Abstract CD200 is an immunosuppressive molecule overexpressed in multiple hematologic malignancies such as B cell chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemia. We previously demonstrated that up-regulation of CD200 on tumor cells suppresses antitumor immune responses and that antagonistic anti-human CD200 mAbs enabled human PBMC-mediated tumor growth inhibition in xenograft NOD/SCID human (hu)-mouse models. Ab variants with effector function (IgG1 constant region (G1)) or without effector function (IgG2/G4 fusion constant region (G2G4)) exhibited high antitumor activity in a human tumor xenograft model in which CD200 was expressed. In this report, we seek to select the best candidate to move forward into the clinic and begin to decipher the mechanisms of tumor cell killing by comparing anti-CD200-G1 vs anti-CD200-G2G4 in two related animal models. In a CD200-expressing xenograft NOD/SCID hu-mouse model where CD200 ligand/receptor interactions are already established before initiating treatment, we find that anti-CD200-G1 is a less effective Ab compared with anti-CD200-G2G4. Separately, in a model that evaluates the effect of the Abs on the immune cell component of the xenograft NOD/SCID hu-mouse model distinctly from the effects of binding to CD200 on tumor cells, we find that the administration of anti-CD200-G1 Abs completely abolished human PBMC-mediated tumor growth inhibition. Along with supporting in vitro studies, our data indicate that anti-CD200-G1 Abs efficiently mediate Ab-dependent cellular cytotoxicity of activated T cells, critical cells involved in immune-mediated killing. These studies suggest important implications regarding the selection of the constant region in anti-CD200 immunotherapy of cancer patients.
Abstract Introduction: PD-1 inhibitors have shown limited efficacy in glioblastoma due to microenvironment immunosuppression and low tumor mutational burden. In GBM, PD-L1 expression is not a predictive marker for response to PD-1 or PD-L1 inhibitors. Multiplex immunostaining panel technology allows for detailed analyses of tumor microenvironment cells and their interaction. Methods: Pre-treatment tumor tissue was collected retrospectively from 27 patients at Columbia University Irving Medical Center with primary glioblastoma who were diagnosed within the past three years, had surgery here, and were either treated with SOC therapy (n= 8) or PD-1 inhibitors at recurrence (n= 19). Multiplex immunofluorescence panels included 1) CD11b/IDO1/HLADR/GFAP, 2) PD1/PD-L1/GFAP, and 3) CD4/CD8/CD25/FoxP3/Ki67/GFAP. Results: Multiplex immunofluorescence panels did not show any correlation with outcomes in patients treated with SOC therapy. Among the 19 patients treated with PD-1 inhibitors, those with more HLA-DR positive cells had worse outcomes (p= 0.02). PD-L1 expression on tumor cells was not predictive of outcomes. There was a correlation trend between PD-1/PD-L1 interaction score (p= 0.08) and outcomes. PTEN loss was correlated with higher Ki67 expression in both tumor cells (p= 0.05) and non-tumors cells (p= 0.03); however, this was not found in Ki67 in CD4+ cells, CD8+ cells, or CD4+CD8+ cells combined. Tumor-associated macrophages, myeloid-derived suppressor cells, CD8+ cells, and CD4+ cells were not significant predictive markers for outcome. Conclusion: Quantitative spatial profiling by multiplex immunofluorescence is feasible in FFPE glioblastoma tissue. More refined and extensive quantitative and spatial microenvironment analyses may allow for the development of biomarkers for immunotherapy in GBM.
Abstract Purpose: Due to profound clinical successes observed with radioligand therapies (RLT) across multiple cancers coupled with advances in drug handling and delivery logistics recently, clinical trials investigating RLTs are on the rise. Ionizing radiation produced by radioisotopes linked to antibodies causes double-stranded breaks either directly or via generation of reactive oxygen species. This type of DNA damage is associated with an increase in proteins mediating cell cycle arrest (e.g., p21/CIP1) and repairing damaged DNA (e.g., CHK2 and γH2Ax). Additionally, proliferating cells (identifiable by Ki67) are most susceptible to ionizing radiation as DNA is exposed during the mitotic phase of the cell cycle. We incorporated these biologically verified markers into a multiplexed fluorescence immunohistochemistry (mFIHC) assay for robust quantitation of the pharmacodynamic activity of RLTs and gain insights into the DNA damage response in on-treatment tumor core biopsies. Study Design: We designed a novel mFIHC assay incorporating antibodies to p21, pCHK2, γH2Ax, Ki67, CK to quantitatively characterize DNA damage across five tumor indications of commercially available ovarian cancer, colorectal cancer, gastric cancer, endometrial (uterine) cancer, and breast cancer formalin-fixed paraffin-embedded (FFPE) tissue blocks. We successfully validated this mFIHC assay using automated staining (Bond RX), imaging (PhenoImager HT), and combined with hypothesis-driven spatial profiling algorithms (e.g., AQUA Technology). Results: Sensitivity and specificity were confirmed on known positive and negative controls. Reproducibility and precision were observed across instruments, operators, and independent experiments for all markers The frequency of p21 ranged from 5% - 8%, pCHK2 from 3% - 36%, pH2Ax from 0% - 8%, Ki67 from 4% - 61%, and CK cells ranged from 25% to 87%. Over 56 unique parameters were evaluated. The prevalence of p21, pCHK2, and γH2Ax ranged from 0% to >30% across five tumor indications tested and were highly concordant. Conclusion: The validated mFIHC assay examines the expression of proteins mediating cell cycle arrest and DNA damage in tumor and stromal cells. This panel may be used to further understand the complexity of DNA damage response pathways in tumor and non-tumor areas in the context of RLT clinical trials. Citation Format: James Santos, Virginia Nyanchama, Vincent Romanet, Ernesta Dammassa, Lisa Kattenhorn, Ying Huang, Jimmie Lim, Xun Li, James Deeds, Lori Iaconis, Emmanuel Pacia, Margaret McLaughlin, Naveen Dakappagari, Jennifer Bordeaux. Quantifying pharmacodynamic markers of radioligand therapies in tumor by multiplex immunofluorescence and automated quantitative analysis (AQUA) algorithms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3623.
Abstract The renaissance of cancer immunotherapies and the positive clinical responses observed with chimeric antigen receptor modified T cells in the hematomalignancy setting has stimulated substantial interest in monitoring immune cell activation and suppression to determine efficacy, prognosis and safety in new agent investigational trials. However, immunophenotyping capacity has been limited and required multi-test tube panels. To address the growing needs of clinical trials, we have developed three fit-for-purpose high complexity (10 or more markers) T-cell phenotyping flow cytometry panels on a qualified LSR FortessaTM platform that enables detection of up to 15 markers in a single test tube. The first panel allows identification of multiple phenotypes along the T-cell differentiation pathway, namely, T-naive (TN), T-stem cell memory (TSCM), T-effector memory (TEM) and T-central memory (TCM) and T-effector memory RA+ (TEMRA). The second panel identifies the most common helper T-cell phenotypes such as Th1, Th2, Treg, and Th17. A third panel determines the functional status of T-cells (e.g., activation vs. suppression) but also enables quantitation of important checkpoint inhibitor expression (e.g., PD-1) on T-cells of interest. These high complexity flow cytometry panels can serve as powerful tools for comprehensive examination of T-cells in a small volume of patient specimen. We believe these new flow cytometry panels will have a substantial impact on the determination of efficacy and safety correlates of immunomodulating agents administered alone or in combination to patients with leukemia. Citation Format: Ghanashyam Sarikonda, Devika Ashok, Anil Pahuja, Jelveh Lameh, Shabnam Tangri, Naveen Dakappagari. High complexity flow cytometry panels to monitor target expression, T-cell activation and suppresssion by novel immunotherapies in hematomalignancy clinical trials. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1302. doi:10.1158/1538-7445.AM2015-1302
Minimal residual disease (MRD) negativity is a key prognostic indicator of outcome in acute lymphocytic leukemia. In the INO-VATE trial (clinicaltrials.gov identifier: NCT01564784), patients with relapsed/refractory acute lymphocytic leukemia who received inotuzumab versus standard chemotherapy achieved greater remission and MRD-negativity rates as well as improved overall survival: hazard ratio 0.75, one-sided P = 0.0105. The current analysis assessed the prognostic value of MRD negativity at the end of inotuzumab treatment. All patients who received inotuzumab (n = 164) were included. Among patients with complete remission/complete remission with incomplete hematologic response (CR/CRi; n = 121), MRD-negative status (by multiparametric flow cytometry) was defined as <1 × 10-4 blasts/nucleated cells. MRD negativity was achieved in 76 patients at the end of treatment. Compared with MRD-positive, MRD-negative status with CR/CRi was associated with significantly improved overall survival and progression-free survival, respectively: hazard ratio (97.5% confidence interval; one-sided P-value) 0.512 (97.5% CI [0.313-0.835]; P = 0.0009) and 0.423 (97.5% CI [0.256-0.699]; P < 0.0001). Median overall survival was 14.1 versus 7.2 months, in the MRD-negative versus MRD-positive groups. Patients in first salvage who achieved MRD negativity at the end of treatment experienced significantly improved survival versus that seen in MRD-positive patients, particularly for those patients who proceeded to stem cell transplant. Among patients with relapsed/refractory acute lymphocytic leukemia who received inotuzumab, those with MRD-negative CR/CRi had the best survival outcomes.
The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19–23, 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) are published in volume 16 of Bioanalysis, issues 8 and 9 (2024), respectively.
