https://openworks.mdanderson.org/sumexp22/1062/thumbnail.jp

Acharya, Sunil, PhD.

Banerjee, Pinaki, PhD

Basar, Rafet, MD

Cui, Yan

Islam, Sanjida

Lin, Paul, MD, PhD

Liu, Dawei

Rafei, Hind, MD, MS

Rezvani, Katy, MD, PhD

Uprety, Nadima

English

OpenWorks @ MD Anderson

20.0010.00 5.002.501.25-20242030405060Effector:Target ratio% specific lysis of CAR NK A cells CAR NK ACAR NK BMultimodal Analysis of the Interaction Between CD70-DirectedChimeric Antigen Receptor Natural Killer Cells and Target Tumors1 Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Atlanta, GA 303322 Stem Cell Transplantation and Cellular Therapy, MD Anderson Cancer Center, Houston, TX 77030Conclusions1. Rafei H, Daher M, Rezvani K. Chimeric antigen receptor (CAR)natural killer (NK)-cell therapy: leveraging the power of innateimmunity. British Journal of Haematology. 2021;193(2):216-230.2. Sanborn KB, Rak GD, Mentlik AN, Banerjee PP, Orange JS. Analysisof the NK cell immunological synapse. Methods in Molecular Biology.2010;612:127-148.3. Jacobs J, Deschoolmeester V, Zwaenepoel K, et al. CD70: AnFlieswasser T, Van den Eynde A, Van Audenaerde J, et al. TheCD70-CD27 axis in oncology: the new kids on the block. Journal ofExperimental & Clinical Cancer Research. 2022;41(1):12.4. Terrén I, Orrantia A, Vitallé J, Astarloa-Pando G, Zenarruzabeitia O,Borrego F. Modulating NK cell metabolism for cancerimmunotherapy. Seminars in Hematology. 2020;57(4):213-224.This project was funded by the RP210028 CPRIT Research TrainingGrant. Thanks to the Department of Experimental Radiation Oncology forproviding me with the opportunity to partake in the CPRIT-CURESummer Program. I would like to express my sincere gratitude to Dr.Pinaki Banerjee, Dr. Hind Rafei, Sanjida Islam, Tosin Banjo, Dr. GoharManzar, and Dr. Katy Rezvani for their incredibly patient mentorship. Iwould also like to thank everyone in the Rezvani lab for their welcomeand dedication to helping trainees succeed. This poster is dedicated tothe memory of Dr. Judy Moyes.ResultsReferencesAcknowledgementsEffector cells: NK cells were harvested fromUCB units, stimulated with IL-2, andexpanded with uAPCs. CAR NK cells wereobtained by retroviral transduction with avector encoding a CD27 receptor andcontaining an IL-15 transgene.Fluorescence tagging and imaging:Samples were stained with shown markersand ran through Amnis imaging flowcytometer and LSRFortessa X-20 CellAnalyzer.Cytotoxicity assay: Standard chromiumrelease assay was performed. Target cellswere radiolabeled with chromium-51 andcocultured with NK cells for 4 hours atvarious effector-to-target ratios. Lysis wasthen determined by measuring chromiumreleased in the supernatant from dying cells.Metabolic analyses: The Cell Mito StressTest and Glycolysis Stress Tests were run onSeahorse XF Analyzer to measure oxygenconsumption rate (OCR) and extracellularacidification rate (ECAR), respectively, of NTand CAR NK cells. Liquid chromatography-mass spectrometry (LC-MS) was conductedat the MD Anderson Metabolomics Core.IntroductionMethodsMM.1S UMRC-3• Natural killer (NK) cells are effectorlymphocytes of the innate immunesystem.• NK cells form conjugates with their targetsto induce cytotoxicity through directcontact.• CD70, the ligand of CD27, is expressedon multiple solid and hematologic cancers.Thus, NK cells transduced to express aCD27 chimeric antigen receptor (CAR)can be used to target CD70-expressingtumor cells.Ø This study aims to compare the conjugateformation, cytotoxicity, and the metabolicfitness of CD27 CAR NK cells andnontransduced (NT) NK cells, in order tobetter understand the interaction betweenCAR NK cells and their tumor targets.Figure 4. (A) Cytotoxicity assays against the NK-susceptible K562 leukemia cells, andagainst CAR NK A cells by sibling CAR NK A cells (HLA-A2+ and HLA-A3-) and CAR NKcells of different HLA haplotype B (HLA-A2+ and HLA-A3+) showing lack of fratricide.(B) Flow cytometry indicating downregulation of CD70 in CD27-transduced CAR NK cells.BA20:1 10:1 5:1 2.5:1 1.25:1020406080100NK : Tumor Specific Lysis (%)20:1 10:1 5:1 2.5:1 1.25:1020406080100Effector:Target ratioSpecific Lysis (%)20:1 10:1 5:1 2.5:1 1.25:1020406080100NK : Tumor Specific Lysis (%)20:1 10:1 5:1 2.5:1 1.25:1020406080100Effector:Target ratioSpecific Lysis (%)0 20 40 60 800100200300400Time (minutes)OCR (pmol/min/106 cells)Oligomycin FCCP R/ANTCD70/IL-15 CAR NK0 20 40 60 800100200300400Time (minutes)OCR (pmol/min/106 cells)Oligomycin FCCP R/ANTCD70/IL-15 CAR NKFigure 3. (A) ECAR of NT and CAR NK cells as measured by Seahorse Glycolysis StressTest. (B) OCR of NT and CAR NK cells as measured by Seahorse Mito Stress Test. (C)Glycolysis (green) and TCA (black) metabolites of NT and CAR NK cells by LC-MS shownas normalized heat map ranked by k-means clustering.BA0 20 40 60 80050100150Time (minutes)ECAR (mpH/min/106 cells)Glucose Oligomycin 2-DG0 20 40 60 800100200300400Time (minutes)OCR (pmol/min/106 cells)Oligomycin FCCP R/ACImmune Synapse Between CAR NK and Tumor CellsFigure 2. Chromium-51 cytotoxicity assays showing higher killing of CD70+ MM.1S andUMRC-3 cell lines by CAR NK cells than by NT NK cells.Cytotoxicity of CAR NK versus CD70+ Tumor CellsCEnhanced Metabolic Fitness of CAR NK CellsCD70 Downregulation Prevents CAR NK Fratricide20:1 10:1 5:1 2.5:1 1.25:1020406080100NK : Tumor Specific Lysis (%)20:1 10:1 :1 2.5:1 1.25:1020406080100Effector:Target ratioSpecific Lysis (%)CAR NK AK562Future Directions• Long-term cytotoxicity and tumorrechallenge assays against CD70+tumors.• Short- and long-term apoptosis assaysagainst HLA-mismatched CAR NK cells.• Metabolic assays and LC-MS of CAR NKcells following coculture with CD70+tumors.Schematic depicting CD27 CAR NK cells actively engaging tumor cells, compared to NT NK cells. CD27 CAR Empowers NK Cell FunctionFigure 1. (A) Map of CD27-based CAR construct. (B) Formation of CAR NK cellimmunological synapse (IS) (shown with white arrows) with CD70-expressing Raji and Karpaslymphoma cell lines. (C) CAR NK cell singlet, and the binding of an NT NK and CAR NK cell toa MM.1S multiple myeloma cell. Shown here is the accumulation of CD27 and CD3ζ at the ISformed by the CAR NK cell, but not the NT NK cell.ABDawei Liu1,2, Pinaki Banerjee, PhD2, Hind Rafei, MD2, Paul Lin, MD, PhD2, Rafet Basar, MD2, Sunil Acharya, PhD2, Nadima Uprety2, Sanjida Islam2, Yan Cui2, Katy Rezvani, MD, PhD2CD70CD27NT NKCAR NKk_means_21.002.00

Multimodal Analysis of the Interaction Between CD70-Directed Chimeric Antigen Receptor Natural Killer Cells and Target Tumors

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1062&context=sumexp22

Multimodal Analysis of the Interaction Between CD70-Directed Chimeric Antigen Receptor Natural Killer Cells and Target Tumors

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