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Enrichment Of Rare Circulating Antigen-Specific Effector T Cells By Mch/Peptide Tetramer Immunomagnetic Sorting And Hyperexpansion For Therapy Of Breast Cancer
Career Catalyst Research
BACKGROUND: Many studies of immunotherapy for breast cancer have focused on vaccines for the prevention of tumor recurrence or treatment of early stage disease. Tumor antigens that have been examined include; MUC-1, NY-ESO-1, HER-2/neu, CEA, and the Mage family. By contrast, for melanoma adoptive transfer of in vitro activated tumor infiltrating lymphocytes (TIL) has demonstrated efficacy against bulky metastatic disease. One potential problem with TIL therapy for breast cancer is that the yield of TIL from a breast tumor sample is low and amplification of TIL in vitro has not been optimized. Moreover, TIL are a mixture of effector cells and various types of suppressor T cells such as CD4+CD25+ Treg cells, Gr1+ immature myeloid cells or CD8+ suppressor T cells. In preliminary experiments, we have confirmed that small starting numbers of antigen-specific effector T lymphocytes can be purified from circulating PBMC of tumor-bearing hosts by depletion of CD62L-high cells with MACS microbeads. Importantly, the purified and amplified CD62L-low cells mediate regression of established metastatic tumors following adoptive transfer. By contrast, adoptive transfer of purified and amplified CD62L-high cells is ineffective, even at high doses. FVB-neuN transgenic mice are highly suited to this proposal because they develop spontaneous breast cancer, overexpress HER-2/neu and have immune tolerance to HER-2/neu.
HYPOTHESIS: We will test the hypothesis that antigen-specific CD62L-low/CD8+ effector T lymphocytes are present in circulation of hosts with transplantable or spontaneous tumors arising in transgenic FVB-neuN mice. We will determine whether tumor-antigen-specific T cells can be further isolated by MHC/peptide tetramer immunomagnetic sorting. We anticipate that the initial yield of such cells will be low therefore they will be subjected to in vitro stimulation with anti-CD3 mAb and IL-2/IL-7 to achieve hyperexpansion. It is possible that antigen-specific CD8+ T cells could have either effector or suppressor functions. This will be analyzed by adoptive transfer to secondary hosts bearing transplantable or spontaneous neu+ tumors. The rationale for this approach is that spontaneous breast carcinomas in FVB-neuN transgenic mice may undergo immunoediting and may induce tumor antigen-specific T cells with immunosuppressive functions such as CD4+CD25+ regulatory T cells or CD8+ suppressor T cells.
SPECIFIC AIMS: 1) To determine whether circulating T cells of hosts bearing transplantable or spontaneous breast tumors in FVB-neuN mice consist of a mixture of antigen-specific CD8+ effector and CD8+ suppressor T cells subsets. 2) To enrich antigen-specific CD8+ T cells through MHC/peptides tetramer immunomagnetic sorting following short-term in vitro cultures, establish cell lines, and test their therapeutic activity in vivo. 3) To analyze differences in the gene expression between antigen-specific CD62L-low/CD8+ effector and CD62L-high/CD8+ suppressor cell lines which are predictive of therapeutic efficacy or suppressive activity.
STUDY DESIGN: 1) Circulating T cells will be obtained from FVB-neu tolerant hosts bearing transplantable tumors or from FVB-neuN mice with spontaneous tumors. The CD62L-low and CD62L-high subsets will be separated then culture-activated in vitro. CD62L-low, CD62L-high, or their combination will be adoptively transferred to recipients bearing subcutaneous tumors and tumor size will be monitored; 2) H-2Dq tetramers bound to different neu-specific peptides will be synthesized. CD62L-low/CD8+ effector and CD62L-high/CD8+ suppressor subsets will be further separated by immunomagnetic sorting with different neu-specific MHC/peptide tetramers then different cell lines will be established and hyperexpanded. The therapeutic efficiency or suppressive activity will be determined for each cell line. 3) The profile of induced gene expression following neu+ tumor cell stimulation will be determined in CD62L-low/CD8+ effector or CD62L-high/CD8+ suppressor cell lines. The mechanisms by which CD62L-high/CD8+ cells inhibit CD62L-low/CD8+ cells in vivo when co-transferred will be investigated by quantitatively measuring trafficking and proliferation of adoptively transferred T cells labeled with fluorescent dyes.
RELEVANCE: One of the key issues in adoptive immunotherapy of cancer is how to enrich antigen-specific effector T cells from a reliable and convenient source and then achieve robust amplification to produce a sufficient yield of effector T cells for adoptive immunotherapy. This proposal will investigate this approach and simultaneously identify mouse neu-specific effective peptides, which may have human homologues. In addition, we will identify genes that are differentially expressed in CD62L-low/CD8+ or CD62L-high/CD8+ subsets. Genes that are specifically up-regulated in the CD62L-low or CD62L-high T cell lines will be hypothesized to involve in effector or suppressor functions. These studies would help predict therapeutic or suppressive efficacy that might be translatable to human cancer immunotherapy protocols.
Breast cancer is a significant cause of cancer-related mortality with 40,000 deaths attributed to this disease in US each year. We propose to conduct pre-clinical studies of T cell-mediated immunotherapy of breast cancer in a relevant mouse model. Many types of tumor contain tumor-infiltrating lymphocytes (TIL) and clinical studies at the National Cancer Institute demonstrated that TIL therapy produced objective responses in approximately 50% of patients with metastatic melanoma refractory to other therapeutic modalities. Therefore, the transfer of ex vivo activated antigen-specific effector T cells represents an attractive modality in the treatment of cancer. However, adoptive immunotherapy with TIL has not been developed for breast cancer in part because these tumors typically contain low numbers of tumor-specific T cells. Moreover, it has been difficult to achieve sufficient proliferation of effector T cells to generate the large numbers required for therapy. At present, one key issue for adoptive immunotherapy is the optimal source of tumor specific effector T cells. In this proposal, we will test the hypothesis that tumor antigen-specific effector T cells can be enriched from peripheral blood. The easy accessibility of peripheral blood and the possibility to obtain large numbers of T cells through apheresis are promising aspects of this approach. Antigen-specific enrichment of effector T cells from the total population of lymphocytes will potentially eliminate CD4+CD25+ Treg cells and antigen-specific or non-specific CD8+ suppressor cells, which have been identified in a wide spectrum of human cancer patients.
In preliminary experiments, we have explored whether we can segregate circulating T cells based on differential expression of L-selectin (CD62L), which is expressed at low levels on tumor-reactive effector T cells vs. high levels on T cells with suppressor activity. Moreover, we have applied methods of in vitro activation of effector T cells from blood with CD3/IL-2/IL-7 to amplify the number of such cells by greater than 100,000-fold. Adoptive transfer of CD62L-low/CD8+ cells eradicated established pulmonary metastases. By contrast, adoptive transfer of much higher numbers of CD62L-high/CD8+ cells had no therapeutic effect. We hypothesize that MHC/peptide tetramer immunomagnetic sorting will allow us to enrich antigen-specific T cells to an even greater purity compared with L-selectin sorting, prior to long-term culture activation. We will synthesize approximately 50 fluorescent MHC class I/peptide tetramers identified as potential CD8+ epitopes derived from the neu oncoprotein. CD62L-low/CD8+ effector and CD62L-high/CD8+ suppressor subsets isolated from peripheral blood of hosts with transplantable or spontaneous neu+ breast tumors will be sorted using different fluorescent MHC/peptide tetramers following short-term culture activation. Positively-selected cells will be further expanded to establish multiple cell lines reactive against specific MHC/peptide tetramers. We will directly test the function of derived T cell lines by in vivo by adoptive transfer of each cell line to hosts bearing transplantable neu+ tumors. In addition, we will analyze their activity against spontaneous tumors in neu+ transgenic FVB/N mice. If some cell lines display suppressor activity the gene expression pattern will be compared with that of effector T cell lines.
Circulating T cells are an important and convenient source for the patients with breast cancer to enrich antigen-specific effector T cells for adoptive immunotherapy. The therapeutic efficacy of T cell lines enriched with neu-specific MHC/peptide tetramers and hyperexpanded with anti-CD3/IL-2/IL-7 will be directly analyzed in vivo to investigate whether they can retain specific anti-tumor activity. Moreover, identification of neu-specific effector peptides may guide the design of a human vaccine for active immunotherapy of breast cancer. In addition, in this proposal, we will analyze whether a subset or the antigen-specific T cells that are isolated from transplantable or spontaneous breast carcinomas in HER-2/neu transgenic mice have suppressor function in addition to effector T cells. It is anticipated that the spontaneous breast cancer model with neu+ overexpression might mimic aspects of poor prognosis breast cancer in humans and provide leads toward development of T cell adoptive immunotherapy of breast cancer.