PD-1 Checkpoint Inhibitors Research Articles

Porphyrin Cholesterol Conjugates for Enhanced Photodynamic Immunotherapy toward Lung Cancer.

Lung cancer is the major cause of cancer death worldwide. Immune checkpoint inhibitors (ICIs) of PD-1/PD-L1 have improved the survival rate in some patients with lung cancer. However, the efficacy of ICIs is limited by the inhibitory tumor immune microenvironment. Herein, we designed porphyrin cholesterol conjugates (TPPC) for synergistic photodynamic therapy (PDT)-immunotherapy for lung cancer. Porphyrin derivatives with great reactive oxygen species (ROS) production efficiency have been applied as photosensitizers in clinics, and cholesterol is one of the main components of the cell membrane. Porphyrin cholesterol conjugates could assemble into nanoparticles (NPs) in the absence of surfactants or amphiphilic polymers. On the other hand, TPPC NP-mediated PDT could accumulate at the tumor site and induce immunogenic cell death to stimulate and recruit antigen-presenting cells to mature and activate T cells, rendering cancer cells more sensitive to ICIs. Importantly, the combination strategy reshapes the tumor immune microenvironment to enhance the antitumor immune response and significantly suppresses the tumor growth and eliminates metastasis. This study offers theoretical guidance for the combination of PDT and ICIs as a potential therapeutic option in lung cancer.

Adjuvant immunotherapy with nivolumab versus observation in completely resected Merkel cell carcinoma (ADMEC-O): disease-free survival results from a randomised, open-label, phase 2 trial

Merkel cell carcinoma (MCC) is an immunogenic but aggressive skin cancer. Even after complete resection and radiation, relapse rates are high. PD-1 and PD-L1 checkpoint inhibitors showed clinical benefit in advanced MCC. We aimed to assess efficacy and safety of adjuvant immune checkpoint inhibition in completely resected MCC (ie, a setting without an established systemic standard-of-care treatment). In this multicentre phase 2 trial, patients (any stage, Eastern Cooperative Oncology Group performance status 0-1) at 20 academic medical centres in Germany and the Netherlands with completely resected MCC lesions were randomly assigned 2:1 to receive nivolumab 480 mg every 4 weeks for 1 year, or observation, stratified by stage (American Joint Committee on Cancer stages 1-2 vs stages 3-4), age (<65 vs ≥65 years), and sex. Landmark disease-free survival (DFS) at 12 and 24 months was the primary endpoint, assessed in the intention-to-treat populations. Overall survival and safety were secondary endpoints. This planned interim analysis was triggered when the last-patient-in was followed up for more than 1 year. This study is registered with ClinicalTrials.gov (NCT02196961) and with the EU Clinical Trials Register (2013-000043-78). Between Oct 1, 2014, and Aug 31, 2020, 179 patients were enrolled (116 [65%] stage 3-4, 122 [68%] ≥65 years, 111 [62%] male). Stratification factors (stage, age, sex) were balanced across the nivolumab (n=118) and internal control group (observation, n=61); adjuvant radiotherapy was more common in the control group. At a median follow-up of 24·3 months (IQR 19·2-33·4), median DFS was not reached (between-groups hazard ratio 0·58, 95% CI 0·30-1·12); DFS rates in the nivolumab group were 85% at 12 months and 84% at 24 months, and in the observation group were 77% at 12 months and 73% at 24 months. Overall survival results were not yet mature. Grade 3-4 adverse events occurred in 48 [42%] of 115 patients who received at least one dose of nivolumab and seven [11%] of 61 patients in the observation group. No treatment-related deaths were reported. Adjuvant therapy with nivolumab resulted in an absolute risk reduction of 9% (1-year DFS) and 10% (2-year DFS). The present interim analysis of ADMEC-O might suggest clinical use of nivolumab in this area of unmet medical need. However, overall survival events rates, with ten events in the active treatment group and six events in the half-the-size observation group, are not mature enough to draw conclusions. The explorative data of our trial support the continuation of ongoing, randomised trials in this area. ADMEC-O suggests that adjuvant immunotherapy is clinically feasible in this area of unmet medical need. Bristol Myers Squibb.

Guiding model-driven combination dose selection using multi-objective synergy optimization.

Despite the growing appreciation that the future of cancer treatment lies in combination therapies, finding the right drugs to combine and the optimal way to combine them remains a nontrivial task. Herein, we introduce the Multi-Objective Optimization of Combination Synergy - Dose Selection (MOOCS-DS) method for using drug synergy as a tool for guiding dose selection for a combination of preselected compounds. This method decouples synergy of potency (SoP) and synergy of efficacy (SoE) and identifies Pareto optimal solutions in a multi-objective synergy space. Using a toy combination therapy model, we explore properties of the MOOCS-DS algorithm, including how optimal dose selection can be influenced by the metric used to define SoP and SoE. We also demonstrate the potential of our approach to guide dose and schedule selection using a model fit to preclinical data of the combination of the PD-1 checkpoint inhibitor pembrolizumab and the anti-angiogenic drug bevacizumab on two lung cancer cell lines. The identification of optimally synergistic combination doses has the potential to inform preclinical experimental design and improve the success rates of combination therapies. Jel classificationDose Finding in Oncology.

Deep learning-based scoring of tumour-infiltrating lymphocytes is prognostic in primary melanoma and predictive to PD-1 checkpoint inhibition in melanoma metastases.

Recent advances in digital pathology have enabled accurate and standardised enumeration of tumour-infiltrating lymphocytes (TILs). Here, we aim to evaluate TILs as a percentage electronic TIL score (eTILs) and investigate its prognostic and predictive relevance in cutaneous melanoma. We included stage I to IV cutaneous melanoma patients and used hematoxylin-eosin-stained slides for TIL analysis. We assessed eTILs as a continuous and categorical variable using the published cut-off of 16.6% and applied Cox regression models to evaluate associations of eTILs with relapse-free, distant metastasis-free, and overall survival. We compared eTILs of the primaries with matched metastasis. Moreover, we assessed the predictive relevance of eTILs in therapy-naïve metastases according to the first-line therapy. We analysed 321 primary cutaneous melanomas and 191 metastatic samples. In simple Cox regression, tumour thickness (p<0.0001), presence of ulceration (p=0.0001) and eTILs ≤16.6% (p=0.0012) were found to be significant unfavourable prognostic factors for RFS. In multiple Cox regression, eTILs ≤16.6% (p=0.0161) remained significant and downgraded the current staging. Lower eTILs in the primary tissue was associated with unfavourable relapse-free (p=0.0014) and distant metastasis-free survival (p=0.0056). In multiple Cox regression adjusted for tumour thickness and ulceration, eTILs as continuous remained significant (p=0.019). When comparing TILs in primary tissue and corresponding metastasis of the same patient, eTILs in metastases was lower than in primary melanomas (p<0.0001). In therapy-naïve metastases, an eTILs >12.2% was associated with longer progression-free survival (p=0.037) and melanoma-specific survival (p=0.0038) in patients treated with anti-PD-1-based immunotherapy. In multiple Cox regression, lactate dehydrogenase (p<0.0001) and eTILs ≤12.2% (p=0.0130) were significantly associated with unfavourable melanoma-specific survival. Assessment of TILs is prognostic in primary melanoma samples, and the eTILs complements staging. In therapy-naïve metastases, eTILs ≤12.2% is predictive of unfavourable survival outcomes in patients receiving anti-PD-1-based therapy. See a detailed list of funding bodies in the Acknowledgements section at the end of the manuscript.

Combination oncolytic virus, radiation therapy, and immune checkpoint inhibitor treatment in anti-PD-1-refractory cancer

BackgroundImmunotherapies are becoming front-line treatments for many advanced cancers, and combinations of two or more therapies are beginning to be investigated. Based on their individual antitumor capabilities, we sought to...

CXCL10 mediates CD8+ T cells to facilitate vessel normalization and improve the efficacy of cetuximab combined with PD-1 checkpoint inhibitors in colorectal cancer

The immunotherapy and anti-EGFR targeted treatment occupying a pivotal position in colorectal cancer (CRC), is still limited to a group of patients who display specific molecular alterations and inevitably escape from resistance, further studies are still needed in colorectal cancer. We found that chemokine ligand 10 (CXCL10) expression correlates with intratumoral CD8+ T cell infiltration and reprograms tumor vasculatures in colorectal cancer. CXCL10 overexpression not only suppressed tumor growth but also increased CD8+ T cell infiltration and induced tumor vascular normalization in vivo. Additionally, the growth inhibition and tumor vascular normalization induced by CXCL10 can be reversed by the depletion of CD8+ T cells in vivo. Mechanically, CXCL10 interacts with VCAN to mediate tumor vascular normalization. The VCAN expression correlated inversely with the expression of CXCL10 and the infiltration of CD8+ T cells in CRC. Elevated CXCL10 expression sensitized colorectal cancer cells to cetuximab/anti-PD1 combination therapy compared with cetuximab or anti-PD1 alone. We propose that CXCL10 could be used to increase the anti-EGFR therapy and immunotherapy effect, targeting both tumor vessels and immune cells in colorectal cancer.

Phase 2 study of HDAC inhibitor tucidinostat in combination with anti-PD-1 antibody in patients with locally advanced or metastatic urothelial carcinoma.

e16563 Background: Tucidinostat (also known as chidamide), a novel histone deacetylase (HDAC) inhibitor, has been approved for treatment of patients (pts) with relapsed or refractory peripheral T-cell lymphoma and advanced hormone receptor-positive breast cancer in China. Tislelizumab (a PD-1 checkpoint inhibitor) is currently approved for pts with PD-L1-high locally advanced or metastatic urothelial carcinoma (UC) who progressed during/following platinum-containing therapy in China. However, the response rate of tislelizumab as monotherapy is limited in UC. The aim of this study was to evaluate the efficacy and safety of tucidinostat combined with tislelizumab in pts with locally advanced or metastatic UC. Methods: This study is an open-label, single-center, phase Ⅱ trial. Patients who previously failed systemic platinum-based chemotherapy with UC were included, regardless of PD-L1 status. Eligible pts received 30 mg oral tucidinostat twice weekly (BIW) in combination with 200 mg tislelizumab by intravenous drip every three weeks (Q3W). This is a Simon's two-stage design. All pts were treated until progression or unacceptable toxicity in two years. The primary endpoint was objective response rate (ORR). The secondary endpoints included progression free survival (PFS), disease control rate (DCR), overall survival (OS), the safety and tolerability. Results: From Jan. 2021 to Sep. 2022, 26 pts were enrolled (median age 62 y, range 31-74y), of whom 24 were evaluable for response per RECIST 1.1 criteria. One pt was lost to follow-up and one withdrew the group due to sudden cerebral infarction. Complete response (CR) was observed in 2 pts, while partial response (PR) was observed in 8 pts, with an ORR of 41.7% (10/24). The DCR was 62.5%. The median PFS was 4.6 months. Most adverse events (AEs) were grade 1 or 2. The most common grade 3 AEs were neutropenia 11.5%, thrombocytopenia 11.5%, leukopenia 7.7%, anemia 7.7%, anorexia 7.7%, creatinine increased 3.8%. One pt experienced grade 4 immuno-pneumonia was permanently discontinued. One pt was on maintenance therapy of tucidinostat without immunotherapy because of interstitial pneumonia. Conclusions: The preliminary data indicate that tucidinostat combined with tislelizumab might be a potentially effective regimen with a manageable safety profile in UC. The study remains ongoing and updated results will be presented later. Clinical trial information: NCT04562311 .

Anti-drug antibodies related to CTLA-4, PD-1, or PD-L1 inhibitors across tumour types: A systematic review.

e14600 Background: Treatment-emergent anti-drug antibodies (ADAs) are postulated to reduce the efficacy of immune checkpoint inhibitors. A systematic review (SR) was conducted to evaluate the incidence of treatment-emergent ADAs with programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors and their impact on treatment outcomes in patients with cancer. Methods: Electronic databases MEDLINE, EMBASE and Cochrane Library were searched in February 2022 for studies reporting incidence of ADAs and the impact of immunogenicity on treatment efficacy in oncology patients treated with PD-1, PD-L1, or CTLA-4 checkpoint inhibitors. Results: Of the 4,612 articles identified, 34 publications reporting on 68 trials were included. An additional 41 and 32 records were identified from clinicaltrials.gov and packaging inserts respectively. In total, 141 trials of 15 checkpoint inhibitors and 16 tumour types were included. There was considerable variability in the reporting of ADAs, and comparisons between trials were challenging due to intra-study heterogeneity. The incidence of ADAs ranged from 0 to 74%. Across trials, in analyses with the largest sample sizes, atezolizumab was associated with the highest incidence of ADAs (29.6%), then nivolumab (12.2%), followed by the remaining treatments (0-4.4%). The incidence of neutralising antibodies (nAbs) mirrored ADA distribution, being highest for atezolizumab (4.3–27.5%) vs other treatments. Only 17 trials reported on ADA impact on treatment outcomes. Of 11 trials examining efficacy, two reported a negative effect of ADAs, eight reported no measurable effect and another had insufficient patient numbers. The impact of ADAs on treatment safety, and pharmacokinetics was also inconclusive. Conclusions: Treatment-emerging ADAs are inconsistently reported across trials and documented in up to 30% of subjects treated with checkpoint inhibitors across indications. Emergence of nAbs follows but does not perfectly mirror ADA titers, raising questions around the functional meaning of different ADAs. Standardised reporting of ADAs and nAbs should be advocated to facilitate more conclusive immunologic and clinical insight as to the relationship with ADAs and response. [Table: see text]

A gut microbiota rheostat to forecast response to PDL1-VEGF blockade in mesothelioma.

e20534 Background: Targeting of the immune inhibitory PD1-PDL1 axis has proven clinically effective in mesothelioma, despite a low somatic mutation burden, and high rate of CDKN2A deletion. Tumour responses to anti PD1 or PDL1 immune checkpoint inhibition are heterogeneous, and the factors underpinning sensitivity remain poorly understood. We therefore addressed this knowledge gap through multi-omic interrogation of tumours from patients enrolled into arm 4 of the Mesothelioma Stratified Therapy umbrella trial (NCT03654833, MiST4), a multi-centre single arm phase IIA trial of atezolizumab and bevacizumab in patients with relapsed mesothelioma. Methods: Next generation sequencing of whole exomes (mesotheliomas and matched germline DNA), transcriptomes, and 16sRNA to profile gut microbiota were undertaken. Spatial phenotyping of the immune landscape employed multiplex immunofluorescence analysis using a 19x depth 4 panel detection. Tumour proportion score (TPS) for PDL1 was assessed using the 22C3 clone, and BAP1, p16ink4a assessed by immunohistochemistry. The MIST4 cohort was dichotomised by best tumour response (50:50) into those patients exhibiting any tumour reduction (R) versus those without (NR). Machine learning (boosting and bagging) was employed to decipher correlates of response. Results: Tumour responses correlated with progression-free survival (PFS, p = 0.0003). Neither PDL1 TPS or CDKN2A expression were predictive. The NR group exhibited a greater degree of genomic instability with higher somatic copy number burden (p = 0.02), homologous recombination deficiency (HRD, p = 0.03), and uniparental disomy (UPD, p = 0.01). Notably the burden of nonsynonymous mutations and neoantigens did not differ significantly between groups. The NR group was transcriptionally enriched for epithelial mesenchymal transition (p &lt; 0.05). Conversely, 16s RNA sequencing revealed higher gut microbial diversity in the R group compared with NR (Shannon index p = 0.009) with R-group enrichment of the type 2 enterotype (provotella 33% vs 9%). R-group enriched genera comprised prevotella, butyricicoccus, bilophilla, Eubacterium ventriosum, whereas the NR group was enriched for erysipelatoclostidium. The log ratio of genera, ie. Log[GR/GNR] was 2-log higher for the R group (p &lt; 0.0001) vs NR group, and was highly predictive of response (with an area under the receiver operator curve of 0.99). Log[GR/GNR] positively correlated with tumour CD8 T cell infiltration (r = 0.6, p = 0.05) and PFS (p = 0.04), but negatively with CD68 monocyte infiltration (p = 0.05), UPD (p = 0.008) and HRD (p = 0.05). Conclusions: We propose a model in which interacting tumour intrinsic and extrinsic factors correlate with response to PDL1-VEGF inhibition in patients with mesothelioma. Gut microbiota composition represents a new, potentially modifiable target with potential to improve immunotherapy outcomes in patients with mesothelioma. Clinical trial information: NCT03654833 .

Efficacy, cellular and molecular determinants of PD-1 checkpoint inhibition in relapsed mesothelioma.

8506 Background: Leveraging adaptive immunity to control mesothelioma is now a standard approach, however the factors that underpin clinical response are poorly understood. Here we report the final analysis of the CONFIRM trial (NCT03063450), a double-blind phase III randomized study of the PD-1 inhibitor nivolumab (N) versus placebo (P) in patients (pts) with unresectable mesothelioma. Genomic, transcriptomic and multiplex spatial phenotypic correlates were explored in mesotheliomas exhibiting either partial response or progressive disease as their best outcome. Methods: Pts with relapsed pleural or peritoneal mesothelioma with ECOG performance status 0–1 were randomized 2:1 to N (3 mg/kg) or P once every 2 weeks until progression, or a maximum of 12 months. Pts were stratified by epithelioid (E) vs non-epithelioid (NE) histology. Co-primary endpoints were progression-free survival (PFS) and overall survival (OS); key secondary endpoints included best overall response, safety and tolerability. PD-L1 tumour proportion score (TPS, Dako22C3) was evaluated. To decipher correlates response to N, blinded multi-omic analysis was conducted in a subset of responders (R, n=16) versus progressors (NR, n=13). Whole exomes (WES) and transcriptomes were sequenced, with immune landscapes profiled by 19x depth 4-panel multiplex immunofluorescence. Acquired resistance was explored by WES in a biopsied responder at the time of progression. Results: Between April 2017-March 2020, 332 pts were randomised to N (n=221) or P (n=111). Median follow up was 37.2m. Baseline characteristics were balanced between arms. Histology: E 88.3%, 3rd line or greater 69.9%. PFS was longer for N vs P (events=324; median, 2.9 vs 1.6 months; HR 0.65; 95% CI, 0.51 – 0.82; P&lt;0.001). Crossover from P was 18.0% (due to widespread availability of N during the covid19 pandemic). OS curves crossed at 32m; 9.5 vs 6.8 months; HR, 0.81; 95% CI, 0.64 – 1.04; P=0.096). Grade 3-4 treatment-related toxicity occurred in 20.4% vs 7.2% of pts; discontinuation occurred in 13.6% (N) versus 9.9%. PD-L1 TPS was not predictive of PFS or OS. The R-subgroup had longer PFS versus NR (319 versus 30 days, Hazard ratio, HR 0.02, p&lt;0.001) and OS (670 vs 122 days HR 0.19, p&lt;0.001). The R-group were enriched for BAP1 inactivation, CD8+ T-cell infiltration, T cell co-stimulation, cytolytic activity and mature tertiary lymphoid structures (TLSs). Conversely, the NR group had significantly more aneuploidy (notably uniparental disomy, UPD), and enrichment of epithelial mesenchymal transition, TGF beta signalling, and E2F transcription. Acquired resistance was associated with increased aneuploidy (UPD) and subclonal evolution including a new DNMT3A c2204A&gt;G mutation. Conclusions: N met its co-primary endpoint of PFS with responding mesotheliomas harbouring a TLS enriched, inflamed tumour microenvironment and stable genome compared to non-responders. Clinical trial information: NCT03063450 .

Medicinal Plants in the Regulation of PD-L1/PD-1 Immune Checkpoint of Various Human Cancer Cells: A Narrative Review

Background: Immunotherapy has garnered attention in cancer treatment following the success of recent trials in solid tumors adopting PD-L1/PD-1 checkpoint inhibition. PD-1 is a T-cell checkpoint molecule that limits autoimmune and auto-inflammatory reactivity in the normal host by suppressing adaptive immune responses. Although PD-L1 expression in the tumor is generally considered a poor prognostic marker, it has been used to screen patients for cancer therapy since it is associated with a positive response to PD-L1/PD-1 blocking antibodies. Outline: This review focuses on the complex interconnections between cancer-reactive and selfreactive immune cells, as well as the potential contribution of a wide range of leading immunomodulatory chemical products from plant-based origins as cancer therapeutics or to foreseeably ameliorate autoimmune diseases. The natural compounds derived from plants should be used as a PD-L1/PD-1 checkpoint modulator to combat cancer cells and other chronic diseases. Conclusion: The significance of herbal plant extracts in the regulation of the PD-L1/PD-1 checkpoint is presented in this review together with the expression of PD-L1 and PD-1 in cancer cells and diseases in human bodies.

The abscopal effect: inducing immunogenicity in the treatment of brain metastases secondary to lung cancer and melanoma.

The phenomenon of radiation therapy (RT) causing regression of targeted lesions as well as lesions outside of the radiation field is known as the abscopal effect and is thought to be mediated by immunologic causes. This phenomena has been described following whole brain radiation (WBRT) and stereotactic radiosurgery (SRS) of brain metastasis (BM) in advanced melanoma and non-small-cell lung cancer (NSCLC). We systematically reviewed the available literature to identify which radiation modality and immunotherapy (IT) combination may elicit the abscopal effect, the optimal timing of RT and IT, and potential adverse effects inherent to the combination of RT and IT. Using PRISMA guidelines, a search of PubMed, Medline, and Web of Science was conducted to identify studies demonstrating the abscopal effect during treatment of NSCLC or melanoma with BM. 598 cases of irradiated BM of melanoma or NSCLC in 18 studies met inclusion criteria. The most commonly administered ITsincluded PD-1 or CTLA-4 immune checkpoint inhibitors (ICI), with RT most commonly administered within 3 months of ICI. Synergy between ICI and RT was described in 16 studies including evidence of higher tumor response within and outside of the irradiated field. In the 12 papers (n = 232 patients) that reported objective response rate (ORR) in patients with BM treated with RT and concurrent systemic IT, the non-weighted mean ORR was 49.4%; in the 5 papers (n = 110 patients) that reported ORR for treatment with RT or IT alone, the non-weighted mean ORR was 27.8%. No studies found evidence ofsignificantly increased toxicity in patients receiving RT and ICI. The combination of RT and ICIs may enhance ICI efficacy and induce more durable responses via the abscopal effect in patients with brain metastases of melanoma or NSCLC.

Secretion of 4-1BB Ligand Crosslinked to PD-1 Checkpoint Inhibitor Potentiates Chimeric Antigen Receptor T Cell Solid Tumor Efficacy.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies but has yet to achieve similar success in solid tumors due to a lack of persistence and function in the tumor microenvironment. We previously reported the augmentation of CAR T cell therapy in an engineered solid tumor model through the secretion of anti-PD-1 single-chain fragment variable region (scFv), as shown by enhanced CAR T cell antitumor efficacy, expansion, and vitality. We have since improved the platform to create a superior cellular product-CAR T cells secreting single-chain trimeric 4-1BB ligand fused to anti-PD-1 scFv (αPD1-41BBL). 4-1BB signaling promotes cytotoxic T lymphocyte proliferation and survival but targeting 4-1BB with agonist antibodies in the clinic has been hindered by low antitumor activity and high toxicity. CAR T cells using 4-1BB endodomain for costimulatory signals have demonstrated milder antitumor response and longer persistence compared to CAR T cells costimulated by CD28 endodomain. We have, for the first time, engineered CD28-costimulated CAR T cells to secrete a fusion protein containing the soluble trimeric 4-1BB ligand. In vitro and in vivo, CAR19.αPD1-41BBL T cells exhibited reduced inhibitory receptor upregulation, enhanced persistence and proliferation, and a less differentiated memory status compared to CAR T cells without additional 4-1BB:4-1BBL costimulation. Accordingly, CAR19.αPD1-41BBL T cell-treated mice displayed significantly improved tumor growth control and overall survival. Spurred on by our preclinical success targeting CD19 as a model antigen, we produced mesothelin-targeting CAR T cells and confirmed the enhanced solid tumor efficacy of αPD1-41BBL-secreting CAR T cells.

Abstract 5464: Predictive biomarkers for PD-1 and PD-L1 checkpoint inhibitor response in NSCLC: An analysis of clinical trial and real-world data

Abstract Importance: Largest analysis combining clinical trial and real-world data (RWD) to assess the clinical utility of biomarkers for anti-PD-1/PD-L1 checkpoint inhibitors (CPI) in NSCLC patients. Objective: To assess the association between tumor PD-L1, TMB and blood analytes with clinical outcomes in patients with advanced NSCLC following CPI therapy. Design: Retrospective cohort study with patients stratified into high and low biomarker groups. Correlation with treatment outcome in the different biomarker groups was investigated and compared between patients treated with CPI versus chemotherapy. Setting: Data derived from electronic health records mostly from community oncology settings were combined with clinical trial data. Participants: Starting with 71850 patients with advanced NSCLC who received CPI or chemotherapy, we selected 24152 patients from an electronic health record-derived de-identified NSCLC clinicogenomic database (CGDB) from a real-world cohort and nine Roche atezolizumab trials. Patients were diagnosed between January 2011 and February 2020 (data collection cut-off date). Main Outcomes and Measures: Drug response was assessed by RECIST criteria for clinical trials and real-world response for RWD. Durable response was defined as having CR/PR without progression during the study period of 270 days. Associations of biomarker levels with treatment outcomes were analyzed using Fisher’s exact test. Results: High expression of PD-L1 on tumors (≥50%) were associated with lower risk of resistance to CPI (odds ratio [OR] 0.21; 95% CI = 0.14, 0.32; P&amp;lt; .001). The association was stronger in patients who had prior chemotherapy than first line CPI, with non-squamous than squamous histology, and smokers than non-smokers. Higher TMB (≥9.57 mut/Mb) was also associated with durable response (OR = 0.41; CI = 0.30, 0.55; P&amp;lt; .001). The combination of high TMB and PD-L1 expression was the strongest predictor of durable response (OR = 0.04; CI = 0.00, 0.20; P&amp;lt; .001). There was no evidence of an association between PD-L1/TMB and response to chemotherapy, suggesting a CPI-specific predictive effect. In contrast, blood analytes were prognostic, having correlation with clinical outcome irrespective of treatment type. Conclusion: This study supports the predictive utility of PD-L1 expression and TMB for NSCLC patients receiving CPI therapy and further elucidates their utility in patient sub-populations. Citation Format: WeiQing Venus So, David Dejardin, Eva Rossmann, Jehad Charo. Predictive biomarkers for PD-1 and PD-L1 checkpoint inhibitor response in NSCLC: An analysis of clinical trial and real-world data. [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 5464.

Abstract 1858: Enhanced delivery of anti PD-1 antibody to liver tumors in oncopig using pressure enabled drug delivery (PEDD) versus a systemic delivery

Abstract Background: Anti-PD-1 checkpoint inhibitors have been developed to mitigate immune suppression by reversing T cell exhaustion. Typically, checkpoint inhibitors are delivered systemically, which may be inadequate due to poor drug distribution and limited penetration into solid tumors due to high intra-tumoral pressure. In such tumors, interstitial fluid pressure and solid stress restricts blood flow and therapeutic agent distribution throughout the tumor stroma. Pressure enabled drug deliveryTM (PEDDTM) has been demonstrated to improve therapeutic delivery in pre-clinical models and in patients. PEDD has also been shown to reduce off-target tissue exposure to embolics while increasing intra-tumoral therapeutic concentration. We hypothesized that PEDD hepatic arterial infusion (HAI) using a TriNav device would enhance anti-PD-1 antibody delivery to porcine liver tumors when compared to systemic delivery. Methods: The study was conducted in transgenic pigs (oncopigs) 35-50kg in weight. Explanted liver tissue was treated to induce oncogene expression and re-implanted in the liver at 4 locations for tumor formation. A fixed dosage (1 mg/animal) of fluorescently labeled InVivoSIM anti-human PD-1 (AB_2894731) checkpoint inhibitor (CPI) was infused in 10ml of PBS at 2 ml/min. The TriNav was positioned within a HA branch supplying a hepatic lobe with a fully formed tumor for local-regional infusion. Systemic infusion was conducted through the femoral vein. Liver tissue was collected 1 hour following infusion and drug levels were quantified by nearIR fluorescence imaging on sequential 1 cm thick sections. NearIR images were overlaid on color images, tumor border delineated, and the mean fluorescent CPI signal was calculated in concentric rings relative to the tumor border. Results: A significant 2.6-to-5.4-fold increase in CPI signal intensity was observed from 15mm within the tumor to 30mm from the outer edge of tumor with PEDD using the TriNav device when compared to systemic delivery (p ≤ 0.05). PEDD delivery also resulted in significant 2.5-to-3.9-fold enrichment from 5mm within the tumor to 10mm surrounding the tumor (relative to normal tissue within the treated lobe, p ≤ 0.05). No significant difference in signal intensity was observed in peri-tumoral regions and a reduction in signal was observed in tissues 5mm to 15mm within the tumor relative to normal tissue (p ≤ 0.05) following systemic CPI infusion. Conclusions: This study demonstrates that HAI delivery of CPI into liver tumors in the oncopig via PEDD using a TriNav device resulted in superior delivery compared to systemic infusion. Importantly, PEDD significantly concentrated delivery of CPI into the tumors and within the rapidly growing margin of the tumors where target immune cells such as lymphocytes are present. Citation Format: David Benjamin Jaroch, Chandra C. Ghosh, Prajna Guha, Steven Katz, Bryan F. Cox, Thomas G. Hullinger. Enhanced delivery of anti PD-1 antibody to liver tumors in oncopig using pressure enabled drug delivery (PEDD) versus a systemic delivery [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 1858.

Abstract 1044: Cell-free DNA concentration as a prognostic biomarker in patients with non-small cell lung cancer under immunotherapy treatment

Abstract The irruption of the immunotherapy for the treatment of non-small cell lung cancer (NSCLC) based on Immune Checkpoint Inhibitors (ICI) PD-1 and PD-L1 inhibitors is considered a treatment revolution. However, only a small percentage of patients benefit with ICI treatment over the long term, and precise biomarkers that can recognize these individuals before or early during treatment have so far eluded. PD-L1 expression and tumor mutational burden (TMB) are the most well studied biomarkers for predicting response to PD- (L)1 blockade-based ICI prior to treatment. TMB is still being clinically assessed whereas PD-L1 has several drawbacks for prediction of persistent benefit. Our aim is to evaluate the basal state and dynamic changes of cell-free DNA (cfDNA) concentration to predict and monitor response in NSCLC patients starting ICI. A total of 248 cfDNA concentration measurements were performed from 87 NSCLC patients. The quantification was done before the start of the treatment, at the second ICI cycle, after 6 and 12 months in treatment, and at progression if it was within the first 12 months. Quality and quantity of the cfDNA was assessed using Qubit High Sensitivity and Bioanalyzer 2100. We first explored the association with response of the basal cell-free DNA concentration using Mann-Whitney-Wilcoxon test. The longitudinal analysis between different time points was tested with Wilcoxon signed-rank tests. Response was ascertained using RECIST parameters at 3, 6, and 12 months. We evaluated the utility of the cfDNA concentration as a prognostic factor using Mantel-Cox test. The response association results indicate that early and long-term response is associated with lower levels of basal cfDNA (p&amp;lt;0.002, 3 months; p&amp;lt;0.001, 6 months; p&amp;lt;0.05, 12 months). The longitudinal analysis could also detect a decrease in cfDNA from the second cycle to the month 6 of treatment in responders (p&amp;lt;0.05), and an increase in non-responders (p&amp;lt;0.05). To identify a prognostic concentration threshold, statistical stratification of cfDNA levels based on the selected maximum rank method of cfDNA levels was used to group them into low and high concentration. Patients with high basal cfDNA concentrations showed both worse FPS (log-rank 3.4x10-03, Medians: Low 12.57 [8.36 - 18.46] High 5.50 [3.03 - 10.06]) and OS (log-rank 3.2x10-05, Medians: Low 21.26 [15.33 - 33.96] High 5.45 [3.10 - 15.66]). With these results, we provide proof-of-principle that specifically detecting tumor cfDNA by PCR or NGS methods would not be necessary to predict the long-term clinical benefit to ICI in NSCLC patients. Moreover, concentration cut-offs can be defined to categorize the patients in potential responders and non-responders before the start of the treatment. This would constitute a cost-effective and easily implementable prognostic tool that could be particularly useful for avoiding loss of treatment windows of opportunities. Citation Format: Isabel Barragan, Elisabeth Perez-Ruiz, Juan Luis Onieva, Maria Garrido-Ramos, Beatriz Martinez-Galvez, Jaime Dubbelman, Emilio Alba, Juan Zafra, Manuel Cobo, Javier Oliver, Antonio Rueda-Dominguez. Cell-free DNA concentration as a prognostic biomarker in patients with non-small cell lung cancer under immunotherapy treatment [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 1044.

Abstract 3274: Novel cell-based bioassays for mono- and multi-specific antibodies targeting PD-1/PD-L1 for combination immunotherapy

Abstract Programmed cell death protein 1 (PD-1) is an immune checkpoint (IC) receptor that negatively regulates T cell function. Blocking the interaction of PD-1 with its ligands PD-L1 or PD-L2 unleashes the immune system and enhances anti-tumor responses. Immunotherapy using PD-1 or PD-L1 blocking antibodies has led to a paradigm shift in cancer drug discovery, due to its durable effect against a wide variety of cancers. However, despite its tremendous clinical success in cancer treatment, a significant fraction of patients and tumor types remain unresponsive to this therapy. This has led to the development of combination therapies linking PD-1 checkpoint inhibitors with inhibitors of several other IC receptors. A major challenge in immunotherapy drug development is the lack of quantitative and reproducible functional assays. Existing methods rely heavily on primary immune cells are highly variable and labor-intensive. Here, we report the development of a suite of cell line-based reporter bioassays for monoclonal antibodies targeting human PD-1/PD-L1, or bispecific antibodies targeting PD-1/PD-L1 and a co-stimulatory receptor (e.g., 4-1BB, OX40, ICOS) or an immune inhibitory receptor (e.g., CTLA-4, LAG-3, TIGIT). A mouse PD-1/PD-L1 assay and a human PD-1/PD-L2 assay are also developed and can be used in parallel with human PD-1/PD-L1 assay to enable preclinical studies and assess assay specificity. Each of these assays consist of two engineered cell lines: a T effector cell line that express a luciferase reporter driven by specific promoter/response elements responding to the intracellular signals mediated by the T cell receptor (TCR), with modulation from PD-1 and the second immune receptor, and an engineered artificial antigen presenting cell (aAPC). Importantly, these combination bioassays demonstrate the synergetic effect of PD-1 blockade with blockade of a second IC inhibitor receptor or activation of a costimulatory receptor, thus reflecting the mechanisms of action for the therapeutic antibodies of interest. The bioassays are sensitive and quantitative, are prequalified according to ICH guidelines, and demonstrate the performance characteristics (specificity, precision, accuracy, and linearity) to be used as potency assays for product release and stability studies during immunotherapy drug development. Citation Format: Jun Wang, Julia Gilden, Denise Garvin, Pete Stecha, Jim Hartnett, Frank Fan, Mei Cong, Jamison Grailer. Novel cell-based bioassays for mono- and multi-specific antibodies targeting PD-1/PD-L1 for combination immunotherapy [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 3274.

Abstract 39: Assessment of a novel method using adult healthy donor mobilized human CD34+hematopoietic stem cells in NCG mice for use in tumor modeling

Abstract Checkpoint inhibitors including anti-PD-1/PD-L1 are a type of immunotherapy that have become the standard of care for cancer patients. Continual focus is placed on developing new treatments for cancer, including breast, lung and colon cancer, which are the most common worldwide. Humanized mice are an important in-vivo animal model that recapitulate crucial aspects of human tumor biology and anti-tumoral immunity allowing for assessment of immunotherapies. Currently these models are primarily created using cord blood, which is in limited supply. This leads to the isolation of low numbers of hCD34+ stem cells, resulting in small cohorts of animals. This limitation represents a challenge in working with humanized mice making it important to consider alternative sources of hCD34+ stem cells. In this study, we compared NCG mice engrafted with an alternative source of adult healthy donor mobilized hCD34+ stem cells to those engrafted with cord blood derived hCD34+ stem cells.After evaluating engraftment kinetics between the two cell sources we examined the anti-tumoral effect of an anti-human PD-1 checkpoint inhibitor in mice bearing three different human tumor xenograft cell lines (CDX) (A549 lung, COLO-205 colon and MDA-MB-436 breast cancer). NCG mice were humanized using either cord blood derived or adult healthy donor mobilized hCD34+ stem cells. Levels of human immune cell engraftment were measured in peripheral blood using multi-color flow cytometry. Humanized NCG mice were implanted subcutaneously with cancer cells, and tumor bearing mice (TBM) were randomized into treatment groups when the average tumor size reached comparable volumes for each tumor type. Vehicle control mice were treated with isotype control IgG antibody, whereas TBM were dosed with anti-hPD-1 antibody. Clinical observations, body weights and tumor growth kinetics were recorded throughout the study. At the time of euthanasia whole blood, spleen and tumor tissues were collected and processed for immune profiling by multi-color flow cytometry. Adult healthy donor mobilized hCD34+ humanized mice have sustained humanization levels out to 34 weeks post-injection and can be successfully engrafted with human CDX tumors. The ability to monitor tumor growth kinetics and observe a response to an anti-hPD-1 checkpoint inhibitor across multiple tumor types indicates that NCG mice humanized with adult healthy donor mobilized CD34+ stem cells are useful when assessing immunotherapies. Future directions of this model will expand beyond monotherapies to include the evaluation of additional treatment methods such as bispecific antibodies and cell-directed therapies. Citation Format: Jenny Rowe, Steven Bronson, Christoph Eberle, Ann Fiore, Bob Mihalek, Stephen Festin. Assessment of a novel method using adult healthy donor mobilized human CD34+hematopoietic stem cells in NCG mice for use in tumor modeling [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 39.

Abstract 575: Brain delivery of clinically suitable nanobioconjugates to inhibit glioblastoma growth through extracellular matrix-immune cell crosstalk

Abstract Introduction: Tumor growth, invasion, and escape from immune surveillance largely depend on cancer microenvironment. Laminins are trimeric proteins and essential components of glioblastoma (GBM) microenvironment/extracellular matrix (ECM). In brain glioma samples from 230 patients, we found a correlation between the overexpression of tumor ECM protein laminin-411 (α4β1γ1) and faster tumor recurrence with shorter patient survival. Laminin-411 is produced by endothelial cells, neutrophils, monocytes, platelets, lymphocytes, and glioma cells and can modulate the immune system. Novel nanotechnology approach to block trimeric laminin-411 and activate of brain local immune system with brain delivery of PD-1 checkpoint inhibitor was developed for future translational application. Methods: Nanobioconjugates (NBC) based on poly (β-L-malic acid, P), were synthesized, characterized and used to intravenously treat mice with intracranial syngeneic GL261 or CT-2A GBM. The lead NBCs P/PEG/LLL(40%)/AON(α4β1)(2.0%)/AP-2(2%) and P/PEG/LLL(40%)/AP-2(2%)/αPD-1(0.2%) contained antisense to laminin-411 α4 and β1 chains, or αPD-1 antibody as well as trileucine (LLL) peptide for endosomal escape and AP-2 peptide for BBB crossing and tumor cell targeting. CRISPR/Cas9 constructs were used to knockdown α4 and β1 laminin chains in GBM ex vivo. Flow cytometry and RNA-seq analyses were performed to evaluate treatment. Results: Laminin-411 depletion with CRISPR/Cas9 and multifunctional NBC in vivo treatment equally suppressed GBM growth and significantly prolonged animal survival. The brain privileged immune system was activated upon treatment with a significant increase of CD3+, CD8+ T cells, NK, IFNγ+ NK cells, and M1 macrophages. RNA-sec analyses after treatment with a combination of NBC suppressing laminin-411 and checkpoint PD-1 showed enhanced anti-tumor effect with upregulation of genes coding for apoptotic Caspase 3 and IFNγ, and reduction of proliferation markers EGFR, c-Myc and Ki-67. Conclusion: Study describes novel GBM treatment strategy via NBCs crossing blood-brain barrier and targeting critical ECM and immune components of tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma. Citation Format: Alexander V. Ljubimov, Rameshwar Patil, Hui Ding, Liron Israel, Eggehard Holler, Julia Y. Ljubimova, Tao Sun, Keith L. Black. Brain delivery of clinically suitable nanobioconjugates to inhibit glioblastoma growth through extracellular matrix-immune cell crosstalk [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 575.

Evidence-based rationale for low dose nivolumab in critically ill patients with sepsis-induced immunosuppression.

A substantial part of critically ill patients suffer from sepsis-induced immunosuppression. Reversal of immunosuppression through PD-1 checkpoint inhibition has been proposed as a treatment strategy to overcome immunosuppression in these patients. The PD-1 inhibitor nivolumab, currently used in treatment of cancer, has been evaluated in phase I/II studies in patients with sepsis, demonstrating tolerability and signs of clinical efficacy. No proper dose finding was performed in these studies and, after a single high dose of 480 mg or 960 mg nivolumab, PD-1 inhibition persisted beyond 90 days in the majority of cases. As the duration of sepsis is ~7-10 days, prolonged PD-1 inhibition may unnecessarily induce longer-term immune-related side effects. Based on previously published pharmacokinetic and pharmacodynamic data of nivolumab, a thorough in silico dose finding study for nivolumab in critically ill patients was performed. We found that volume of distribution and clearance of nivolumab were not higher in patients with sepsis compared to the cancer population for which nivolumab is currently approved and showed profound variability. We found that with a single dose of 20 mg nivolumab, the PD-1 receptor occupancy is predicted to stay above the 90% threshold for a median of 23 days (90% prediction interval of 7-78 days). We propose to investigate this dose in critically ill patients as a potential safe and cost-effective pharmacotherapeutic intervention to treat sepsis-induced immunosuppression.