PD-1 Checkpoint Inhibitors Research Articles

Stereotactic body radiotherapy extends the clinical benefit of PD-1 inhibitors in refractory recurrent/metastatic nasopharyngeal carcinoma

PurposeEmerging evidence shows that immune checkpoint inhibitors lead to durable responses in a variety of cancers, including nasopharyngeal carcinoma (NPC), however, combination approaches (i.e., stereotactic body radiation therapy, SBRT) are required to extend this benefit beyond a subset of patients. This study retrospectively evaluated eight recurrent/metastatic NPC patients, to investigate how radiation could potentiate PD-1 checkpoint inhibition therapy.MethodsBetween September 2016 and July 2017, eight consecutive cases with histologically confirmed PDL1-positive status, for which prior standard therapy had been ineffective (five patients), were treated at our institution and Macao Clinics and two patients had disease progression within 6 months of completion of definitive chemoradiation, or one patient refused to receive chemoradiotherapy. All received PD-1 inhibitors first, seven of them accepted SBRT with an unmodified PD-1 inhibitors regimen after first evaluation as they were unresponsive to PD-1 inhibitors alone. Treatment was discontinued as long as patients were experiencing a clinical benefit in the opinion of the physicians and at least five cycles were given before stoppage.ResultsMedian follow-up time was 56.7 months. The confirmed objective response rate based on RECIST-v1.1 at first evaluation was 12.5% (1/8). For the seven cases who received SBRT, six of them experience an objective response (6/7, 85.7%) after SBRT. Only one patient showed rapid progress and die within 95 days after the initiation of SBRT intervention. Three patients who did not have all lesions exposed to irradiation were available to evaluate the incidence of an abscopal effect, however, it did not occur as expected. Median PFS and OS for the seven patients were 8.0 and 30.8 months after SBRT intervention, respectively. Two-year OS as indicated was 71.0%.ConclusionsPD-1 inhibitors combined with SBRT demonstrated promising antitumor activity in patients with PD-L1 positive RM-NPC. Patients may benefit from continue immunotherapy beyond disease progression when SBRT was introduced.

Symptomatic melanoma metastases in the brain: are we using all therapy options?

In recent years, significant advances have been made in systemic therapy for patients with metastatic melanoma of the skin, resulting in an increase in one-year overall survival (OS) from 25 to 85% and 5-year OS from less than 10 to 60% in certain patient subgroups. Approximately 50% of patients with metastatic skin melanoma are diagnosed with metastatic brain lesions in the course of the disease. Modern drug therapy for metastatic brain lesions is slowly but surely proving to be effective. Thus, in the presence of a mutation in the BRAF gene, BRAF inhibitor monotherapy provides an intracranial objective response rate (iORR) of 25 to 40%, whereas BRAFi + MEKi combined targeted therapy (CTT) achieves already 58% iORR, including in patients with symptomatic metastases to the brain. However, the duration of responses achieved on targeted therapy (TT) is shorter than for extracranial disease prevalence. On the other hand, regardless of the presence of a BRAF mutation, immunotherapy (PD-1 monotherapy) achieves a response in approximately 20-22% of patients, but these responses are more durable, although fewer than on TT. The combination of CTLA-4 + PD-1 checkpoint inhibitors produces long-lasting responses with a iORR of 51-54%. However, the achievement of these results and an overall increase in life expectancy with immunotherapy is mostly possible in symptom-free patients and in patients receiving low or no doses of glucocorticosteroids (10 mg or less on prednisolone). Therefore, for symptomatic patients, especially those whose tumors have been identified with a BRAF mutation, a combination of targeted therapy, which would quickly achieve an objective response in 58%, and anti-PD1/PDL1 immunotherapy, which is likely to increase the duration of the response achieved and give a chance for a sustained remission, looks promising. This article provides an overview of key studies and our own experience with the triple combination in metastatic brain lesions.

Combination of Whole-Body Baseline CT Radiomics and Clinical Parameters to Predict Response and Survival in a Stage-IV Melanoma Cohort Undergoing Immunotherapy.

Simple SummaryThe use of immunotherapeutic agents significantly improved stage-IV melanoma patients’ overall progression-free survival. To identify patients who do not benefit from immunotherapy, both clinical parameters and experimental biomarkers such as radiomics are currently being evaluated. However, no radiomic biomarker is widely accepted for routine clinical use. In a large cohort of 262 stage-IV melanoma patients given first-line immunotherapy treatment, we investigated whether radiomics—based on the segmentation of all baseline metastases in the whole body—in combination with clinical parameters offered added value compared to the usage of clinical parameters alone in a machine-learning prediction model. The primary endpoints were response at three months, and survival rates at six and twelve months. The study indicated a potential, but non-significant, added value of radiomics for six-month and twelve-month survival prediction, thus underlining the relevance of clinical parameters.Background: This study investigated whether a machine-learning-based combination of radiomics and clinical parameters was superior to the use of clinical parameters alone in predicting therapy response after three months, and overall survival after six and twelve months, in stage-IV malignant melanoma patients undergoing immunotherapy with PD-1 checkpoint inhibitors and CTLA-4 checkpoint inhibitors. Methods: A random forest model using clinical parameters (demographic variables and tumor markers = baseline model) was compared to a random forest model using clinical parameters and radiomics (extended model) via repeated 5-fold cross-validation. For this purpose, the baseline computed tomographies of 262 stage-IV malignant melanoma patients treated at a tertiary referral center were identified in the Central Malignant Melanoma Registry, and all visible metastases were three-dimensionally segmented (n = 6404). Results: The extended model was not significantly superior compared to the baseline model for survival prediction after six and twelve months (AUC (95% CI): 0.664 (0.598, 0.729) vs. 0.620 (0.545, 0.692) and AUC (95% CI): 0.600 (0.526, 0.667) vs. 0.588 (0.481, 0.629), respectively). The extended model was not significantly superior compared to the baseline model for response prediction after three months (AUC (95% CI): 0.641 (0.581, 0.700) vs. 0.656 (0.587, 0.719)). Conclusions: The study indicated a potential, but non-significant, added value of radiomics for six-month and twelve-month survival prediction of stage-IV melanoma patients undergoing immunotherapy.

Abstract 1116: Spatial distribution of granzyme B to improve the predictive power of response to immunotherapy in operable lung cancer

Abstract Purpose Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers. There is a strong rationale for incorporating immunotherapy into the treatment of early-stage NSCLC, given the breakthrough results with PD-1 checkpoint inhibitors in advanced-stage NSCLC. How immunotherapy should be implemented in patients who are operable is still unclear. Most of the efforts so far to identify clinically useful biomarkers do not preserve spatial information and leave us blind to the critical source of information revealed in the cell-to-cell biology of the tumor microenvironment (TME). In order to overcome these limitations, we used spatial biomarkers assays that preserve this critical information about which cells are influencing treatment response. Method Frozen sections from retrospectively collected surgically resected NSCLC (adenocarcinoma and squamous cell carcinoma) tumors treated with adjuvant pembrolizumab therapy were used. Patients were classified in two groups according to their Objective Response Rate (ORR): Complete Response (CR) and Progression Disease (PD) for spatial transcriptomic and proteomics assays. Associated to RNA In situ hybridization (ISH) technology, the NanoString GeoMx™ Digital Spatial Profiling (DSP) technology was used to determine immune markers within compartment specific defined by fluorescence localization [tumor (panCK) and leucocytes (CD45)]. Wilcoxon, Linear Mixed Model and Mann-Whitney U statistic tests were used to evaluate differences between ORR groups. Results Using the spatial analysis, we identified 4 protein markers from immune cell profiling and immune activation status modules independently associated with benefit from single-agent PD-1 checkpoint blockade in spatial context. High expression of CD4, CD40, Granzyme B and HLA-DR were significantly associated with all favorable clinical outcome, whereas upregulation of extracellular matrix (ECM) proteins related to pro-tumoral reprogramming of the stroma were associated with immunotherapy resistance. We also validated the DSP finding that high granzyme level in the stroma compartment was predictive for response to check point inhibitor in the same set of patients using ISH immunofluorescence, strengthening this marker relevance in antitumor immunity and as a predictive biomarker candidate for immunotherapy in NSCLC. Conclusion This work identifies a number of relevant spatial candidate predictors of immunotherapy outcome in spatial context for operable lung cancer that show promise for future validation in larger independent cohorts. Citation Format: Corinne Ramos, Ophelie Lemieux, Adele Ponzoni, Jonathan Stauber. Spatial distribution of granzyme B to improve the predictive power of response to immunotherapy in operable lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1116.

Abstract 3180: Targeting LRG1 to normalize tumor vasculature and enhance therapeutic efficacy

Abstract In recent years improving tumor vascular function, to render the tumour microenvironment less permissive and to improve delivery of therapeutics, has gained traction due to a growing body of supportive evidence. Identifying suitable targets that are tractable, ubiquitous and safe, however, has proven to be more challenging. Almost a decade ago we reported that a secreted glycoprotein, leucine-rich alpha-2-glycoprotein 1 (LRG1), was induced in ocular neovascular complications and contributed to the formation of dysfunctional neovessels1. More recently, we have reported that LRG1 is expressed in experimental and human tumors, and that its inhibition with a function-blocking antibody improves outcome in multiple primary2 and metastatic3 models of cancer. Crucially, we found that LRG1 blockade normalizes tumor vessels and enhances the efficacy of cisplatin, adoptive T cell and checkpoint inhibitor therapy, and that a humanized version of our blocking antibody named Magacizumab effectively inhibits tumour growth both alone and as an antibody-drug-conjugate4.Using animal models and in vitro assays we present here further data in support of LRG1 as a promising target for the treatment of solid cancers. We have further investigated the effects of LRG1 blockade on immune cell infiltration using flow cytometric and immunohistochemical analysis. In subcutaneous B16 melanoma-bearing mice treated with a PD1 checkpoint inhibitor, antibody blockade of LRG1 significantly enhanced the infiltration of CD3+ and CD8+ T cells, with the latter exhibiting a more activated phenotype as evidenced by higher GrzB, reduced PD1hi expression and increased proliferation. We also observed a reduction in the Tregs:Th ratio and a higher number of MHCII+ cells. These outcomes were also observed in LLC tumors alongside a reduction in the number of infiltrated neutrophils. In preliminary studies, where we investigated LRG1 blockade in the Rag1 mouse, we observed no effect suggesting that the downstream mode of action is mediated by impacting the immune system. Finally, to test the development of our humanized anti-LRG1 antibody Magacizumab, that only recognizes human LRG1, we demonstrated its efficacy in B16F0 tumors grown in a human LRG1 knock-in mouse. These studies provide compelling evidence that LRG1 is a novel, legitimate and potentially efficacious target for the treatment of various human solid cancers.

Abstract 1113: NUC-3373 potentiates immune-mediated cytotoxicity of CRC cells

Abstract Background: NUC-3373 is a phosphoramidate modification of fluorodeoxyuridine-monophosphate (FUDR-MP), the active anti-cancer metabolite of fluorouracil (5-FU), which binds and inhibits thymidylate synthase (TS), disrupting DNA synthesis and repair. NUC-3373 is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown, reduce generation of toxic metabolites, and deliver higher levels of active metabolite FUDR-MP to tumors. Our previous work demonstrated that NUC-3373 causes colorectal cancer (CRC) cell lines to release damage associated molecular patterns (DAMPs)- molecular signals that activate circulating immune cells leading to immunogenic cell death (ICD). NUC-3373 promotes activation of co-cultured NK-92 MI cells, a natural killer cell line, by upregulating degranulation and IFN-γ production and downregulating the immune checkpoint molecule TIGIT. This study aims to test the hypothesis that NUC-3373 induced DAMPs promote ICD in co-cultures of CRC cells with peripheral blood mononuclear cells (PBMCs). We also explore the dynamics of PD-L1 and cytokine expression and combination with a PD-1 checkpoint inhibitor. Methods: HCT116 cells were exposed to increasing concentrations of NUC-3373 or vehicle control for 24 hours prior to co-culture with healthy patient-derived PBMCs. At the time of co-culture, cells were treated with 10 υg/ml nivolumab to assess the effect of an anti-PD-1 antibody being combined with NUC-3373. CRC and PBMC co-cultures were continued for up to 72 hours. Gene expression of PD-L1 and cytokines (TNF-α, IFN-γ, and IL-2) was assessed by RT-PCR. PD-L1 surface expression was determined by flow cytometry. Confluence was determined by automated cytometry analysis (Celigo) and cell viability of CRC cells was determined by sulforhodamine-B assay (SRB). Results: NUC-3373 increased the gene expression of cytokines and PD-L1 over 48 hours of co-culture. Surface expression of PD-L1 was increased on both PBMCs and CRC cells in co-culture, as well as on CRC cells cultured without PBMCs, indicating that global increase in PD-L1 is NUC-3373 mediated. CRC cells pre-treated with NUC-3373 and co-cultured with PBMCs displayed a reduction in cell viability compared to monocultured CRC cells. Furthermore, CRC and PBMC co-cultures treated with anti-PD-1 antibody showed increased cytotoxicity when CRC cells were pre-treated with NUC-3373. Conclusions: NUC-3373 induces DAMPs and PD-L1 expression in CRC cells and promotes pro-immune cytokine production from PBMCs resulting in ICD in vitro. Furthermore, the addition of anti-PD-1 antibody to co-cultures of NUC-3373-treated CRC cells and PMBCs enhanced this ICD. With an improved clinical safety profile (NCT03428958), including less haematologic toxicity, and a more convenient dosing regimen than 5-FU, NUC-3373 is an attractive combination partner for immune checkpoint inhibitors. Citation Format: Oliver James Read, Jennifer Bré, David J. Harrison. NUC-3373 potentiates immune-mediated cytotoxicity of CRC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1113.

New approaches in palliative systemic therapy of anal squamous cell carcinoma.

Metastatic and locally recurrent inoperable anal squamous cell carcinoma (ASCC) belong to rare tumours - ASCC accounts for about 2% of gastrointestinal tumours. Synchronous metastatic involvement is usually confirmed in approximately 15% of patients and about 20 % of patients develop relapse in the form of distant metastases after curative treatment. The current standard palliative systemic treatment is a PF regimen composed of 5-fluorouracil (5-FU) with cisplatin (cDDP) or a combination of carboplatin with paclitaxel. The aim of this study is to summarize other options for systemic palliative care in ASCC, including the results of relevant clinical trials performed with new or modified regimens. Triple combinations with taxanes have yielded promising treatment results, and in particular, the inclusion of the DCF regimen (cDDP + 5-FU + docetaxel) in first-line treatment results in a significant increase in treatment responses with good treatment tolerance. Another promising option for palliative care is immunotherapy with checkpoint inhibitors PD-1 or PD-L1, which can be successfully used in palliative systemic treatment or in curative regimens. Epidermal growth factor receptor inhibitors remain the subject of research, whose role in the primary or palliative treatment of ASCC is not entirely clear. Compared to standard chemotherapy, available data confirm the treatment outcomes improvement in the use of triple combination DCF with docetaxel or immunotherapy with checkpoint inhibitors in the treatment of metastatic ASCC.

Abstract 4239: Development of multi-targeting immunomodulatory SeekR RNA therapeutics to overcome resistance to PD-1 blockade

Abstract T-cell exhaustion describes the dysfunctional state of effector T cells in the tumor microenvironment that results from chronic and prolonged exposure to antigen. It also represents an important mechanism of clinical resistance to immune checkpoint blockade. Recently, the transcription factor NR4A1 has been shown to be upregulated in tumor-specific T cells and plays a role in driving exhaustion during chronic antigen stimulation of T cells. Pro-oncogenic activities of NR4A1 have long been observed in various solid tumors including colorectal and breast cancers, and co-expression of NR4A1 with known immune checkpoint markers such as PD-L1 have also been observed in various cancer cell lines. These findings have identified NR4A1 as an important target for overcoming resistance to cancer immunotherapy. We hypothesize that multi-targeting to simultaneously block PD-1 signaling and silence NR4A1 gene expression can reverse T-cell exhaustion and expand the clinical benefit of PD-1 blockade in immune therapy resistant solid tumors. Using our SeekR™ RNA therapeutics platform, we combined aptamer binders to PD-1 with siRNA against NR4A1, andNR4A1 and evaluated their ability to reverse T-cell exhaustion and re-activate T-cell based anticancer immunity in solid tumors. Specifically, we created SeekR™ therapeutic RNA molecules with dual flanking RNA aptamers binders to PD-1 and other checkpoint inhibitors, connected by a double stranded bridge that encodes for siRNA silencers that target multiple immunomodulatory genes, such as NR4A1. The novel anti-PD-1 RNA aptamer component serve to: A) direct and deliver the SeekR™ RNA oligo therapeutics to exhausted T-cells that express PD-1 receptors; and B) internalize the siRNA component into the T-cells. The siRNA components of the SeekR™ serve to silence NR4A1 which lead to reversal of the cytotoxic T-cell exhausted phenotype following chronic in-vitro stimulation. These results demonstrate that dual targeting of PD-1 and NR4A1 has the potential to reverse T-cell exhaustion and the potential to overcome cancer immunotherapy resistance in the clinic. Citation Format: Marvin O'ketch, Victoria A. Love, Jeffrey A. Kiefer, David W. Lee, Lizette A. Castaño, Spyro Mousses, David O. Azorsa. Development of multi-targeting immunomodulatory SeekR RNA therapeutics to overcome resistance to PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4239.

Abstract CT147: Phase 1 study of recombinant interleukin 15 (rhIL-15) in combination with checkpoint inhibitors nivolumab and ipilimumab in subjects with refractory cancers

Abstract Introduction: Recombinant human interleukin 15 (rhIL-15) has been shown to stimulate the activation and expansion in the number of effector T lymphocytes and natural killer cells in patients. The addition of the immune checkpoint inhibitors (ICI) CTLA-4 and PD-1 has shown increased antitumor efficacy in preclinical models, and the triplet is hypothesized to enhance anti-tumor immune response by augmenting effector cell expansion, differentiation, cytotoxic activity, and immune checkpoint inhibition. Results of the lead-in safety arms of rhIL-15/ipilimumab (Ipi) or rhIL-15/nivolumab (N) doublets were previously reported (O’Sullivan et al. AACR. 2019) and we now report results for the dose escalation phase of the triplet combination (NCT03388632). Methods: This phase 1, open-label, 3+3 dose escalation trial enrolled adult patients (pts) with measurable metastatic or refractory solid tumors and ECOG performance status ≤2. Prior therapy with 2 of the 3 study agents was permitted. Pts were treated on three dose levels (DL 1-3) of rhIL-15 (0.5, 1, or 2 mcg/kg/day) administered subcutaneously on days (D)1-8 and D22-29 of a 6-week cycle for the first 4 cycles only. Nivolumab (240mg IV) was administered on D8, 22, 36 and ipilimumab (1mg/kg IV) on D8 of every cycle. Response was assessed by RECIST 1.1. Upon progression, pts could continue for response assessment utilizing iRECIST if clinically stable and without toxicity. Treatment-induced changes in circulating and tumor T cell activation, signaling, and the PD-L1 checkpoint will be assessed with validated pharmacodynamic biomarker assays. Results: A total of 17 pts were enrolled on the triplet dose escalation phase; 15 pts were evaluable for response and 6 pts had received prior treatment with a PD-1/PD-L1 inhibitor. Median time on treatment (MTT) was 12 weeks (range 3-101). A confirmed partial response (PR) was observed in a pt with intrahepatic cholangiocarcinoma (MSI-H) after 3 cycles (total time on treatment 101 weeks). Across all dose levels, 7 pts (46.7%) had stable disease (SD) that prolonged their time on treatment with MTT of 20.4 weeks; 7 pts (46.7%) experienced progressive disease (PD) with MTT of 6.4 weeks. To better profile toxicity, 5 pts were replaced due to incomplete dosing of C1. Dose limiting toxicities at DL 3 (rhIL-15 at 2 mcg/kg/day) were photosensitivity and rash. Main drug-related adverse events included grade 4 lymphopenia (n=1) and grade 3 colitis, hyperthyroidism, hyponatremia, myocarditis, neutropenia, and photosensitivity (n=1, each). No pts discontinued therapy due to toxicity events. No deaths occurred on study. Conclusions: The recommended phase 2 dose (RP2D) of rhIL-15 is 1 mcg/kg/day when administered in combination with N+Ipi. The triplet dose expansion phase with the RP2D is currently accruing and includes biomarker studies in blood and tumor biopsies to assess the tumor microenvironment. This study was funded in part by NCI Contract HHSN261200800001E. *In memorium: Dr. Thomas Waldmann7. Citation Format: Sarah J. Shin, Geraldine O'Sullivan Coyne, Howard Streicher, Naoko Takebe, Ashley Bruns, Elad Sharon, Richard Piekarz, Lamin Juwara, Larry Rubinstein, Ralph Parchment, Kristin Fino, King L. Fung, Katherine Ferry-Galow, Arjun Mittra, Abdul Rafeh Naqash, Kevin Conlon, James H. Doroshow, Alice P. Chen. Phase 1 study of recombinant interleukin 15 (rhIL-15) in combination with checkpoint inhibitors nivolumab and ipilimumab in subjects with refractory cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT147.

Abstract 1875: Novel cell-based bioassays for monoclonal antibody and bispecific molecules in PD-1 blockade monotherapy and combination therapy

Abstract Programmed death 1 (PD-1) is an immune checkpoint (IC) receptor that negatively regulates T-cell immune function. Blocking the interaction of PD-1 with its ligand PD-L1 unleashes the immune system and enhances anti-tumor responses. Immunotherapy using PD-1 blockade 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 combining PD-1 checkpoint inhibitors with a broad range of clinically active treatments including those targeting other IC receptors. A major challenge in immunotherapy drug development is the lack of quantitative and reproducible functional assays as existing methods rely heavily on primary immune cells and are highly variable. Here, we report the development of a suite of cell line-based reporter bioassays for monoclonal antibody targeting human PD-1/PD-L1, or bispecific molecules target 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 address preclinical study and 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), PD-1 and the second IC receptor, and an artificial antigen presenting cells (aAPCs). Particularly, the combination bioassays are able to measure the synergetic effect of PD-1 blockade with a second IC inhibitor receptor blockade or a costimulatory receptor activation. These reporter-based assays reflect the mechanisms of action for the therapeutic drugs of interest and are sensitive and quantitative. They are prequalified according to ICH guideline and demonstrated the performance characteristics (specificity, precision, accuracy, and linearity) to be used as potency assay for product release and stability studies during immunotherapy drug development and manufacture. Citation Format: Jamison Grailer, Julia Gilden, Jun Wang, Denise Garvin, Pete Stecha, Jim Hartnett, Steven Edenson, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Novel cell-based bioassays for monoclonal antibody and bispecific molecules in PD-1 blockade monotherapy and combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1875.

Abstract 695: Patient derived organoids model lung adenocarcinoma metastases

Abstract Introduction: While lung cancer patient-derived xenografts (PDX) models predict response to therapy, PDX tumors rarely metastasize thus offering little personalized assessment of the biology of metastases. We hypothesize that the development of an in vivo patient-derived organoid (PDO) model will improve our understanding of the underlying genomic and transcriptomic alterations that help drive the biology of metastases for specific lung adenocarcinoma (LUAD) histologic subtypes. Methods: Fresh LUAD specimens were processed and cultured using optimized organoid culture conditions. Established PDOs were characterized by histologic analyses, RNA-sequencing and whole genomic sequencing (WGS). Response to therapy of PDOs was evaluated by treatment with combined chemotherapies, targeted therapies, or co-culturing with patient-matched peripheral blood mononuclear cells (PMBC) in vitro and/or in vivo. The metastatic capabilities of PDOs were assessed in vivo using intracardiac injection and scoring for tumor outgrowth in distant organs using in vivo bioluminescence imaging system. Metastatic tumors from specific organs were isolated, PDOs reestablished, and those tumors underwent additional genomic and transcriptomic characterization. Phylogenetic analyses of tumor metastatic progression and putative metastasis markers were investigated by WGS and RNA-sequencing. Results: We successfully generated 14 PDOs from early-stage (I-II) primary LUAD specimens and 12 from human LUAD metastatic sites (brain, bone and pleural effusion). All PDOs recapitulate genetic alterations and transcriptomic signatures of their original tumors. Moreover, we found that individual PDOs exhibit distinct responses to different chemotherapy regimens and targeted therapies. Pre-treatment of PDOs with low doses of platinum-based doublet chemotherapy regimens activated patient-matched tumor specific T cells and enhanced PD-1 checkpoint inhibitor Nivolumab induced CD8+ T cell-mediated tumor killing. Importantly, utilizing our in vivo PDO metastasis model we successfully developed a platform to evaluate: 1) metastatic potential, 2) response to targeted therapies, 3) tumor evolution with respect of genomic instability, tumor phylogeny, and colonization, and 4) molecular signatures promoting tumor metastases. Conclusion: Our PDO system reliably models human LUAD in vitro and in vivo, by recapitulating histologic architectures, genomic alterations and transcriptomic signatures of the parent LUAD. Using LUAD PDOs we have successfully created multiple platforms to assess response to chemo- and targeted- therapies, develop an in vivo PDO metastasis model to assess tumor evolution, and use of patient matched PMBCs and PDOs to assess immune-priming strategies for individual patient tumors. Citation Format: Yuan Liu, David Jones. Patient derived organoids model lung adenocarcinoma metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 695.

Abstract 1647: Assessment of a novel tumor model using adult patient mobilized human CD34+ hematopoietic stem cells in NCG mice

Abstract Background: Inhibitors of the PD-1/PD-L1 checkpoint pathway were among the first FDA approved immunotherapies and are now among the standard of care for cancer patients. In vivo animal models that recreate crucial aspects of human tumor biology and anti-tumor immunity are needed for evaluating new therapy developments across cancer types. In 2019, we reported an animal model using non-adult (cord blood derived) hCD34+ hematopoietic stem cells, considered the standard cell source, for humanizing the immune system of NCG mice. In this study we assessed a mouse model using adult patient mobilized hCD34+ stem cells as an alternative source and evaluated engraftment and tumor modulation using checkpoint inhibitors. Study Details: We evaluated the anti-tumor effects of an anti-human PD-1 checkpoint inhibitor on basal lung cell adenocarcinoma (A549) in NCGs humanized with adult patient mobilized hCD34+ stem cells. Humanized mice were implanted subcutaneously with A549 cells, and tumor bearing mice (TBM) were randomized into treatment groups when the average tumor size reached a volume of ~80-120 mm3. Vehicle control mice were treated with isotype control IgG antibody, whereas A549 TBM were dosed with anti-hPD-1 antibody biweekly (10 mg/kg) for three weeks. Clinical observations, body weights and tumor growth kinetics were recorded throughout the study. At study termination, whole blood, spleen and tumor tissue were collected and processed for immune profiling by multi-color flow cytometry. Results: Anti-hPD-1 monotherapy significantly inhibited A549 tumor growth. NCG mice injected with adult patient mobilized hCD34+ stem cells sustained engraftment. At study termination common human leukocytes were distributed in peripheral blood, spleen and tumors of surviving mice. Phenotyping of the cancer microenvironment revealed the presence of lymphoid (T-lymphocytes and subsets including Tregs) and myeloid immune cells (DC, TAM) with the latter lineage less detectable overall. PD-1 checkpoint blockade on average increased CD8+ T-cell infiltration and decreased Treg frequencies among the TIL population. Generally, human cytokines (IFN-γ, IL-2 and TNF-α) were secreted by viable tumor-infiltrating total T-cells (CD3+) and subsets (CD4+ and CD8+) following ex vivo PMA/Ionomycin stimulation in the presence of Brefeldin A, demonstrating the capability to produce polyfunctional responses. Conclusions: This study demonstrates that adult patient mobilized hCD34+ cells can successfully reconstitute NCG mice. An immune modulating anti-tumor response was elicited with tissue infiltration patterns of human leukocytes overall comparable to those found in cord blood derived hCD34+ humanized NCG mice. These findings make this model utilizing NCG mice engrafted with adult patient mobilized hCD34+ stem cells a viable approach for cell transfer studies when assessing targeted immuno-oncology therapies. Citation Format: Jenny Rowe, Christoph Eberle, Ann Fiore, Robert Mihalek, Brianna Schoen, Stephen Festin. Assessment of a novel tumor model using adult patient mobilized human CD34+ hematopoietic stem cells in NCG mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1647.

Abstract 310: Harness small peptide to improve anti-cancer effect of PD-1 checkpoint inhibitor in melanoma-bearing obese mice

Abstract Obesity-associated chronic inflammation and adipokine increase the cancer risk through promotion of tumor growth and malfunction of immune cells. Leptin, an adipokine abundantly found in obesity, is positively correlated with PD-1 expression and up-regulation of PD-1 on immune cells leads to immune cell exhaustion. PD-1 blockade (checkpoint inhibitor) has shown promising anti-tumor efficacy in patients with obesity through direct effects on the up-regulated PD-1. However, there are still need for improving the response rate and the efficacy of PD-1 blockage. In our previous study, we have verified that prohibitin binding peptide (PBP) inhibits glucose metabolism of cancer cells, resulting in alterations in pro-tumor tumor microenvironment (TME) to pro-immune TME. The pro-immune TME reinvigorates anti-cancer activity of immune cells and results in better suppression of tumor growth. Based these findings, we hypothesized that PBP would improve the anti-cancer effect of PD-1 checkpoint inhibitor on cancer with obesity. Melanoma was embedded in lean or obese mice, then the mice were assigned to one of following groups: i) PBS, ii) PBP, iii) anti-PD-1 antibody (aPD-1) and iv) combination of PBP and aPD-1 (PBP+aPD-1). The PBP+aPD-1 group showed improved anti-cancer efficacy in tumor-bearing obese mice (not in lean mice) compared to single aPD-1 injection. Immune cells in PBP+aPD-1 injected obese mice showed the more activated characteristics in terms of its cytokine secretion and phenotype. In addition, the serum leptin level was decreased upon PBP treatment, indicating that the PBP-mediated decrease in the serum leptin level attributed the rejuvenation of immune cells through the balanced PD-1 expression on immune cells. PBP improves the anti-cancer efficacy of aPD-1 on tumor with obesity through recovering anti-cancer activity of immune cells that is driven, at least in part, by a decrease in the glucose metabolism of cancer cells and the circulating leptin levels. Consequently, combination of PBP and aPD-1 would have the great potential to treat cancer patients with obesity. Citation Format: Seungmin Han, Kelsey E. Decker, Young-Wook Won. Harness small peptide to improve anti-cancer effect of PD-1 checkpoint inhibitor in melanoma-bearing obese mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 310.

Evaluation of the use of COVID-19 vaccines in patients treated with immunotherapy.

e18781 Background: Immunotherapy can boost a patient’s immune system to bring about anti-tumor activity and may theoretically exaggerate the inflammatory immune response to infection or vaccinations. Conversely, vaccination may enhance immune activity in patients on immunotherapy. Currently, there are limited safety data in patients treated with immunotherapy who received COVID-19 vaccines. Methods: An IRB-approved retrospective review of Dana-Farber Cancer Institute patients treated with PD-1 checkpoint inhibitors and received COVID-19 vaccines was performed. The primary endpoint was the incidence rate of immune-related adverse events (irAEs) pre- and post- COVID-19 vaccinations. An Electronic Health Record search identified all patients who completed COVID-19 vaccines between 12/1/2020 and 3/31/2021 and received anti-PD-1 monotherapy between 10/1/2020 and 5/31/2021. Medical record review identified timing, occurrence, and grading of irAEs as defined by clinicians or Common Terminology Criteria for Adverse Events (CTCAE), version 5.0. Results: A total of 229 patients were included in the study. The most common underlying cancers were lung (32%) and melanoma (26%). The majority of patients had Pfizer-BioNTech COVID-19 vaccine; 64% of all patients received the third dose of vaccine. The median duration of treatment was 15 months, and median duration of follow-up was 19 months. Among the study cohort, 120 irAEs occurred before vaccination (any grade); 30 (25%) were grade 1, 35 (29%) were grade 2, 11 (9%) were grade 3 or 4, and 44 (37%) were ungraded. The main types of irAEs were skin toxicities (37% of all irAEs) followed by thyroid dysfunction (22%) and colitis (11%). Of the 75 irAEs that occurred after vaccination, 24 (32%) were grade 1, 16 (21%) were grade 2, 16 (21%) were grade 3 or 4, and 19 (26%) were ungraded. The main irAEs types were skin toxicity (31% of all irAEs) followed by thyroid dysfunction (13%) and hepatic toxicity (12 %). The all-grade pre- and post-vaccine irAE incidence rates per 100 patient-months were 4.13 and 5.09, respectively. Within this cohort, there were 0.96 (95% CI -0.362-2.284, p = 0.15) more irAEs post vaccine per 100 patient-months of anti-PD-1 therapy. Conclusions: The data weakly suggested that there is a higher incidence of irAEs post COVID-19 vaccine; however, the comparison did not achieve statistical significance. Further analysis of other patient characteristics will help to determine if any specific patterns are observed.[Table: see text]

The ImPrint immune signature to identify patients with high-risk early breast cancer who may benefit from PD1 checkpoint inhibition in I-SPY2.

514 Background: The remarkable increase of novel Immuno-Oncology drugs in many malignancies has led to the need for biomarkers to identify who would benefit. Various predictive biomarkers have been developed (PD-1/PD-L1 expression, mutations in mismatch repair genes and microsatellite instability, tumor mutational burden and immune infiltration), none have consistently predicted efficacy. The I-SPY2 consortium qualified several expression-based immune biology related signatures that predict response to PD1 checkpoint inhibition. Here we assessed whole transcriptome data of high-risk early-breast cancer (EBC) patients who received Pembrolizumab within the neoadjuvant biomarker-rich I-SPY2 trial (NCT01042379), aiming to migrate the I-SPY2 research findings to a robust clinical grade platform signature to predict sensitivity to PD1 checkpoint inhibition. Methods: Whole transcriptome microarray data were available from pre-treatment biopsies of 69 HER2- patients enrolled in the Pembrolizumab (4 cycles) arm of the I-SPY2 trial. All patients had a High-Risk 70-gene MammaPrint profile. Pathologic complete response (pCR) was defined as no residual invasive cancer in breast or nodes at the time of surgery. Of the 69 patients, 31 had a pCR (12 HR (hormonal receptor)+HER2-, 19 Triple Negative (TN)), while 38 (28 HR+HER2-, 10 TN) had residual disease (RD). To identify the most predictive genes associated with pCR, gene selection was performed comparing pCR and RD groups by iteratively splitting the dataset in training and test, balancing for HR status. Due to limited sample size, leave one out cross validation was used for performance assessment. Genes with effect size > 0.45 were considered significant. Results: A signature of 53 genes, named ImPrint, was identified with overall sensitivity and specificity > 90% and > 80% for predicting pCR to pembrolizumab in all patients. Sensitivity and specificity in TN were > 95% and ≥70%, and in HR+HER2- > 80% and > 85%, respectively. The Positive Predictive Value (PPV) is 77% for the HR+HER2- subgroup. Biological annotation of the 53 genes showed that over 90% of the genes have known immune system related functions, of which 63% were previously known to be involved in immune response (including genes coding PD-L1 and PD-1, as well as those identified in I-SPY2). Conclusions: In the signature development phase, ImPrint predicts pCR to Pembrolizumab in a set of 69 high risk EBC with high sensitivity and specificity. The signature features genes with immune-related functions known to be involved in immune response indicating that it might aid identifying patients with an immune-active phenotype. Importantly, ImPrint appears effective in identifying a subset of HR+HER2- patients who could benefit from immunotherapy. External validation in independent dataset(s) is ongoing and will be presented at the time of the meeting.

A bivalent Tim-3/PD-1 bispecific antibody for the treatment of PD-1 antibody resistant or refractory NSCLC.

e14597 Background: Immune checkpoint inhibitors (ICI) PD-1/PD-L1 antibody are key drugs for the treatment of non-small cell lung cancer (NSCLC). Bispecific antibody is one of the strategies aimed at the clinical needs for NSCLC patients who are resistant to or refractory from ICI treatment. Tim-3, one of the next generation of ICB targets, is co-expressed on exhausted T cells with PD-1. It is also expressed by innate immune populations, including NK and DC. Dual blocking PD-1 and Tim-3 not only on T cells but also on DC, NK cells may achieve better clinical benefit. Methods: A bivalent to both Tim-3 and PD-1 bispecific antibody (Bis5) was developed and is in Phase I clinical trials for NSCLC patients who are resistant to or refractory from PD-1 antibody treatment. Results: Bis5 showed affinity of 5-8 nM to both Tim-3 and PD-1. Moreover, Bis5 showed better cell activity than Tim-3 and PD-1 antibody combination to activated T cell as well as NK and DC. Bis5 showed 77%-88% tumor inhibition which is close to PD-1 antibody alone in MC38 model. Neither PD-1 antibody or PD-1 and Tim-3 antibody combination show any activity in CT26 model while Bis5 showed significant tumor inhibition activity and doubled the survival rate. The highest non-severe toxicity dose (HNSTD) was 200mpk in monkeys. ADA were 33.3% (2/6), 83.3% (5/6), and 0.0% (0/6) at doses of 2, 10, and 50 mpk, respectively. The T1/2 were 31.7-66.5 h for doses 2-50 mpk. Conclusions: A Phase I, multicenter, open-label study to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity and preliminary efficacy of Bis5 in patients with advanced and/or metastatic solid tumors has started. Seven cohorts (0.1, 0.3, 1, 3, 6, 10, 15 mg/kg) are planned to be enrolled sequentially in the dose escalation part. In the expansion part, a cohort group of 10 patients is planned for post PD-1 treated NSCLC as the second or third line treatment.

OMIP-084: 28-color full spectrum flow cytometry panel for the comprehensive analysis of human γδ T cells.

Using full spectrum flow cytometry, we designed a 28-color panel for the analysis of markers known to be associated with the γδ T cell immune response. This panel allows the classification of γδ T cell subsets via specific V gene usage (Vγ9, Vδ1, Vδ2, and Vδ3) of their T cell receptor (TCR) and according to their functional differentiation. Phenotypical surface receptors to distinguish different stages of cell maturation included CD45RA, CD27, CD28, CD127, CD57, and CD16; chemokine receptors CXCR6, CCR5, CCR6, and CX3CR1; NK-associated markers NKG2A, NKG2D, CD56, and CD161, checkpoint-inhibitor PD-1, and activating receptors CD38 and CD25. T cell lineage markers for the analysis of αβ T cells (CD4 and CD8) and MAIT cells (Vα7.2) were also included. This optimized multicolor panel allows a comprehensive immune-profiling of all main human γδ T cell subsets and is suitable for longitudinal or exploratory analysis of γδ T cell development and γδ T cell dynamics in clinical cohorts.

Use of novel fluorescent dyes enables in depth analysis of checkpoint inhibitors CTLA-4 (CD152) and PD-1 through spectral flow cytometry

Abstract PD-1 and CTLA-4 (CD152) are prominent checkpoint inhibitors in T cell responses and their inhibition has emerged as a successful route to boost anti-tumor immune responses in cancer patients. Clinical trials involving CAR-T cell therapy with anti-CTLA-4 and PD-1 antibodies seem to be improving the tumor directed T cell response. Regulation of surface vs cytoplasmic CTLA-4 pools is crucial to balancing stimulatory and inhibitory signals in T-cell responses. Surface CTLA-4 contributes to tumor escape by inhibiting T cell responses, and upregulation of CTLA-4 expression from the cytoplasm to the surface is highly regulated. Therefore, it might be of interest to monitor these CTLA-4 pools as this might indicate the efficacy of CAR-T cell therapeutic approaches. Here we show how novel fluorescent dyes enable the composition of a spectral flow cytometry panel to interrogate the presence of surface versus cytoplasmic CTLA-4 pools and the dynamics of PD-1 expression in a single panel. Combining these parameters into a single panel allows for less workflow steps and permit conservative sample use.

Real-world data on the incidence of immune-related adverse events associated with anti-PD-1/PD-L1 treatment in Russia

Background. PD-1 and PD-L1 immune checkpoint inhibitors (ICI) show efficacy in many tumor types, and the indications for their use continue to expand. Meanwhile, treatment with ICI is often accompanied by specific immune-related adverse events (irAEs). The real-world data of the incidence of irAEs in Russian patients is lacking. Objectives. The study aimed to characterize the frequency and profile of irAEs registered in Russian patients. Materials and methods. The retrospective study included patients with various solid malignancies who received immunotherapy in N.N. Petrov National Research Institute of Oncology or other oncological institutions of St. Petersburg during 2018–2021. The following treatment regimens were used: monotherapy with anti-PD-1 monoclonal antibody (nivolumab, pembrolizumab or prolgolimab), anti-PD-L1 monoclonal antibody (atezolizumab) or combination therapy with anti-PD-1 and anti-CTLA-4 antibodies (nivolumab and ipilimumab). The frequency and spectrum of irAEs were evaluated depending on the treatment regimen. Results. Any-grade irAEs were registered in 58 out of 178 (32.6%) analyzed patients. The most common adverse events were liver toxicity (11.2%) and thyroid dysfunction (10.1%). Severe irAEs (≥3 grade) occurred in 15.7% of cases. There was one fatal outcome due to grade 5 immune-mediated hepatitis. Combined immunotherapy was associated with a higher frequency of immune-related toxicity compared to monotherapy anti-PD-1/PD-L1 monoclonal antibodies (70.6% vs 23.6%, respectively, p<0.0001). Conclusions. The reported frequency of adverse events of PD-1 and PD-L1 inhibitors in Russian patients was lower than in the majority of the foreign studies.

Hypoxia-inducible factor-1α inhibition augments efficacy of programmed cell death 1 antibody in murine prostatic cancer models.

This study was designed to explore whether hypoxia-inducible factor-1α (HIF-1α) inhibitor could enhance immunotherapy efficacy in prostate cancer. Western blot was used to detect the expression of HIF-1α in the tumor and peritumor tissues from prostate cancer patients. The analysis from Cancer Genome Atlas database was used to show an association between HIF-1α expression and survival rate in prostate cancer patients. Murine prostate cell-derived xenograft (CDX) model was set up in both nude mice and BALB/c mice to observe the therapeutic effect of HIF-1α inhibitor IDF-11774. Protein expression of HIF-1α, as well as changes in the immune microenvironment, was detected. Moreover, the synergistic antitumor effect of IDF-11774 and PD-1 antibody was detected in another murine prostate cancer model. HIF-1α was found to have higher expression in prostate cancer tumor tissue than in peritumor tissue, and the expression level was negatively correlated with survival rate (P = 0.0157). HIF-1α inhibitor IDF-11774 reduced tumor volume and exhibited better efficacy in BALB/c mouse model (P < 0.0001) with normal immune system, with the same suppression level against HIF-1α. HIF-1α inhibitor reduced CD45+CD11b+Gr-1+ myeloid-derived suppressor cells (P = 0.0027) and CD45+ CD11b+F4/80+CD206hi M2 macrophages (P = 0.0059) but increased the abundance of CD45+CD3+CD8+ T cells (P = 0.0002) and CD45+CD3+CD4+ T cells (P = 0.0001) in tumor-infiltrating immune cells. The same synergistic effect was observed in RM-1 murine prostate CDX tumor model. HIF-1α inhibition augmented the antitumor efficacy of immune checkpoint inhibitor PD-1 antibody in murine prostate cancer models, probably through modulating the immunosuppressive microenvironment.