Abstract 512: Tetramer and pentamer assays for antigen-specific lymphocytes: Implementation in clinical trials
Authors: Xue Chengsen , Zhong Weiguo , Cho Yeji , Cuomo Joanne , Swenson Christina D. , Closkey Thomas W. Mc 
Published: July 2021

Abstract
The success of inhibiting PD1 on T cells for cancer immunotherapy initiated strong interest in studying therapeutic antibodies to alter the adaptive immune system. As the knowledge of neoantigens, oncogenes, and autoantigens is rapidly accelerating, the search for antigen-specific lymphocytes for better treatment outcomes is increasing. A tetramer or pentamer assay is an essential tool to immunophenotype and enumerate antigen-specific lymphocytes. These assays offer a powerful technique able to monitor immune status, using peripheral whole blood as a matrix, in contrast to invasive tissue biopsies. However, the assays have a number of obstacles to overcome in clinical research. Over several years, we have developed and validated tetramer and pentamer assays with both successes and failures.
Rare events and precision: Clinical studies require robust reproducibility and high precision. As a standard in judging the acceptability of a flow cytometric biomarker test, the majority of tests are set up to have 20-30% CV between results. However, if a biomarker is expressed in less than 1% of a target population, the criteria can fail. The tetramer assay requires precision analyses that are even more stringent. These cells are rare events among millions of hematopoietic cells. The precision requirements may be at one hundredth percent or lower. A successful sign for the assay is that over a one-year period during the course of the clinical trial, some biomarkers remain at a stable level.
Increasing signals and reduction of background noise: Unlike single marker based antibodies, however, tetramers and pentamers are highly sensitive. It is critical, therefore, to increase signals and reduce background noise. A combination of 7AAD (to remove dead cells) and incorporation of a dump channel (to remove non-target populations) significantly improves the precision of the assays.
Data analyses and management: With new and powerful flow cytometers, a single sample can be labelled with multiple surface markers along with fluorescent tetramer or pentamers. The file size can reach megabytes when two to five million cells are tagged. This creates a bottleneck for data analysis and analytical software, especially in an assay with a panel of multiple tubes. The solution is to cut non-essential cell numbers including those with high side-scatter (SSC) numbers in half and to re-export data from cytometers.
Controls setup: The most challenging part of these assays is to have strict controls, both for reagents and cells. Customized reagents and a source for characterized specific peripheral blood cells (PBMC) with HLA-A markers are necessary for developing and validating these complex assays. In conclusion, tetramer and pentamer assays targeting antigen-specific lymphocytes are deliverables for cancer immunotherapy research, cancer vaccine development and for use in studying other autoimmune diseases, such as type 1 diabetes.
Citation Format: Chengsen Xue, Weiguo Zhong, Yeji Cho, Joanne Cuomo, Christina D. Swenson, Thomas W. Mc Closkey. Tetramer and pentamer assays for antigen-specific lymphocytes: Implementation in clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 512.