Joseph N. Blattman, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Adoptive immunotherapy with large numbers of in vitro expanded antigen-specific CDS T cells has the potential to become a novel and safe treatment modality for cancer or viral infections, but efficacy is often limited. One obstacle is insufficient in vivo survival and retention of function by donor T cells, which can often be sustained by cotransfer of CD4 helper T cells or by IL-2 administration to substitute for CD4 help. However, specific helper CD4 T cells have not been identified for many tumors, CD4 T cells are often limiting and can be pose additional problems during treatment of HIV infection, and prolonged high-dose IL-2 supplementation can be both inefficient and toxic. Another major obstacle to adoptive immunotherapy can be the failure of transferred T cells to be effectively activated by viral and tumor targets in vivo. T cells specific for tumors often have low affinity TCRs, malignant or infected targets can have impaired antigen processing machinery or accessory signals, and prolonged recognition of target cells can induce T cell tolerance. Thus, increasing the capacity of CDS T cells for IL-2 production and autocrine IL-2 mediated proliferation, reducing the threshold for CDS T cell activation, or preventing induction of tolerance in antigen-specific CDS T cells should significantly enhance the efficacy of adoptive immunotherapeutic strategies. One potential means to increase CDS T cell avidity and function or sustain autocrine IL-2 proliferation and survival is to reduce inhibitory signals in transferred CDS T cells mediated by intracellular regulatory proteins. We have identified Cbl-b, SHP-1, and SOCS-1 as potential targets to enhance CDS T cell aviditiy and function. CDS T cells from mice deficient in these genes results in increased T cell reactivity and costimulation independent IL-2 production. The major goals of this project are to determine if decreasing the negative regulation of activation signals in CDS T cells by Cbl-b, SHP-1, or SOCS-1 by expression of dominant negative versions of these proteins, or by reducing endogenous levels of these proteins via expression of specific siRNA, can improve T cell avidity and function and render these cells more effective in therapy of chronic viral infections or malignancy in mouse models. Additionally, we will determine if abrogating inhibition by these regulatory proteins can enhance the avidity, proliferation and survival of human CDS T cells in preclinical studies. [unreadable] [unreadable] Chronic viral infections and cancer represent major public health concerns. Adoptive immunotherapy with T cells has the potential to become an effective and broadly applicable approach for the treatment of viral infections and malignancy. However, the efficacy of transferred T cells is limited by insufficient in vivo survival and function. These studies will determine if genetically modifying T cells to improve function and survival can enhance adoptive immunotherapy approaches in mouse models of chronic viral infection or malignancy as well as the survival and function of human virus and tumor specific T cells in preclinical studies. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Currently, there are few methods available to analyze the evolution of tumor heterogeneity; micro-dissection of tumors only provides information on major species of malignant cells but is unable to detect rare therapy-resistant subclones that have the potential to regenerate tumors. Identification of these rare cells by single-cell isolatio and sequencing is both time-consuming and prohibitively expensive. Recent next-generation deep sequencing studies have demonstrated the clinical relevance of clonal heterogeneity within individual cancers, but currently rapid and cost-effective methods to measure and track the rates of co-occurrences of mutations in cell populations do not exist. Therefore, the development of rapid, flexible, single- cell technologies with the capacity to identify heterogeneous mutations of multiple genes in individual cells within bulk populations is critical for the development of effective targeted therapies that prevent tumor relapse. To overcome this challenge, we propose to adapt novel nanomolecular scaffolds (termed DNA origami) to transfect tumor cells and capture mRNA encoding known-tumor suppressor genes. We propose to test the specificity of these scaffolds in breast tumor cell lines and primary breast tumor with known mutations in p53, PTEN, and PIK3CA genes. This approach will allow the rapid quantitation of genomic diversity and evolutionary order in cells from solid tumors for improved targeting of rare malignant subclones.

Project Summary Next generation sequencing platforms have revolutionized modern approaches for understanding a wide variety of biological processes, including immune responses and cancer. However, the diversity of the cells involved in these processes has important implications for understanding biologic outcomes. For instance, the diversity of T cell receptors on lymphocytes during responses to virus or cancer can have dramatic effects on disease progression. Conversely, the diversity of cancer cells or virus populations has important implications for successful control of disease. Therefore, a critical hurdle in these situations is the ability to provide single-cell analysis techniques coupled with high-throughput next generation sequencing, to adequately measure the diversity of cells. Unfortunately, current single-cell analysis approaches are either unfeasible for large cell populations, too expensive, and/or require specialized equipment that is not available to most labs. To address this problem, we have engineered DNA origami nanostructures that are able to specifically bind and protect two different mRNA within transfected cells, and use novel molecular approaches facilitated by the constrained geometry of the mRNA bound to DNA origami to generate bi-cistronic amplicons for use in paired-end high-throughput next generation sequencing. Importantly, the mRNA from individual cells remain physically linked throughout this process, so linked sequences are from individual cells. In this proposal we develop this approach for quantitating the diversity of clonally-distributed TCR? and TCR? T cell receptors in lymphocyte populations. We have shown that we can transfect polyclonal populations of T cells with high efficiency, isolate DNA origami nanostructures with bound TCR mRNA from transfected T cells, and generate CDR3 amplicons for Illumina 2x250 paired-end deep sequencing reactions to obtain linked TCR? and TCR? sequence information from individual T cells without the need for single cell sorting. We propose to validate and use the developed DNA origami nanostructures to provide the first estimate of total TCR diversity in the naïve T cell repertoires of mice. This technology will be useful for downstream application to a wide variety of biologic processes, by relatively simple modifications to the DNA origami nanostructure probe sequences, including single-cell analysis of other diverse lymphocyte populations, including other T cell subsets or antibody producing B cells, as well as single cells analysis of heterogeneous tumors or diverse microbial communities. Moreover, because this approach utilizes equipment found in most modern molecular biology laboratories, it can be easily adopted by many researchers for these analyses.