bluuebunny/crossref_metadata_2025 huggingface dataset:
Successful tumor-infiltrating lymphocyte (TIL) growth from uveal melanoma (UM) using a three-signal (3.0) method.
Authors: Pelster Meredith , Forget Marie-Andree , Gruschkus Stephen K. , Haymaker Cara L. , Bernatchez Chantale , Hwu Patrick , Amaria Rodabe Navroze , Gombos Dan S. , Patel Sapna Pradyuman
Published: May 2020

3027

Background: Metastatic UM is a rare cancer with poor response rates to systemic therapy. Adoptive transfer of patient-specific TIL may represent the best strategy for treatment. TIL are harvested from primary or metastatic tumors and initially expanded in culture with high dose IL-2 prior to undergoing rapid expansion protocol and therapeutic administration. Here, we report improved rates of initial expansion using a previously described TIL 3.0 method which utilizes dual agonistic antibodies to TCR and 4-1BB (Urelumab) for stimulation, respectively, with high dose IL-2, compared to the traditional method. Methods: Between 2006 and 2019, patients were consented for TIL harvest from either primary or metastatic UM tumors. Demographics, clinical features, and outcomes of the TIL initial expansion were collected. Success rates, number of cells expanded, and days in culture for the two methods were analyzed using partially overlapping samples t-tests and z-tests. Results: There were 85 harvests and expansions from 76 patients using the traditional method and 32 expansions from 30 patients using TIL 3.0. Initial TIL expansion was successful in 97% of TIL 3.0 harvests compared to 35% for the traditional method (p < 0.001). More TIL were expanded with TIL 3.0 compared to the traditional method (291.3 million cells vs. 88.6 million cells, p < 0.001), and fewer days were required in culture (18.5 vs. 29.0, p < 0.001). Both primary UM harvests and metastatic harvests were more successful with TIL 3.0 (90% vs. 12% for primary, p < 0.001, and 100% vs. 42% for metastatic, p < 0.001). Conclusions: Expansion of UM tumors via the TIL 3.0 method led to successful growth in 97% of harvests. Therapeutic administration to patients with TIL 3.0 is under active investigation. [Table: see text]
Modulation of inhibitory signals in CAR T cells leads to improved activity against glioblastoma.
Authors: Sanber Khaled , Nawas Zeid , Salsman Vita , Gad Ahmed , Matthew Pretty , Lee Ciaran , Joseph Sujith , Ahmed Nabil , Hegde Meenakshi
Published: May 2020

3031

Background: Early clinical trials have demonstrated the safety of chimeric antigen receptor (CAR) T cells targeting glioblastoma (GBM), however, their efficacy remains limited by multiple obstacles including the immunosuppressive tumor microenvironment. Adoptively transferred CAR T cells remain susceptible to inhibition via the engagement of co-inhibitory receptors on their surface such as PD1, BTLA, CTLA4 and LAG3. The subsequent recruitment of Src homology region 2 containing protein tyrosine phosphatase 2 (SHP2) by these receptors to the immune synapse may represent a common mechanism of T cell inhibition, as SHP2 can de-phosphorylate key signaling molecules that mediate T cell activation (including CD28 and CD3ζ). We hypothesized that SHP2 deletion will simultaneously offset the effects of multiple co-inhibitory receptors, thereby improving the anti-tumor activity of CAR T cells. Methods: Electroporation of sgRNA/Cas9 ribonucleoprotein complexes into human T cells was used to knockout (KO) SHP2. Retroviral vector transduction was used to express a clinically-utilized second generation CAR (with a CD28 endodomain) targeting HER2. The phenotype of wild-type (WT) and SHP2KO CAR T cells was evaluated with mass cytometry and flow cytometry. Their anti-tumor function was tested in vitro using the xCELLigence assay (an impedance-based cytotoxicity assay), and in vivo, in an orthotopic xenograft mouse model of GBM. Results: Efficient and reproducible depletion of the SHP2 protein in human T cells was verified using western blotting. The Inference of CRISPR Efficiency (ICE) Assay confirmed efficient editing of the PTPN11 gene encoding SHP2. An anti-HER2 CAR was efficiently expressed in the SHP2KO T cells. SHP2 deletion did not significantly affect CAR T cell expansion, proliferation or baseline phenotype. However, following co-culture with HER2+ LN229-GBM cells, the CD8+ central memory (CCR7+ CD45RA-) and effector memory (CCR7- CD45RA-) subsets were enriched to a greater extent in the SHP2KO CAR T cells. The pattern of cytokine co-expression varied between donors in a single-cell analysis comparing SHP2KO to WT CAR T cells after encountering LN229 cells. Functionally, SHP2KO CAR T cells derived from the majority of healthy donor and patient peripheral blood eliminated LN229 cells more rapidly in vitro. In an orthotopic mouse model of GBM, SHP2KO CAR T cells showed better early control of established LN229 xenografts and improved survival in comparison to WT CAR T cells. Conclusions: SHP2 deletion in CD28ζ.CAR T cells improves their anti-tumor activity.
CYAD-101: An innovative non-gene edited allogeneic CAR-T for solid tumor cancer therapy.
Authors: Prenen Hans , Dekervel Jeroen , Anguille Sébastien , Hendlisz Alain , Michaux Alexandre , Sotiropoulou Panagiota A , Gilham David Edward , Mauen Sébastien , Snykers Sarah , Cerf Emilie , Lonez Caroline , Flament Anne , Lehmann Frederic , Van Cutsem Eric
Published: May 2020

3032

Background: In contrast to autologous CAR-T cell therapies, allogeneic donor-derived CAR-T cells can be banked and used in a timely fashion overcoming the critical time delay of just in time autologous cell manufacture. CYAD-101 is an allogeneic CAR-T that uses a non-gene edited peptide-based technology (TIM) to control graft versus host disease (GvHD) combined with a NKG2D-based CAR. Pre-clinical studies confirmed that CYAD-101 maintained CAR-directed anti-tumor activity in the absence of the induction of GvHD. Clinical grade CYAD-101 cells were produced for the phase 1 alloSHRINK trial (NCT03692429). Methods: A bank of clinical grade CYAD-101 cells was generated through two production runs using a single donor apheresis. Together, the bank generated > 53 billion CYAD-101 cells suitable for the entire dose escalation segment and short expansion phase of the trial (15 patients in total). Both runs showed high consistency with the CYAD-101 product generated composed mainly of CD4+ T cells (>85%) with a transduction level of > 92%, low relative expression of CD69/CD25 and largely absent expression levels of PD-1/LAG-3. The CYAD-101 cells were predominantly (>80%) CD45RA−/ CD62L−/ CD27− suggestive of an effector memory T cell population. Results: Upon co-culture with target K562 cells, CYAD-101 readily produced IFN-γ that was blocked by a NKG2D blocking antibody confirming specificity of the CAR. CYAD-101 cells showed in vitro cytotoxicity against tumor cells and produced an array of Th1 (IFN-γ, IL-2 and TGF-β) and Th2 (IL-4, IL-5) cytokines. Importantly, minimal IFN-γ was produced upon TCR stimulation while stimulation with a non-TCR mitogen (PMA + ionomycin) lead to high levels of IFN-γ. Together, these data show that clinical grade CYAD-101 cells were able to functionally respond through the CAR but showed minimal TCR-driven activation. Fifteen refractory metastatic CRC patients who had previously failed at least one line of oxaliplatin—containing therapy were treated with three doses of CYAD-101 cells given on Day 3 of three successive FOLFOX chemotherapy cycles. Updated clinical results continue to demonstrate an encouraging clinical activity (2 patients with partial response and 9 with stable disease) and the absence of GvHD in the context of CYAD-101 cell engraftment. Conclusions: These early clinical results demonstrate the safety and tolerability of a non-gene edited predominantly CD4+ CAR-T therapeutic approach. The initial observations of clinical activity in metastatic CRC patients warrants the continued development of this therapy. Clinical trial information: NCT03692429 .
Sequential monitoring of circulating stromal cells from blood is predictive of progression in NSCLC patients undergoing anti-PD-L1 therapy after definitive chemoradiation therapy.
Authors: Adams Daniel , Augustyn Alexander , He Jianzhong , Qiao Yawei , Xu Ting , Liao Zhongxing X. , Gardner Kirby P , Ali Amama , Gironda Daniel J , Tang Cha-Mei , Lin Steven H.
Published: May 2020

3051

Background: Cancer Associated Macrophage-Like cells (CAMLs) are a recently described circulating stromal cell common in the peripheral blood of patients with solid tumors. In non-small cell lung carcinoma (NSCLC), patients with CAMLs ≥50µm after completion of chemoradiation therapy (CRT) have been shown to have worse progression free survival (PFS). However, with the recent addition of anti-PD-L1 therapies in conjunction with CRT as standard of care, it has never yet to be evaluated whether CAMLs remain predictive for monitoring progression in NSCLC patients post anti-PD-L1 therapy. Methods: A 2 year single blind prospective study was undertaken to test the relationship of ≥50µm CAMLs to PFS based on imaging in lung patients before and after induction of CRT and PD-L1. We recruited 104 patients with pathologically confirmed unresectable NSCLC Stage II (n = 14), Stage III (n = 83), Stage IV (n = 3), and locally recurrent disease (n = 4). Baseline (BL) blood samples were taken prior to start of therapy. A second time point blood sample (T1) was taken at the end of radiotherapy (~40 days). A third time blood sample (T2) was taken after induction of anti-PD-L1 therapy (e.g. Imfinizi, Keytruda, etc.). Blood was filtered by CellSieve filtration and CAMLs were quantified. Analysis by CAML size of < 49 µm or ≥50 µm was used to evaluate PFS hazard ratios (HRs) by censored univariate & multivariate analysis. Results: CAMLs were found in 87% of samples averaging 2.9 CAMLs/7.5mL sample. At BL, CAMLs ≥50 µm had similar PFS to patients with < 50 µm CAMLs (HR = 1.1 95%CI 0.6-1.95 p = 0.8661). However, after CRT (T1), patients with CAML size ≥50 µm had worse PFS (HR = 3.2, 95%CI 1.8-5.8 p = 0.0002). After induction of anti-PD-L1 therapy (T2), patients with ≥50 µm CAMLs also had worse PFS (HR = 2.8 95%CI 1.5-5.4 p = 0.0037). CAML size at BL was not accurate at predicting progression within 24 months; however ≥50 µm CAMLs after CRT or after 1 cycle of anti-PDL1 therapy was 71% accurate at predicting progression of disease. Conclusions: Our data suggests that in NSCLC, ≥50 µm CAMLs after completion of CRT or appearing after induction of anti-PD-L1 therapy appears to predict progressive disease. If validated, additional studies are needed to determine if CAMLs can serve as a significantly prognostic blood based marker for predicting survival in NSCLC patients early in the treatment regime.
Anti-CTLA-4 probody BMS-986249 alone or in combination with nivolumab in patients with advanced cancers: Initial phase I results.
Authors: Gutierrez Martin , Long Georgina V. , Friedman Claire Frances , Richards Donald A. , Corr Bradley , Bastos Bruno R. , Uemura Marc Isamu , Conkling Paul R. , Moreno Victor , Edenfield William Jeffery , Becerra Carlos Roberto , Piha-Paul Sarina Anne , Peterson Amy C. , Brown Michelle , James Lee P. , Zheng Ming , Jiang Jieying , Kollia Georgia , Swijter Ailsa , Ascierto Paolo Antonio
Published: May 2020

3058

Background: Blockade of the CTLA-4 pathway with ipilimumab (IPI) ± nivolumab (NIVO; anti–PD-1) is an effective treatment for a variety of cancers. To optimize the risk-benefit profile of CTLA-4–directed therapy, a Probody therapeutic technology platform (Pb-Tx, CytomX Therapeutics) was used to generate BMS-986249, a peptide-masked version of IPI that is unmasked by tumor-associated proteases. Pb-Tx may localize CTLA-4 activity to the tumor, minimize systemic toxicity, and allow for higher doses of anti–CTLA-4 ± anti-PD-1. In preclinical studies, BMS-986249, given at similar doses, showed comparable intratumoral and reduced peripheral pharmacodynamic activity relative to IPI (Engelhardt, AACR 2020). Here, we present the initial results of the first-in-human phase 1/2 study of BMS-986249 ± NIVO in pts with advanced (adv) cancers (NCT03369223). Methods: During dose escalation, pts received BMS-986249 at or above the approved doses of the parent molecule using a Q4W or Q8W dosing schedule as monotherapy (240–2400 mg Q4W or 1600 mg Q8W; ≈ 3–30 mg/kg vs approved 3 mg/kg Q3W IPI) or in combination (240–1200 mg Q4W or 800 mg Q8W) + NIVO 480 mg Q4W. Safety and pharmacokinetics (PK) were evaluated. Efficacy is being assessed in the dose-expansion phase. Results: As of December 7, 2019, 82 anti–CTLA-4 naive pts with various adv cancers received BMS-986249 ± NIVO (mono, n = 39; combo, n = 43). Median age 60 (25–78) y; 95% pts had prior systemic therapy. TRAEs occurred in 59% of pts (Gr 3/4, 23%) with mono and 74% of pts (Gr 3/4, 30%) with combo. Diarrhea was the most common any-Gr TRAE (mono, 23%; combo, 21%) and Gr 3/4 TRAE (mono, 15%; combo, 7%). Rates of Gr 3/4 TRAEs increased with higher doses of BMS-986249 but were substantially reduced with Q8W schedule (eg, 800 mg Q4W, 18%; 1600 mg Q4W, 60%; 1600 mg Q8W, 9%). Most TRAEs resolved, no Gr 5 TRAEs occurred. The peptide-masked intact probody accounted for most (73%) of the systemic BMS-986249-related species; elimination of the probody indicated involvement of both catabolism and cleavage processes. Conclusions: BMS-986249 ± NIVO displayed a clinically manageable safety profile, allowing assessment of comparably higher BMS-986249 dose intensity (240-1200 mg; ≈ 3-15 mg/kg) + NIVO (480 mg Q4W, full dose) than that tested with IPI + NIVO. The types of TRAEs were consistent with CTLA-4 blockade, and the overall data align with the proposed Pb-Tx mechanism of action. The preclinical and clinical data support the ongoing randomized BMS-986249 + NIVO expansion in pts with adv melanoma, in addition to other adv tumors. Clinical trial information: NCT03369223 .
A first-in-human phase I study in patients with advanced and/or refractory solid malignancies to evaluate the safety of ATOR-1015, a CTLA-4 x OX40 bispecific antibody.
Authors: Yachnin Jeffrey , Ullenhag Gustav J. , Carneiro Ana , Nielsen Dorte , Rohrberg Kristoffer Staal , Kvarnhammar Anne Månsson , Schultz Lena , Bågeman Erika , Wennersten Camilla , Russell Charlotte Astrid
Published: May 2020

3061

Background: ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody developed to be a next generation CTLA-4 antibody with enhanced immune activation and tumor-directed activity for improved efficacy and reduced toxicity. Methods: The primary objective of the study is to determine the maximum tolerated dose (MTD) and/or the recommended phase 2 dose (RP2D) in patients with advanced solid malignancies. Safety and tolerability of ATOR-1015 are assessed by adverse events (AEs), vital signs, ECG, laboratory evaluations and physical examinations. Secondary objectives include pharmacokinetics (PK), immunogenicity and clinical efficacy. Clinical efficacy is assessed using Response Evaluation Criteria in Solid Tumors for immune-based therapeutics (iRECIST). The study is designed with single patient cohorts for doses below 100 mg followed by a modified 3+3 design (NCT03782467). Intra-patient dose escalation is allowed. ATOR-1015 is administered intravenously every other week as a single agent until confirmed progressive disease, unacceptable toxicity or withdrawal of consent. Results: From March 2019 to February 2020, 15 patients have been exposed to ATOR-1015. The median age of the patients is 52 years (range 40-72). The following cancer types have been included: colorectal cancer (n=8), uveal melanoma (n=2), pancreatic cancer (n=2), ovarian cancer (n=2), and cholangiocarcinoma (n=1). Patients received a median of 6 prior lines of therapy (range 3-16). Dose levels from 0.043 mg to 200 mg have been evaluated and declared safe. Dose escalation is ongoing, and 400 mg is under evaluation. The median time on study was 8 weeks (range 2.1-34.3). Four patients are on study and eleven patients have discontinued treatment . Reasons for discontinuation include clinical deterioration (n=7), death due to disease progression (n=2), confirmed disease progression (n=1) and investigator´s decision (n=1). Six of the 15 patients experienced drug-related AEs which were grade 2 or less. Infusion-related reactions (IRR) were reported in four patients. One of those four also had abdominal pain and mediastinal burning sensation. The IRR symptoms were predominantly rash. One patient had vitiligo, and one had rash. No dose-limiting toxicities have occurred. Preliminary PK data show dose-proportional kinetics up to 200 mg. Conclusions: The dosing of ATOR-1015 has been safe and well-tolerated up to 200 mg. Dose escalation continues and the current dose level under evaluation is 400 mg. Clinical trial information: NCT03782467 .
Statin treatment improves response to anti-PD1 agents in patients with malignant pleural mesothelioma and non-small cell lung cancer.
Authors: Cantini Luca , Pecci Federica , Hurkmans Daan , Copparoni Cecilia , Aerts Sophie , Belderbos Robert A , Cornelissen Robin , Dumoulin Daphne P , Fiordoliva Ilaria , Rinaldi Silvia , Aerts Joachim , Berardi Rossana
Published: May 2020

3074

Background: After progression to standard chemotherapy, only a small proportion of malignant pleural mesothelioma (MPM) and non-small cell lung cancer (NSCLC) patients (pts) benefit from anti-programmed cell death (PD-1) treatment. Combination strategies might improve response. In pre-clinical models, statins showed vaccine adjuvant activities and synergized with anti-PD1 agents. In this multi-center study, we evaluated the impact of baseline statin treatment in MPM and NSCLC pts. Methods: We separately examined MPM and NSCLC pts treated with anti-PD1 monotherapy after progression to standard chemotherapy at two European academic institutions. As control cohort, MPM pts treated with first-line chemotherapy were also examined. Pts receiving statins at start of treatment were compared with those who did not. Objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were analyzed. Results: A total of 287 patients were examined. Twenty-seven out of 80 (34%) MPM and 36 out of 130 (20%) NSCLC pts received statins at start of anti-PD1 treatment. The most common statins were simvastatin, atorvastatin and rosuvastatin. In MPM pts, statin use was associated with improved ORR (22% versus 5%, P = 0.05), longer PFS (median 6.7 versus 2.4 months, hazard ratio (HR) 0.42, 95% confidence interval (CI) 0.23–0.77, P < 0.01), and longer OS (median not reached versus 6.0 months, HR 0.43, 95% CI 0.21–0.85, P = 0.01). In NSCLC pts, statin use was associated with improved ORR (40% versus 22%, P = 0.04), longer PFS (median 7.8 versus 3.6 months, HR 0.59, 95% CI 0.37–0.97.2, P = 0.03) but similar OS (median 13.1 versus 10.1 months, HR 0.79, 95% CI 0.49–1.28, P = 0.30). At multivariate analyses, after adjusting for ECOG performance status (PS) and histological subtype, the impact of statins remained significant for ORR, PFS and OS in MPM and for PFS in NSCLC. Conversely, no association between statin use and outcomes was found in 77 MPM pts treated with first-line chemotherapy. Conclusions: This study shows that statin use at start of anti-PD1 treatment improves response to anti-PD1 agents in MPM and NSCLC pts who progressed to standard chemotherapy in routine clinical practice. This association could not be found in MPM pts treated with first-line chemotherapy, thus suggesting a synergy between statins and anti-PD1 agents. Prospective studies are needed to confirm whether the combination of statin and anti-PD1 therapy could improve outcome in pts with poorly immunogenic thoracic malignancies.
Novel blood-based biomarker predicting severe toxicity in melanoma anti-CTLA-4 immunotherapy treatment.
Authors: Chat Vylyny , Ferguson Robert , Morales Leah , Monson Kelsey , Esteva Eduardo , Moran Una , Pavlick Anna C. , Ascierto Paolo Antonio , Osman Iman , Weber Jeffrey S. , Kirchhoff Tomas
Published: May 2020

3077

Background: Immune checkpoint inhibition (ICI) has improved clinical outcomes of metastatic melanoma (MM). However, 65-80% of treated patients experience immune-related adverse events (irAEs), urging for the availability of personalized and easy-access clinical biomarkers. We have previously shown that germline genetics related to host immunity affects ICI response and MM survival. In this study, we investigated if germline immune-related expression quantitative trait loci (ieQTLs) may predict ICI-induced irAEs in MM. Methods: Through a comprehensive interrogation of a healthy twin-cohort expression dataset (MuTHER), we identified 40 ieQTLs most significantly associated with the expression of 382 immune-related genes. These germline variants were genotyped using the MassARRAY system in anti-CTLA-4-treated MM patients, collected as part of a multi-institutional collaboration. Using multivariable logistic regression models, we tested the association of 40 ieQTLs with irAEs in a discovery cohort of 97 MM patients followed by a validation in additional cohort of 97 anti-CTLA-4 treated patients. Results: We found rs7036417 significantly associated with severe anti-CTLA-4 irAEs in the discovery (OR = 6.18; 95%CI = 1.61-23.74; p = 0.007) and validation (OR = 6.73, 95%CI = 1.42-31.86; p = 0.02) cohorts. Pooled analysis showed that carriers of two rs7036417 alternate alleles (TT) have a 6-fold increased risk of developing severe irAEs (OR = 6.11; 95% = 2.26-16.56;p = 0.0003). This association was not observed with ICI response or survival. The alternate allele of rs7036417 is associated with higher expression of SYK (spleen-associated tyrosine kinase), suggesting that elevated SYK contributes to developing severe irAEs. Conclusions: We report that rs7036417, an ieQTL in SYK, associates with an increased risk of severe irAEs, independent of ICI efficacy. SYK plays an important role in B-cell/T-cell expansion and increased pSYK has been reported in patients with rheumatoid arthritis or systemic lupus erythematosus. Based on our data, the over-expression of SYK likely explains the biological mechanisms of the association between rs7036417 and anti-CTLA4 irAEs. These findings propose a novel blood-based baseline biomarker stratifying the patients at increased risk of severe irAEs, with a clinical effect substantially surpassing those observed for currently available predictors. Our ongoing studies are currently investigating SYK eQTL as a novel target in toxicity-reducing therapies.
Prevalence of human leukocyte antigen-B27 supertype in the context of positively charged neoepitopes and association with PD-L1 as an immune escape mechanism.
Authors: Fares Charlene Marie , Cummings Amy Lauren , Theisen Matthew Karl , Gukasyan Jaklin , Lind-Lebuffe Jackson P , Akingbemi Wisdom O , Li Debory Y , Brodrick Paige M , Yessuf Nawal M , Barksdale Chantal Ava , Wong Deborah J.L. , Lisberg Aaron Elliott , Garon Edward B.
Published: May 2020

3083

Background: Recent evidence suggests efficacy of immune checkpoint blockade may be influenced by human leukocyte antigen (HLA)-B. HLA-B27 supertype has an electronegative binding pocket which favorably binds and displays neoepitopes harboring positively charged amino acids (AAs). Based on immune surveillance, we postulate that B27 tumors that have favorable neoepitopes should face negative selective pressure, and B27 tumors with favorable neoepitopes that develop could be more likely to upregulate immune escape mechanisms. Here we evaluate the relationship between prevalence of B27 and positively charged neoepitopes and assess association between positively charged neoepitopes and expression of PD-L1. Methods: TCGA datasets from head and neck squamous cell (HNSC), lung squamous cell (LUSC), and melanoma (SKCM) patients were evaluated. HLA alleles were determined with OptiType and supertype was based on 2008 criteria. Nonsynonymous mutations were annotated with Ensembl VEP and VAtools. pVAC-Seq using NetMHCpan algorithm predicted neoepitopes 9 AAs in length. Favorable B27 neoepitopes were defined as those having new positively charged AA substitutions (H/K/R) from negative or uncharged wildtype AAs. RNA-seq data for the PD-L1 gene were normalized on transcripts per million and log2 transformed. Linear regression tests were performed between PD-L1 gene expression values and fraction of nonsynonymous mutations resulting in neoepitopes with new positively charged AAs in patients with B27. Results: Data from 497 HNSC, 494 LUSC, and 468 SKCM patients were analyzed. B27 was observed in 20.1%, 23.2%, and 26.5% of HNSC, LUSC, and SKCM patients, respectively, with a significant difference seen between HNSC and SKCM by chi-square test (χ² = 5.14, p = .023). Of new charged AAs resulting from nonsynonymous mutations, 76.3% in HNSC, 74.0% in LUSC, and 72.0% in SKCM were positively charged (p < .05 between all histologies, paired t-tests). In B27 patients, association between PD-L1 gene expression and fraction of neoepitopes with new positively charged AAs was seen in HNSC (r = 0.25 p = .036) and SKCM (r = 0.30 p = .007), but not LUSC (r = -0.12 p = .296). Conclusions: With increasing fraction of positively charged neoepitopes, a decrease in prevalence of B27 was observed, suggesting improved binding and immune elimination of tumors with favorable neoepitopes. In B27 tumors that develop despite having favorable neoepitopes, upregulation of PD-L1 could be a putative mechanism to evade immune detection.
Effect of Kaiso on immune signaling of breast cancer exosomes.
Authors: Dean-Colomb Windy Marie , Ahmed Shakir , Karanam Balasubramanyanam , Wang Honghe , Davis Melissa , Grizzle William E. , Gardner Kevin , Yates Clayton
Published: May 2020

3088

Background: Exosomes are communication vesicles that act as mediators of intracellular transfer of genetic information, an important role in intercommunication between tumor cells and immune cells. However, the mechanism underlining this cell-cell communication is not well understanding, particularly in African American breast cancer patients. Recently, our lab has demonstrated that Kaiso, a novel bi-modal transcription factor is highly expressed in African American breast cancer and notably, high Kaiso expression correlates with breast cancer aggressiveness and the disparity in survival outcomes of breast cancer patients of African American compared to European American patients. However, the differential expression and biological consequences of Kaiso in immune signaling of breast cancer exosomes has not been studied yet. Herein we demonstrate the biological role of Kaiso in immune signaling in breast cancer exosomes. Methods: In this study we utilized Nanostring immune profiling technology along with multiple in vitro and in vivo assays were used to study the role of Kaiso in breast cancer immune escape. Results: Nanostring pan cancer immune profiling demonstrated that European American breast cancer exosomes exhibited higher expression of TILs markers, T cell activation markers and CD8+T Cells markers compared to African American (p < 0.05, FDR), while we observed an increase in the expression of the anti-phagocytic molecule CD47 in breast cancer patient exosomes of African American compared to European American patients. In addition to that CD47 and SIRP-α (Signal Regulatory Protein) are highly expressed in Kaiso-scrambled MDA-MB-231 cells (sh-Scr) and exosomes, whereas THBS1, which is a regulator of CD47 expression and is regarded as angiogenesis inhibitor is significantly increased in sh-Kaiso MDA-231 cells and exosomes. Additionally, we observed that Kaiso directly binds methylated sequences in the promoter region of CD47 and THBS1 by ChIP assay. Furthermore, in vivo sh-Kaiso cells injected into athymic mice exhibited delayed tumor formation after four weeks with smaller tumor size as compared to sh-SCR cells (p < 0.05), and we observed higher expression of THBS1 with lower expression of CD47 and SIRP-α molecules by IHC and exosomes isolated from in vivo tumors (p < 0.05), indicating that Kaiso is associated with macrophage mediated immune escape. Conclusions: These findings demonstrate the important role of kaiso in immune signaling through exosomes which may be related with more aggressive cancer phenotype in breast cancer, especially in African Americans.
Single vector multiplexed shRNA provides a non-gene edited strategy to concurrently knockdown the expression of multiple genes in CAR T cells.
Authors: Gilham David Edward , Bornschein Simon , Springuel Lorraine , Michaux Alexandre , Steklov Mikhail , Breman Eytan , Sotiropoulou Panagiota A
Published: May 2020

3103

Background: Engineered T cells expressing chimeric antigen receptors (CAR) are now delivering clinically relevant results in patients with advanced hematological malignancies. One critical area for future development is to modulate gene expression thereby endowing the engineered T cell with specific desired features that enhance anti-tumor activity. Methods: Short-hairpin RNA (shRNA) were cloned individually or multiplexed within micro-RNA scaffolds that enabled the co-expression of the individual shRNA with a CAR and a selectable marker all driven by a PolII promoter within a single retroviral vector. Primary human T cells transduced with the CAR-shRNA vectors were selected, expanded in vitro, subjected to negative selection to eliminate any remaining TCR+ cells and examined for target gene expression and functional activity. Results: A 500bp DNA fragment incorporating a shRNA-specific for CD3ζ cloned into a retroviral vectoreffectively knocked down expression of CD3ζ in transduced BCMA-specific CAR T cells. The consequent reduction of cell surface TCR expression resulted in minimal cytokine production upon TCR stimulation in vitro providing a potential allogeneic CAR T approach. These CAR T cells showed no demonstrable evidence of GvHD induction when infused in NSG mice yet maintained BCMA-specific CAR activity in KMS-11 and RPMI-8226 established myeloma models. Initial studies further confirmed that two shRNA could be expressed from a single retroviral vector to modulate the expression of multiple genes. Further engineering of the microRNA framework reduced the size of the transgene load to 394bp while enabling the expression of up to 4 shRNA within a single vector. shRNA specific for CD3ζ, beta-2-microglobulin, CD52 and diacylglycerol kinase alpha were engineered into the framework downstream of a CD19-CAR. Transduced Jurkat cells showed concurrent knockdown of the respective gene products at the mRNA and protein levels. Conclusions: A first-in-human clinical trial evaluating the first-generation single shRNA-vector in the context of a BCMA-targeting CAR as a non-gene edited approach to allogeneic CAR T cell therapy will be initiated in 2020. The proof of principle study here shows that multiple shRNAs are active within a single viral vector thereby avoiding the need for bespoke individual clinical reagents to target multiple genes. The multiplexed shRNA vector system is now in further development to explore whether this strategy can enhance the therapeutic potential of CAR T cells.
A pilot study of Bruton’s tyrosine kinase inhibitor ibrutinib alone and in combination with PD-1 inhibitor nivolumab in patients with metastatic solid tumors.
Authors: Benner Brooke , Quiroga Dionisia Marie , Good Logan , Sun Steven , Savardekar Himanshu , Duggan Megan Christina , Konda Bhavana , Verschraegen Claire F. , Kendra Kari Lynn , Shah Manisha H. , Rupert Robert , Monk Paul , Shah Hiral A. , Noonan Anne M. , Bixel Kristin Leigh , Hays John L. , Behbehani Gregory , Pietrzak Maciej , Carson William Edgar , Wesolowski Robert ,   ASCO Authors Group
Published: May 2020

3111

Background: Myeloid-derived suppressor cells (MDSC) are expanded in cancer and promote immune suppression. We have shown that ibrutinib inhibits migration and immunosuppressive function of MDSC. Moreover, the combination of ibrutinib and a PD-L1 inhibitor has been found to have synergistic anti-tumor effects in a multiple solid tumor mouse models. Therefore, we conducted a pilot study testing the combination of ibrutinib and nivolumab in patients with metastatic solid tumors. Methods: Sixteen patients with advanced solid tumors were recruited to this trial. Ibrutinib was dosed as an oral single agent, starting 7 days prior to cycle 1 of nivolumab and given until cycle 1, day 8 of nivolumab. Nivolumab was administered intravenously on days 1 and 15 on 28-day cycles. Patients had blood samples collected prior to initiation of ibrutinib, day 1 of cycle 1, day 8 of cycle 1, day 1 of cycle 2, and at the time of disease progression. From these specimens, we measured circulating MDSC levels, other circulating immune subsets, T cell proliferation, and cytokines/chemokines levels. Circulating MDSC levels were measured by mass spectrometry. T cell function was evaluated by CFSE to monitor proliferating cells by dye dilution and cytokine/chemokine levels were measured with a U-PLEX assay. Data were analyzed using two-tailed, paired Student's t-tests to assess statistical significance. Results: An increase in circulating MDSC (22% to 28%; SD 9.158) levels was observed following 7 days of single-agent ibrutinib compared to baseline. However, in combination therapy, MDSC levels decreased (19%; SD 13.17) prior to cycle 2. Despite increasing levels of circulating MDSC, T cell function improved throughout the study. Furthermore, plasma levels of chemokines associated with MDSC recruitment and migration significantly decreased with ibrutinib treatment (IL-12, CCL2, CCL3, and CCL4). Of the 16 patients, four achieved a partial response and four achieved stable disease. Median progression free survival was 3.5 months and median overall survival was 11.5 months. Conclusions: The combination of ibrutinib and nivolumab was well tolerated, demonstrated early signs of immune modulation, and showed preliminary signs of promising clinical activity in patients with metastatic solid tumors. Clinical trial information: NCT03525925 .
Gene signature of antigen processing and presentation machinery (APM) as highly predictive of response to checkpoint blockade in lung cancer and melanoma.
Authors: Thompson Jeffrey C. , Davis Christiana , Hwang Wei-Ting , Deshpande Charuhas , Jeffries Seth , Langer Corey J. , Albelda Steven M.
Published: May 2020

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Background: Treatment of non-small cell lung cancer (NSCLC) with immune checkpoint blockade (ICB) has resulted in striking clinical responses, but only in a subset of patients (pts), underscoring the need to identify genomic and molecular determinants of immune evasion. Limited data exist on the potential role of alterations in HLA Class I antigen processing and presentation machinery (APM) in mediating response to ICB. Methods: We conducted a retrospective cohort study analyzing transcriptional profiles from pre-treatment tumor samples of chemotherapy-refractory advanced NSCLC pts treated with ICB. RNA was analyzed using the AmpliSeq transcriptomic platform. An APM signature was generated utilizing 8 genes associated with antigen processing ( B2M, CALR, NLRC5, PSMB9, PSME1, PSME3, RFX5, HSP90AB1) and was examined for its association with response to therapy and progression-free and overall survival (PFS, OS). The APM signature was then evaluated in two independent melanoma cohorts treated with ICB. Results: We analyzed pre-treatment tumor samples from 51 advanced NSCLC pts treated with ICB, median age 64 (range 31-92), smokers (n = 43), adenocarcinoma (n = 31). There were 23 responders and 28 non-responders. The APM signature was significantly higher in responders compared to non-responders (average z-score 2.69 vs. -2.49, p = 0.0001). An APM score above the median value for the entire cohort was significantly associated with improved PFS (HR 0.24, 95% CI, 0.12-0.47, log-rank = 0.001) and OS (HR 0.34, 95% CI, 0.18-0.67, log-rank = 0.005). The APM score was significantly correlated with the well-validated T-cell-inflamed resistance gene expression profile (GEP) score (R2 = 0.32, p < 0.0001). However, the APM score demonstrated improved ability to predict response to ICB relative to the GEP score with AUCs of 0.83 and 0.69, respectively. In an independent cohort of 14 high-risk resectable stage III/IV melanoma pts treated with neoadjuvant anti-PD1 therapy, upregulation of genes involved in antigen processing was associated with improved disease free survival (HR: 0.08, 95% CI, 0.01-0.50, p = 0.0065). In an additional independent melanoma cohort of 28 metastatic pts treated with ICB, a higher APM score was associated with improved overall survival (HR 0.31, 95% CI, 0.09-0.89, log-rank = 0.044). Conclusions: Our data demonstrate that defects in antigen presentation may be an important feature in predicting outcomes to ICB in both lung cancer and melanoma.
A machine learning-based approach for the inference of immunotherapy biomarker status in lung adenocarcinoma from hematoxylin and eosin (H&E) histopathology images.
Authors: Batenchuk Cory , Chang Huan-Wei , Cimermancic Peter , Yi Eunhee S. , Sadhwani Apaar , Velez Vanessa , Patel Hardik L. , Behrooz Ali , Tekiela Kamilla , Findlater Robert , Hanks Debra , Kulig Kimary
Published: May 2020

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Background: The current standard work-up for both diagnosis and predictive biomarker testing in metastatic non-small cell lung cancer (NSCLC), can exhaust an entire tumor specimen. Notably, gene mutation panels or tumor mutation burden (TMB) testing currently requires 10 tissue slides and ranges from 10 days to 3 weeks from sample acquisition to test result. As more companion diagnostic (CDx)-restricted drugs are developed for NSCLC, rapid, tissue-sparing tests are sorely needed. We investigated whether TMB, T-effector (TEFF) gene signatures and PD-L1 status can be inferred from H&E images alone using a machine learning approach. Methods: Algorithm development included two steps: First, a neural network was trained to segment hand-annotated, pathologist-confirmed biological features from H&E images, such as tumor architecture and cell types. Second, these feature maps were fed into a classification model to predict the biomarker status. Ground truth biomarker status of the H&E-associated tumor samples came from whole exome sequencing (WES) for TMB, RNAseq for the TEFF gene signatures or reverse-phase protein array for PD-L1. Digital H&E images of NSCLC adenocarcinoma for model development were obtained from the cancer genome atlas (TCGA) and commercial sources. Results: This approach achieves > 75% accuracy in predicting TMB, TEFF and PD-L1 status, offers a way to interpret the model, and provides biological insights into the tumor-host microenvironment. Conclusions: These findings suggest that biomarker inference from H&E images is feasible, and may be sufficiently accurate to supplement or replace current tissue-based tests in a clinical setting. Our approach utilizes biological features for inference, and is thus robust, interpretable, and readily verifiable by pathologists. Finally, biomarker status inference from a single H&E image may enable testing in patients whose tumor tissue has been exhausted, spare further tissue use, and return test results within hours to enable rapid treatment decision-making to maximize patient benefit.
The HLA Ligand Atlas: A novel immuno-oncology resource for T-cell antigen discovery.
Authors: Marcu Ana , Bichmann Leon , Kuchenbecker Leon , Backert Linus , Kowalewski Daniel J , Freudenmann Lena-Katharina , Löffler Markus W. , Lübke Maren , Walz Juliane , Velz Julia , Moch Holger , Regli Luca , Silginer Manuela , Weller Michael , Schlosser Andreas , Kohlbacher Oliver , Stevanovic Stefan , Rammensee Hans-Georg , Neidert Marian Christoph
Published: May 2020

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Background: The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system, which is the basis for T cell-dependent tumor rejection. Therefore, a comprehensive map of the entirety of both HLA class I- and class II-presented peptides from various benign tissues is a highly sought after resource, as it enables the definition of tumor-association on the immunologically pivotal level of the HLA ligandome. Methods: Human tissue samples were snap frozen post mortem during autopsy. The study was approved by the local IRB. HLA ligands were immunopurified and characterized using an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC Nano UHPLC System. Data acquisition was performed as technical triplicates in data-dependent mode, and data were analyzed using the containerized, computational pipeline MHCquant. Results: In this work, we describe the HLA Ligand Atlas, a comprehensive collection of matched HLA class I and class II ligandomes from 29 non-malignant tissues and 13 human subjects (208 samples in total), covering 38 HLA class I, and 17 HLA*DRB alleles and comprising 48,381 HLA class I and 16,146 HLA class II peptides. Nearly 50% of HLA ligands have not been previously described. The HLA Ligand Atlas is publicly available as a raw data resource, but also in the form of a user-friendly web interface that allows users to quickly formulate complex queries against the data set. Both downloadable data and the query interface are available at www.hla-ligand-atlas.org. Conclusions: This data set provides a valuable tool for research in diverse fields such as systems biology, general immunology, autoimmune disease and organ transplantation. Most importantly, the HLA Ligand Atlas provides essential information for translational applications in immuno-oncology. The knowledge of HLA ligands from benign tissues strongly supports the informed design of proteogenomic HLA-dependent target discovery approaches.
Effect of chemokine signaling signatures on resolving discrepancy between TMB and checkpoint expression.
Authors: Szeto Christopher , Veerapaneni Saihitha , Parulkar Rahul , Reddy Sandeep K. , Rabizadeh Shahrooz , Benz Stephen Charles
Published: May 2020

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Background: Tumor mutation burden (TMB) and PD1/L1 expression are independent biomarkers for immune checkpoint blockade therapy, as seen in the Checkmate227 trial. Here we explore whether chemokine activity, an intermediate step between neoantigen presentation and immune-infiltration, can resolve this lack of association between existing biomarkers. We further use this novel biomarker to corroborate recent findings from Crowther et al. for a role of MHC class 1-related gene ( MR1) downregulation in immune evasion. Methods: 1,395 clinical samples from the NantHealth database with matched tumor:normal whole exomes and deep whole-transcriptomic sequencing (> 200M reads) were available for analysis. The most common indications in the cohort were Breast (18%), Colon (9.8%), Lung (7.8%), Soft-tissue/Sarcomas (7.7%), and Pancreatic (6.1%). TMB was calculated by counting non-synonymous exonic mutations as per Rizvi, 2015. Immune-infiltration and chemokine signaling were inferred from RNAseq expression of published immune-cell-specific genesets (Bindea, 2013) and chemokine ligands (Nagarsheth, 2017) respectively. Significant associations between TMB, chemokine activity, immune-infiltration, and checkpoint expression were analyzed by ranksums test and corrected for multiple-hypothesis testing using Benjamini-Hochberg adjustment. Results: As expected, TMB and PD1/L1 mRNA expression were not correlated in this cohort (r = 0.08 and r = 0.07 respectively). 36.3% of patients classified as highly immune-infiltrated by unsupervised clustering of immune-cell scores, and this subgroup significantly overexpressed all 11 targetable checkpoint genes analyzed including PD1, PDL1, CTLA4, IDO1, and VISTA (adj. p 9.7e-68 to 4e-168). There was no association between immune-infiltrated samples and TMB (t = 0.9, p = 0.35). Twice as many patients classified as chemokine-active (70.0%) and there was significant agreement between immune-infiltrated and chemokine-active patients (OR = 34.8, p = 6.5e-81). Interestingly, there was a weak but significant association between high chemokine-activity and increased TMB (t = 3.3, p = 0.001). Within patients that were chemokine-active but lacked immune-infiltration, MR1 expression was significantly depleted (t = -10.7, p = 1e-26). Conclusions: Chemokine signatures can help resolve discordance between TMB and checkpoint expression. Analysis of discordance between chemokine-active but immune-depleted tumors may aid in identifying targets for converting from cold to hot tumor microenvironments.
A shared tumor-antigen RNA-lipoplex vaccine with/without anti-PD1 in patients with checkpoint-inhibition experienced melanoma.
Authors: Loquai Carmen , Hassel Jessica Cecile , Oehm Petra , Derhovanessian Evelyna , Jabulowsky Robert Alexander , Gold Maike , Schwarck-Kokarakis Doreen , Attig Sebastian , Cuk Katarina , Vogler Isabel , Sikorski Julian , Leierer Melanie , Mitzel-Rink Heidrun , Miederer Matthias , Grabbe Stephan , Utikal Jochen , Pinter Andreas , Kaufmann Roland , Sahin Ugur , Tureci Oezlem
Published: May 2020

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Background: Cancer vaccines are considered unsuitable for patients with advanced tumours and have not been clinically successful. Methods: Lipo-MERIT is an ongoing phase 1/2 trial (NCT02410733) with melanoma FixVac, a liposomal RNA vaccine targeting four non-mutant shared tumour-associated antigens (TAAs) (MAGE-A3, NY-ESO-1, tyrosinase, TPTE). Patients with stage IIIB-C and IV melanoma are eligible. The trial comprises 7 dose escalation and 3 dose expansion cohorts, the latter with FixVac alone or combined with anti-PD1. Eight doses of FixVac are administered i.v. weekly/bi-weekly followed by optional continued monthly treatment. This abstract summarizes the findings of an exploratory interim analysis (cut-off JUL2019) of 89 patients. Results: 42 of 89 patients had measurable disease at baseline and were eligible for assessment of best objective overall response. All but one patient were stage IV and had undergone previous lines of treatment, 41 patients were checkpoint-inhibitor (CPI)-experienced, and 35 had been exposed to both anti-CTLA4 and anti-PD1 therapy. In the vaccine monotherapy group (n = 25) three patients experienced a partial response (PR) and 7 patients had stable disease (SD). An additional patient showed a complete metabolic remission of metastatic lesions based on [18F]-FDG PET/CT imaging. In the group of patients treated with melanoma FixVac and PD1 blockade, 6 of 17 patients developed a PR. Patients with PR showed induction of poly-epitopic and strong CD4+ and CD8+ T cell immunity against the vaccine antigens. The number of antigen-specific cytotoxic T cells in some responders reached up to low 2-digit percentages of circulating CD8+ T cells and was maintained at high levels by continued vaccination. Overall, 75% of the 50 patients tested by ex vivo IFNg ELISpot analysis and all 20 patients tested by IFNg ELISpot after in vitro stimulation showed vaccine-induced immune responses against at least one vaccine antigen. Typically, antigen-specific T cells ramped up within the first 4-8 weeks to single-digit and low double-digit percent fractions of circulating CD8+ T cells. Immune responses were of effector memory phenotype and their strength and frequency did not depend on disease status at baseline (measurable versus non-measurable disease), on vaccine dose or on treatment (FixVac alone versus in combination with anti-PD1). Conclusions: FixVac alone and in combination with anti-PD1 mediates durable objective responses in pre-treated, CPI experienced patients with advanced progressing melanoma. Clinical trial information: NCT02410733 .