adverse events (AE) were grade 1-2 and manageable. The grade 3-4 treatment-related AE included elevated transaminase (3 [10.0%]), nausea (1 [3.3%]), pulmonary embolism (1 [3.3%]) and vomiting (1 [3.3%]). No treatment-related deaths occurred. Conclusions: Systemic toripalimab combined with intrahepatic OrienX010 injection has shown remarkable long PFS, OS and ORR in melanoma pts with liver metastases with manageable toxicity. Clinical trial information: Clinical trial information: NCT04206358 .

Abstract 5075: Antitumor activity of liposomal formulation of eribulin combined with anti-human PD-1 antibody using hPBMC-humanized mouse models

Abstract Eribulin (ERI) has been reported a microtubule dynamics inhibitor with unique tumor microenvironment modulations such as vascular remodeling and reversal of epithelial-mesenchymal transition activities. In the previous meeting (AACR2022), we reported that ERI and liposomal formulation of ERI (ERI-LF) have immunomodulatory activity that induces CD8+ T cells via its vascular remodeling activity and ERI-LF has greater immunomodulatory activity than ERI. Furthermore, we also reported that these immunomodulatory activities contribute combination antitumor activity of ERI and ERI-LF with anti-PD-1 antibody (Ab) using a P-glycoprotein-knockout 4T1 mouse breast cancer syngeneic model; however, evaluation of antitumor activity of immune-checkpoint inhibitor (ICI) using mouse cancer syngeneic model has limitation because numbers and cancer types of mouse cancer cell lines are limited. In this study, we developed humanized mouse model bearing both human cancer cell lines and human peripheral blood mononuclear cells (hPBMC) to evaluate antitumor activity of ICI for various cancer types. Furthermore, we also evaluated combination antitumor activity of ERI-LF with anti-human PD-1 Ab using this hPBMC-humanized mouse models to verify generality of this combination. We first isolated hPBMC from healthy volunteers and the obtained hPBMC was intravenously injected into NOG mice. To confirm transplantation of hPBMC in the mice, we performed flow cytometric analysis of peripheral blood of mice in a time course manner. In the peripheral blood of humanized mice, chimera ratio of hCD45+ cells gradually increased until 5 weeks after hPBMC-injection and then decreased after 6 weeks after hPBMC-injection. Because tumor growth of some cancer cell lines was rejected by hPBMC transplantation, we evaluated some human cancer cell lines that are able to grow in hPBMC-humanized condition and selected MKN45 and NCI-H526 cell lines for human gastric cancer and small cell lung cancer models, respectively. Finally, we verified combination antitumor activity of ERI-LF with anti-human PD-1 Ab in hPBMC-humanized MKN45 and NCI-H526 models. In both models, combination of ERI-LF with anti-human PD-1 Ab showed significantly stronger antitumor activity compared with each monotherapy. In summary, we demonstrated that combination of ERI-LF with anti-PD-1 Ab is effective for gastric and small cell lung cancers, suggesting that this combination therapy is effective for various cancer types. Currently, Phase 1b/2 clinical trial of ERI-LF plus nivolumab in patients with selected solid cancers including gastric and small cell lung cancers (NCT04078295) is underway. Citation Format: Yuki Niwa, Taro Semba. Antitumor activity of liposomal formulation of eribulin combined with anti-human PD-1 antibody using hPBMC-humanized mouse models. [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 5075.

The safety of CAR-T cells and PD-1 antibody combination on an experimental model

Chimeric antigen receptor (CAR) T cells and PD-1 antibodies (PD-1 Ab) are emergent immunotherapies with unprecedented efficacy. The presence of PD-1 on T cells contributes to hypofunction of CAR-T therapy and inhibition of PD-1 enhances anti-cancer effect of CAR-T cells. Therefore, the combination of CAR-T cells and PD-1 antibody is a promissing strategy for cancer treatment. This study aims to establish our in-house CAR-T cells and evaluate the safety of CAR-T cells in combination with PD-1 antibody in animals. The toxicity of CD19-CAR-T cells was examined using Swiss Webster mice. Four mouse groups were treated with control, CAR-T, PD-1 antibody or CAR-T + PD-1 antibody. Mice's overall status was monitored and recorded. At the end-point, hematological and biochemical indices were quantified, histopathology of liver and kidney was evaluated by pathologists. The relative abnormal ratio and absolute values were compared between groups. We generated our in-house CAR-T cells and used them for safety evaluation in mice. The increase in mouse weight was observed in all groups after treatment and the weight was comparable between groups. The hematological, biochemical and histopathological parameters were equivalent between groups, except for liver grain degeneration occurred in treatment groups. Thus, CAR-T cells, PD-1 Ab and their combination were safe in mice. We successfully produced our in-house CAR-T cells and the combination of our CAR-T cells and PD-1 antibody was safe in mice with comparable values of hematopoietic indices, liver and kidney functions. Longer follow-up might be necessary to evaluate their effect on the liver.

Prevention Treatment with Pd-1 Ab Can Increase the Infiltration of Tissue Resident Memory T Cells and Delay the Development of Esophageal Carcinogenesis

Prevention Treatment with Pd-1 Ab Can Increase the Infiltration of Tissue Resident Memory T Cells and Delay the Development of Esophageal Carcinogenesis

Adjuvant Anti-PD-1 Antibody Therapy for Advanced Melanoma: A Multicentre Study of 78 Japanese Cases.

Anti-PD-1 antibodies (Abs) are among the optimal adjuvant therapies for melanoma at high risk of recurrence, especially BRAF wild-type melanoma, but the anti-tumour effects of anti-PD-1 Abs in the adjuvant setting for acral melanoma have not been evaluated previously. The aim of this study was to analyse the efficacy and safety profiles of anti-PD-1 Ab monotherapy in the adjuvant setting in an Asian population including a high ratio of acral melanoma. The efficacy and safety profiles of anti-PD-1 Ab monotherapy in the adjuvant setting were retrospectively analysed in 78 Japanese patients with advanced melanoma, including 31 cases (40%) of acral melanoma. Overall relapse-free survival was 60.3% (47 of 78 cases, 95% confidence interval (CI) 49.2-70.4%), and 39.7% of patients (31 of 78 patients, 95% CI 29.6-50.8%) relapsed during the adjuvant PD-1 Ab treatment. Six cases (7.9%) discontinued the protocol due to serious adverse events. One case (1.3%) discontinued the protocol due to trauma. The relapse-free survival of acral melanoma was 25.8%, whereas that of high cumulative sun damage was 60.0%, and that of low cumulative sun damage was 57.1%. The acral type had a significantly lower 12-month relapse-free survival than other cutaneous types (p = 0.029). The acral type appeared to be an independent prognostic factor on multivariate analysis (p = 0.015). Adverse events due to anti-PD-1 antibody were observed in 37.1% overall. The results of this study suggest that anti-PD-1 Ab therapy in the adjuvant setting is less effective for acral melanoma than for other cutaneous types.

Hyperbaric oxygen facilitates teniposide-induced cGAS-STING activation to enhance the antitumor efficacy of PD-1 antibody in HCC

BackgroundEmerging evidence indicates that the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) axis plays a pivotal role in intrinsic antitumor immunity. Previous studies demonstrate that the conventional chemotherapy agent, teniposide,...

PD-1/PD-L1 Checkpoint Inhibitors Are Active in the Chicken Embryo Model and Show Antitumor Efficacy In Ovo.

Simple SummaryCancer immunotherapy, also known as immuno-oncology (IO), has made impressive progress in recent decades and is becoming an essential approach for cancer treatments. For IO drug development, a pertinent preclinical model is indispensable for the rapid and efficient transition from preclinical evaluation through to clinical progress. To date, rodents represent the most-often used models for preclinical evaluation. However, their use presents several drawbacks, including ethical constraints, and time-consuming and costly experiments, which could slow down IO drug development. The aim of our study was to assess the use of the chicken embryo (in ovo) model as an alternative in vivo model for evaluating IO drugs. We confirmed in ovo the anti-tumor efficacy of programmed cell death protein-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) checkpoint inhibitors based on the Chicken Chorioallantoic Membrane (CAM) assay, revealing the pertinence of the chicken embryo model in its use for IO research.(1) Purpose: To assess the use of the chicken embryo (in ovo) model as an alternative in vivo model for immuno-oncology (IO) drug development, focusing on programmed cell death protein-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) immune checkpoint inhibitors. (2) Methods: First, the presence of immune cells in the model was detected through the immunophenotyping of chicken peripheral blood mononuclear cells (PBMCs) based on fluorescence activated cell sorting (FACS) analysis and the immunohistochemistry (IHC) analysis of in ovo tumor-infiltrating lymphocytes. Second, the cross-reactivity between one anti-human PD-1 Ab, pembrolizumab (KEYTRUDA®), and chicken PD-1 was verified through the labelling of chicken splenocytes with pembrolizumab by FACS analysis. Third, the blockade effect of pembrolizumab on chicken PBMCs was assessed in vitro through cytotoxicity assay based on MTT. Fourth, the CAM assay was used to estimate the anti-tumor performance of pembrolizumab through the analyses of tumor growth and chicken immune cell infiltration in tumors. Finally, the efficacy of several PD-1 or PD-L1 inhibitors (nivolumab, atezolizumab and avelumab) on tumor growth was further assessed using the CAM assay. (3) Results: The presence of CD3+, CD4+, CD8+ T lymphocytes and monocytes was confirmed by FACS and IHC analyses. During in vitro assays, pembrolizumab cross-reacted with chicken lymphocytes and induced PD-1/PD-L1 blockade, which permitted the restoration of chicken T-cell’s cytotoxicity against human lung cancer H460 tumor cells. All these in vitro results were correlated with in ovo findings based on the CAM assay: pembrolizumab inhibited H460 tumor growth and induced evident chicken immune cell infiltration (with significant chicken CD45, CD3, CD4, CD8 and CD56 markers) in tumors. Furthermore, the potency of the CAM assay was not limited to the application of pembrolizumab. Nivolumab, atezolizumab and avelumab also led to tumor growth inhibition in ovo, on different tumor models. (4) Conclusions: The chicken embryo affords a physiological, immune reactive, in vivo environment for IO research, which allows observation of how the immune system defense against tumor cells, as well as the different immune tolerance mechanisms leading to tumor immune escape. The encouraging results obtained with PD-1/PD-L1 inhibitors in this study reveal the potential use of the chicken embryo model as an alternative, fast, and reliable in vivo model in the different fields of IO drug discovery.

CeNtuRIOn: Rucaparib (R) with nivolumab (N) and ipilimumab (I) in patients (pts) with relapsed ovarian cancer (ROC)—Results of an initial safety cohort.

5577 Background: There is an urgent need to improve outcomes for ROC pts; progression free (PFS)/overall survival (OS) are 4-9 months (mo), 12-20mo respectively. ̃50% ROC pts harbour homologous repair deficiencies (HRD), identified by BRCA mutations (m) & Loss of Heterozygosity (LoH). R, a PARP inhibitor (PARPi) has demonstrated efficacy when used as maintenance after chemotherapy (CT), and is superior to CT if used as treatment for ROC BRCAm pts. However repeating PARPi, even in highly selected BRCAm/HRD pts has limited benefit (1.5-2.5mo extra PFS). Single agent immunotherapy (PD-1/PD-L1/CTLA4 antibodies (Ab)) in ROC pts has been disappointing. Response rate to combined PD-1/CTLA4 Ab in ROC pts is 31.3%. In vitro evidence suggests that combinations of PD-1/CTLA4 Ab /PARPi may be more effective. CeNtuRIOn aims to explore the activity/toxicity of repeat PARPi (R) versus R+I (CTLA4 Ab) versus R+I+N (PD-1 Ab). Here we report safety data from the run-in cohort of 15 ROC pts treated with the triplet combination, RNI. Methods: Eligible pts had received ≥1 and <3 lines CT, were > 3 to <12 months from last platinum CT. Only 1 pt could experience a dose limiting toxicity (DLT) over initial 6 weeks of treatment. Pts received N 240mg q14 days, I 1mg/kg q42 days intravenously for up to 12mo and R orally, 600mg bd continuously. All bar 1 of 7 pts (recruited 06/19–12/19) received N+I to meet DLT evaluability, but 5 of remaining 6 received < 43% R during safety period. 6 further pts were recruited (08/20-06/21), with ≥60% starting dose R in first 6 weeks now required for evaluability. Review of toxicity/tolerability prompted reduction of R starting dose to 400mg bd and ipilimumab limited to 4 cycles for the last 4 pts. Results: Of all 15 recruits, median age was 64. Adverse events (AE)s are detailed in Table. 7 pts were evaluable for safety assessment: n = 4 started on 600mg bd and n = 3 on 400mg bd R. Median R dose intensity in these 7 was 82.5%. There was 1 DLT: G3 nephritis on day 18. 1 pt died of E. Coli sepsis related to colitis. Outcome data will be presented. Conclusions:R (400mg bd)+N+I (4 cycles only) will be taken forward to phase II. Pts will be randomised 1:1:2 to single agent R, R+I or RNI. Pts recruited to R alone are eligible for 2nd randomisation (1:1) to add I or N+I at progression. Clinical trial information: ISRCTN10490346. [Table: see text]

Analysis of overall survival (OS) and relapse-free-survival (RFS) in the phase 1b clinical trial of anti–PD-1 ab (toripalimab) plus intralesional injection of OrienX010 in stage Ⅳ melanoma with liver metastases.

9551 Background: Advanced melanoma with liver metastasis has reduced response to anti-PD-1 monotherapy with ORR of 4.3% in a pooled analysis, and initial results of the phase 1b trial, systemic toripalimab combined with intrahepatic OrienX010 injection - a HSV-1-derived oncolytic virotherapy with expression of GM-CSF had shown its efficacy. Here we report the RFS and OS outcomes. Methods: Eligible pts included those over 18 with injectable liver metastasis confirmed by biopsy with or without extra-hepatic metastasis; the ocular melanoma and brain metastasis were excluded. Pts received intravenous toripalimab Q2W combined with ultrasound guided intratumoral injection of OrienX010 Q2W (8×107 pfu/ml, 10ml per injection) until intolerance or disease progression per iRECIST criteria. Liver biopsy would be performed at baseline and first tumor evaluation (8-12weeks). The primary endpoint was toxicity; secondary endpoints included ORR, DCR and PFS. Results: From Jul 2019 to Jan 2022, 23 pts were eligible and enrolled. Baseline characteristics: median age 69 yrs; primary: mucosal 60.9%, cutaneous 13.0%, unknown primary 13.0%, acral 13.0%; gene mutation status: Braf 17.4%, Nras 4.3%; 69.6% got extra-hepatic metastasis: regional or distant lymph node 56.3%, lung 37.5%, bone 31.3%; LDH＞ULN 26.1%; median size of injected lesions: 35mm(10-94mm); median number of liver metastasis: 7(1-10); median number of injection: 10 (3-36). Among these pts, 20 pts could be evaluated for efficacy. The median PFS was 7.0 months (95%CI: 4.3-9.7 months) and the median OS was 18.6 months (95%CI: 13.4-23.7 months) with a median follow-up time of 19.8 months (range, 0.9-29.7). The global ORR by investigator was 15% (3/20), DCR 50% (10/20); the response rate was 35%(7/20) for injected lesions, 27.8%(5/18) for non-injected lesions in liver, and 26.7% (4/15) for extra-hepatic metastases. Biopsies of 15 pts for injected lesions at 8 to 12 weeks after first injection were examined to determine the situation of TIL infiltration. Among them, the median PFS of the pts (7/15) with impressive TIL infiltration (Brisk n = 4 and Nonbrisk n = 3 according to the definition of AJCC 8th edition) was 7.8 months (95%CI: 2.8-12.8 months) versus 4.1 months (95%CI: 0-9.1 months) of the pts without impressive TIL infiltration. For pts（21.7%( 2 PR and 3 SD)）with no melanoma cells residual by immunohistochemistry in biopsies the median PFS was 13.8 months (95%CI: 4.0-23.6 months), and it was much longer than that of other pts which was 5.6 months (95%CI: 2.4-8.8 months). The median OS of the pts with no melanoma cells was 19.7 months (95%CI: 7.5-31.9 months). Conclusions: Systemic toripalimab combined with intrahepatic OrienX010 injection has shown remarkable long PFS and OS in melanoma pts with liver metastases. Clinical trial information: NCT04206358.

Role of an anti-gastrin vaccine (PAS) alone and in combination with a PD-1 antibody on growth and metastases of gastric cancer.

334 Background: Gastric cancer is a leading cause of cancer-related deaths worldwide. Many of those with advanced gastric cancer are responsive to immune checkpoint antibody therapy, although the median survival even with these new agents is less than 12 months. The gastrointestinal peptide, gastrin, has been shown to stimulate growth of gastric cancer through the cholecystokinin-B receptor (CCK-BR) and the inhibition of gastrin’s action results in decreased growth. In the current investigation we studied the effects of a cancer vaccine, Polyclonal Antibody Stimulator (PAS) that targets the gastrin peptide on growth of gastric cancer in mice. Methods: RNA was extracted from NCC-S1 and YTN-16 murine gastric cancers and analyzed by qRT-PCR for gastrin, CCK-BR, and PD-L1 expression. YTN-16 (5x106) cells were injected subcutaneously in (N = 40) syngeneic female C57BL/6 mice. Mice were divided into 4 groups of equal tumor size and treated with PBS (control), PD-1 antibody (PD-1 Ab) 50 μg, PAS 250μg, or the combination of the PD-1 Ab and PAS given at one week after tumor inoculation (week 0) and at weeks 1, 3, and 6. A PAS booster was also given at week 9.Tumor growth rate was monitored weekly with calipers. At the end of treatment, tumors were excised, weighed and analyzed with Masson’s trichrome stain for fibrosis, and for Ki67, CD8 T-cells, and M2-polarized macrophages by immunohistochemistry. Metastases were counted and confirmed by histology. Results: Gastrin, CCK-BR, and PD-L1 expression were confirmed by qRT-PCR in the gastric cancer cells. PAS monotherapy (P = 0.023) or PAS in combination with PD-1 Ab (P = 0.0003) resulted in significantly slowed tumor growth and with no metastases observed. Tumor weights were 40-52% smaller in mice treated with PAS or the combination therapy compared to PBS or PD-1 Ab, respectively, but this difference did not reach significance (P = 0.09). PD-1 Ab monotherapy did not change tumor size compared to PBS-treated control mouse tumors. Ki67 staining was decreased by 62-67% and fibrosis staining was decreased by 51-61% in tumors of mice treated with PAS monotherapy or in combination with PD-1 Ab, respectively (P < 0.0001). PAS monotherapy resulted in a 4-fold increase of CD8+ T-cells and a 56% decrease in M2-polarized macrophages (P = 0.0001) compared to control tumors. PAS and PD-1 Ab combination therapy resulted in an additive effect with 29% more CD8+ T-cells (P < 0.05) and 54% fewer M2-polarized macrophages (P < 0.0001) compared to tumors of PAS monotherapy-treated mice. Conclusions: Treatment of mice with an anti-gastrin vaccine, PAS, slows growth of gastric cancer and prevents metastases. PAS vaccination improves the effectiveness of PD-1 Ab therapy in part by decreasing tumor fibrosis and altering the tumor immune cell signature. This study suggests that addition of PAS to immune checkpoint antibody therapy in gastric cancer may be beneficial.

The CCK-B receptor: A novel target for therapy of hepatocellular carcinoma.

466 Background: Today, the fastest growing cause of cancer-related death is hepatocellular carcinoma (HCC). Treatment with immune checkpoint antibodies has shown promise in HCC, but the response is still low at about 15%-20% as monotherapy. Our research laboratory discovered that the cholecystokinin-B receptor (CCK-BR) becomes over-expressed in the injured/ inflamed liver and is up-regulated in HCC. The primary aim of this study is to evaluate the effects of a CCK-BR antagonist, proglumide, alone and in combination with a PD-1 antibody (PD-1 Ab) on survival in HCC tumor-bearing mice and the tumor infiltrating T-cells. The secondary aim was to evaluate the frequency of CCK-BR expression in human HCC. Methods: RNA was extracted from murine RIL-175 HCC cells and subjected to qRT-PCR for gene expression of CCK-BRs, gastrin, and PD-L1. C57BL/6 mice (N = 40) were injected with RIL-175 HCC cells (100,000) subcutaneously. After one week when the mice had palpable tumors, they were divided into 4 groups (N = 10ea) of equal tumor size. Treatment included: controls-untreated drinking water; proglumide (0.1mg/ml) in drinking water; PD-1Ab (50µg, ip, days 7, 14, 21 with untreated water); and combination therapy with PD-1 Ab (as above) and proglumide water. Mice were euthanized when tumors reached a diameter of 20mm according to IACUC guidelines. Tumors were stained with a CD8 antibody to evaluate CD8+ tumor infiltrating T-cells. CCK-BR protein expression in human liver tissues and human HCC was evaluated by immunohistochemistry using a tissue microarray (US Biomax). Results: RIL-175 murine HCC cells exhibited increased expression of CCK-BR, gastrin, and PD-L1 by qRT-PCR. Survival of mice treated with the combination of proglumide and the PD-1 Ab was double that of the control mice and greater than proglumide or PD-1 Ab monotherapy. Compared to tumors of control mice, tumors of mice receiving proglumide monotherapy exhibited a 5-fold increase in CD8+ cells (P = 2.5x10-8), and there was a 12-fold increase in CD8+ T-cells in tumors of mice treated with the combination therapy (P = 4.7x10-17). In the human liver tissue microarray, immunoreactivity for the CCK-BR was negative in the normal human liver tissues and increased with the grade of HCC tumor (P = 0.0045) with 84.6% of grade-3 human HCC tissues staining positive for CCK-BR. Conclusions: The CCK-BR may provide a novel target for therapy in HCC. Treatment with the CCK-BR antagonist, proglumide, in combination with a PD-1 Ab appears to be synergistic improving the survival of mice by increasing the number of intratumoral CD8+ T-cells.

Gastrin Vaccine Alone and in Combination With an Immune Checkpoint Antibody Inhibits Growth and Metastases of Gastric Cancer.

Gastric cancer is a leading cause of cancer-related deaths worldwide. Recently, clinical studies have demonstrated that many of those with advanced gastric cancer are responsive to immune checkpoint antibody therapy, although the median survival even with these new agents is less than 12 months for advanced disease. The gastrointestinal peptide gastrin has been shown to stimulate growth of gastric cancer in a paracrine and autocrine fashion through the cholecystokinin-B receptor (CCK-BR), a receptor that is expressed in at least 56.6% of human gastric cancers. In the current investigation, we studied the role of the gastrin-CCK-BR pathway in vitro and in vivo as well as the expression of the CCK-BR in a human gastric cancer tissue array. CCK-BR and PD-L1 receptor expression and gastrin peptide was found in two murine gastric cancer cells (NCC-S1 and YTN-16) by qRT-PCR and immunocytochemistry. Treatment of NCC-S1 cells with gastrin resulted in increased growth. In vivo, the effects of a cancer vaccine that targets gastrin peptide (polyclonal antibody stimulator—PAS) alone or in combination with a Programed Death-1 antibody (PD-1 Ab) was evaluated in immune competent mice (N = 40) bearing YTN-16 gastric tumors. Mice were treated with PBS, PD-1 Ab (50 µg), PAS (250 µg), or the combination of PD-1 Ab with PAS. Tumor growth was significantly slower than controls in PAS-treated mice, and tumor growth was decreased even more in combination-treated mice. There were no metastases in any of the mice treated with PAS either alone or in combination with PD-1 Ab. Tumor proliferation by the Ki67 staining was significantly decreased in mice treated with PAS monotherapy or the combination therapy. PAS monotherapy or combined with PD-1 Ab increased tumor CD8+ T-lymphocytes and decreased the number of immunosuppressive M2-polarized tumor-associated macrophages. CCK-BR expression was identified in samples from a human tissue array by immunohistochemistry confirming the clinical relevance of this study. These results confirm the significance of the gastrin-CCK-BR signaling pathway in gastric cancer and suggest that the addition of a gastrin vaccine, PAS, to therapy with an immune checkpoint antibody may decrease growth and metastases of gastric cancer by altering the tumor microenvironment.

388 A trial to evaluate the safety, immunogenicity, and clinical activity of a helper peptide vaccine plus PD-1 blockade (Mel64, PATHVACS: PD-1 antibody and T-helper vaccine and correlative studies)

BackgroundA multipeptide vaccine containing 6 melanoma-associated peptides to stimulate helper T cells (6MHP) is safe and immunogenic and has clinical activity. A phase I/II trial was designed to evaluate safety and immunogenicity of 6MHP vaccines plus PD1 blockade.MethodsParticipants with measurable advanced melanoma, age ≥ 18 years, and ECOG performance status 0–1 were administered 6MHP vaccine intradermally and subcutaneously in an incomplete Freund’s adjuvant on days (D) 1, 8, 15, 43, 64, and 85. Pembrolizumab 200 mg was administered intravenously every 3 weeks for up to two years. Biopsies of accessible tumors at baseline and D22 were analyzed by multiparameter immunofluorescence histology. Primary endpoints were safety (CTCAE 4.03) and immunogenicity (ex vivo IFNγ ELIspot assay). Secondary and exploratory endpoints included changes in the tumor microenvironment (TME), and clinical outcomes.ResultsTwenty-two eligible participants were enrolled and treated, including 6 naïve to PD-1 Ab and 16 anti-PD-1 Ab-experienced. Median follow-up was 20 months. Treatment-related adverse events (any grade) experienced by >20% were injection site reactions, fatigue, anemia, nausea, fever, bruising, and rash. Treatment-related dose limiting toxicities (grade 3 elevated AST, skin ulcer, or uveitis) were observed in 3 (14%), which did not cross the study safety bound. Objective clinical responses were observed in 23% (1 CR, 4 PR), including 4/6 anti-PD-1 Ab-naive (67%) and one 1/16 anti-PD1 Ab-experienced (6%). Four participants (18%) had SD as best radiographic response (18%), all in the Ab-experienced cohort. T cell responses to 6MHP were detected in seven participants (32%) by week 13 and were associated with clinical response (CR/PR 80% vs. SD/PD 18%; p = 0.01). Overall survival was prolonged for anti-PD-1 Ab naïve vs experienced (p = 0.0048), for those with T cell response (p = 0.045, landmark analysis after week 13), and for those with objective response (p = 0.0148). TME evaluation in 12 participants revealed significant increases by D22 in the densities (per mm2) of CD8+ T cells (p = 0.0186), CD20+ B cells (P = 0.002), and Tbet+ cells (p = 0.034).ConclusionsIn patients with advanced melanoma, combined treatment with the 6MHP vaccine plus pembrolizumab was safe, increased intratumoral T and B cells, as well as Th1 (Tbet+) cells, and induced T cell responses that were associated with objective response and with overall survival. The promising objective response rate and overall survival in patients naive to PD1 blockade supports consideration of a larger study to assess definitive benefit in that clinical setting.AcknowledgementsWe acknowledge the support of Merck for providing pembrolizumab at no charge, and the University of Virginia Cancer Center Support grant P30 CA044579 for support of shared resource facilities.Trial RegistrationThe clinical trial Mel64 (PATHVACS) is registered with Clinicaltrials.gov (NCT02515227).Ethics ApprovalThe clinical trial Mel64 (PATHVACS) was performed with IRB (#18174) and FDA approval (IND #10825) and is registered with Clinicaltrials.gov (NCT02515227). Written informed consent was obtained from each participant prior to participation in the study.

Programmed death (PD)-1/PD-ligand 1 blockade mediates antiangiogenic effects by tumor-derived CXCL10/11 as a potential predictive biomarker.

Immune checkpoint inhibitor (ICI) programmed death (PD)‐1/PD‐ligand 1 (PD‐L1) blockade has been approved for various cancers. However, the underlying antitumor mechanisms mediated by ICIs and the predictive biomarkers remain unclear. We report the effects of anti‐PD‐L1/PD‐1 Ab in tumor angiogenesis. In syngeneic mouse models, anti‐PD‐L1 Ab inhibited tumor angiogenesis and induces net‐like hypoxia only in ICI‐sensitive cell lines. In tumor tissue and serum of ICI‐sensitive cell line‐bearing mice, interferon‐γ (IFN‐γ) inducible angiostatic chemokines CXCL10/11 were upregulated by PD‐L1 blockade. In vitro, CXCL10/11 gene upregulation by IFN‐γ stimulation in tumor cell lines correlated with the sensitivity of PD‐L1 blockade. The CXCL10/11 receptor CXCR3‐neutralizing Ab or CXCL11 silencing in tumor cells inhibited the antiangiogenic effect of PD‐L1 blockade in vivo. In pretreatment serum of lung carcinoma patients receiving anti‐PD‐1 Ab, the concentration of CXCL10/11 significantly correlated with the clinical outcome. Our results indicate the antiangiogenic function of PD‐1/PD‐L1 blockade and identify tumor‐derived CXCL10/11 as a potential circulating biomarker of therapeutic sensitivity.

Immune related adverse events and treatment discontinuation in patients older and younger than 75 years with advanced melanoma receiving nivolumab or pembrolizumab

BackgroundProgrammed cell-death 1 antibodies (PD-1 Ab) improve overall survival (OS) for patients with advanced melanoma in trials; however, safety data in patients ≥75 years are lacking. The prognostic significance of and risk factors for PD-1 Ab discontinuation due immune related adverse events (irAE) are also uncertain. MethodsPatients with advanced melanoma receiving frontline PD-1 Ab at British Columbia Cancer outside of clinical trials between 10/2015–10/2019 were retrospectively analyzed. The incidence and subtypes of irAE were compared between age subgroups <75 and ≥ 75 years. Univariable logistic regression identified variables associated with treatment discontinuation within four months of PD-1 Ab initiation. Cox proportional hazard regression models were used to determine factors significantly associated with OS. Results302 patients were identified, of whom 126 (41.7%) were ≥ 75 years. During all follow-up, 15.9% of patients experienced irAE grade 3/4 and 27.2% of the cohort stopped PD-1 Ab due to immune toxicity. irAE incidence, hospitalization, and need for steroids by the four-month landmark were similar amongst age groups. Advanced age was not associated with risk of PD-1 Ab discontinuation from irAE on logistic regression. For the entire cohort, pre-treatment factors associated with shorter OS on multivariable analysis were ECOG performance status ≥1, M1d stage, lactate dehydrogenase >224, and neutrophil/ lymphocyte ratio ≥ 5. On four-month landmark multivariable analysis, treatment discontinuation due to irAE was significantly associated with worse OS. ConclusionPatients aged ≥75 years experienced similar irAE rates and treatment discontinuation for immune toxicity compared to younger patients. As PD-1 Ab discontinuation due to irAE was associated with shorter OS, efforts to treat irAE early are warranted to potentially avoid therapy cessation.