Linda L. Parker

A Phase I Study on Adoptive Immunotherapy Using Gene-Modified T Cells for Ovarian Cancer

Purpose: A phase I study was conducted to assess the safety of adoptive immunotherapy using gene-modified autologous T cells for the treatment of metastatic ovarian cancer. Experimental Design: T cells with reactivity against the ovarian cancer–associated antigen α-folate receptor (FR) were generated by genetic modification of autologous T cells with a chimeric gene incorporating an anti-FR single-chain antibody linked to the signaling domain of the Fc receptor γ chain. Patients were assigned to one of two cohorts in the study. Eight patients in cohort 1 received a dose escalation of T cells in combination with high-dose interleukin-2, and six patients in cohort 2 received dual-specific T cells (reactive with both FR and allogeneic cells) followed by immunization with allogeneic peripheral blood mononuclear cells. Results: Five patients in cohort 1 experienced some grade 3 to 4 treatment-related toxicity that was probably due to interleukin-2 administration, which could be managed using standard measures. Patients in cohort 2 experienced relatively mild side effects with grade 1 to 2 symptoms. No reduction in tumor burden was seen in any patient. Tracking 111In-labeled adoptively transferred T cells in cohort 1 revealed a lack of specific localization of T cells to tumor except in one patient where some signal was detected in a peritoneal deposit. PCR analysis showed that gene-modified T cells were present in the circulation in large numbers for the first 2 days after transfer, but these quickly declined to be barely detectable 1 month later in most patients. An inhibitory factor developed in the serum of three of six patients tested over the period of treatment, which significantly reduced the ability of gene-modified T cells to respond against FR+ tumor cells. Conclusions: Large numbers of gene-modified tumor-reactive T cells can be safely given to patients, but these cells do not persist in large numbers long term. Future studies need to employ strategies to extend T cell persistence. This report is the first to document the use of genetically redirected T cells for the treatment of ovarian cancer.

Natural variation of the expression of HLA and endogenous antigen modulates CTL recognition in an In vitro melanoma model

Increasing attention has been devoted to elucidating the mechanism of lost or decreased expression of MHC or melanoma‐associated antigens (MAAs), which may lead to tumor escape from immune recognition. Loss of expression of HLA class I or MAA has, as an undisputed consequence, loss of recognition by HLA class I–restricted cytotoxic T cells (CTLs). However, the relevance of down‐regulation remains in question in terms of frequency of occurrence. Moreover the functional significance of epitope down‐regulation, defining the relationship between MHC/epitope density and CTL interactions, is a matter of controversy, particularly with regard to whether the noted variability of expression of MHC/epitope occurs within a range likely to affect target recognition by CTLs. In this study, bulk metastatic melanoma cell lines originated from 25 HLA‐A*0201 patients were analyzed for expression of HLA‐A2 and MAAs. HLA‐A2 expression was heterogeneous and correlated with lysis by CTLs. Sensitivity to lysis was also independently affected by the amount of ligand available for binding at concentrations of 0.001 to 1 mM. Natural expression of MAA was variable, independent from the expression of HLA‐A*0201, and a significant co‐factor determining recognition of melanoma targets. Thus, the naturally occurring variation in the expression of MAA and/or HLA documented by our in vitro results modulates recognition of melanoma targets and may (i) partially explain CTL–target interactions in vitro and (ii) elucidate potential mechanisms for progressive escape of tumor cells from immune recognition in vivo. Int. J. Cancer: 80, 781–790 (1999).

Expansion and characterization of T cells transduced with a chimeric receptor against ovarian cancer.

Adoptive immunotherapy with genetically modified T lymphocytes is being utilized in clinical trials for the treatment of a broad range of diseases including cancer and HIV infection. To improve on these treatments, and to better understand their mechanisms of action, it is necessary to develop techniques to generate large numbers of cells and characterize the functional heterogeneity of the cells produced. In this study, patient peripheral blood lymphocytes were transduced with a chimeric antigen receptor (MOv-gamma) derived from a mouse monoclonal antibody against folate-binding protein, which is overexpressed on many ovarian cancers. Thus, irrespective of their original specificity, normal human T lymphocytes were redirected to react against ovarian cancer cells. Lymphocytes from five patients were transduced and grown to large numbers, with a median expansion of more than 7000-fold. When proliferation was inadequate, the cells were expanded by stimulation utilizing anti-CD3, IL-2, and irradiated allogeneic PBMCs. The cells maintained their functional ability to recognize ovarian cancer over several months. Cloning of transduced cells was undertaken to determine the level of gene expression and function of individual cells making up the bulk population. Transduced CD4(+) and CD8(+) cell clones were isolated from the bulk and demonstrated antitumor activity. These clones had a diverse repertoire with respect to secretion of cytokines, and individual clones maintained their cytokine profile on subsequent expansion. These studies establish the feasibility of consistently generating large numbers of gene-modified tumor-reactive lymphocytes, with a stable and diverse cytokine repertoire, that could be utilized for patient treatment.

Large-Scale Depletion of CD25+ Regulatory T Cells from Patient Leukapheresis Samples

The ability to selectively enrich or deplete T lymphocytes of specific phenotype and function holds significant promise for application in adoptive immunotherapy protocols. Although CD4+ T cells can have an impact on CD8+ T-cell effector function, memory, and maintenance, a subset of CD4+ T cells, CD25+ regulatory T cells (Treg), can regulate peripheral self tolerance and possess the ability to suppress antitumor responses. The authors report the ability to selectively deplete CD25+ Treg cells from patient leukapheresis samples using a clinical-grade, large-scale immunomagnetic system. Using leukapheresis samples containing up to 1.3 × 1010 white blood cells, efficient depletion of Treg cells was measured by flow cytometric analysis of CD25 expression and FOXP3 expression on post-depletion products. Remnant CD25+ cells could not be detected in CD25-depleted products after short-term culture in IL-2 or enriched following secondary immunomagnetic selection for CD25+ cells, confirming that efficient depletion had occurred. In parallel to efficient enrichment of CD25− cells, immunomagnetic selection resulted in the recovery of Treg cells, since CD25+ lymphocytes removed during depletion were primarily composed of CD4+ T cells that expressed high levels of FOXP3 and possessed suppressive activity against autologous TCR-stimulated CD4+ CD25− T cells in vitro. These results show that selective separation of functional CD25+ Treg cells from large-scale samples can be performed in large scale under clinical-grade conditions with sufficient selection, recovery, viability, ability to expand, and function for potential use in adoptive immunotherapy.

Treatment of Patients With Metastatic Cancer Using a Major Histocompatibility Complex Class II–Restricted T-Cell Receptor Targeting the Cancer Germline Antigen MAGE-A3

Purpose Adoptive transfer of genetically modified T cells is being explored as a treatment for patients with metastatic cancer. Most current strategies use genes that encode major histocompatibility complex (MHC) class I-restricted T-cell receptors (TCRs) or chimeric antigen receptors to genetically modify CD8+ T cells or bulk T cells for treatment. Here, we evaluated the safety and efficacy of an adoptive CD4+ T-cell therapy using an MHC class II-restricted, HLA-DPB1*0401-restricted TCR that recognized the cancer germline antigen, MAGE-A3 (melanoma-associated antigen-A3). Patients and Methods Patients received a lymphodepleting preparative regimen, followed by adoptive transfer of purified CD4+ T cells, retrovirally transduced with MAGE-A3 TCR plus systemic high-dose IL-2. A cell dose escalation was conducted, starting at 107 total cells and escalating at half-log increments to approximately 1011 cells. Nine patients were treated at the highest dose level (0.78 to 1.23 × 1011 cells). Results Seventeen patients were treated. During the cell dose-escalation phase, an objective complete response was observed in a patient with metastatic cervical cancer who received 2.7 × 109 cells (ongoing at ≥ 29 months). Among nine patients who were treated at the highest dose level, objective partial responses were observed in a patient with esophageal cancer (duration, 4 months), a patient with urothelial cancer (ongoing at ≥ 19 months), and a patient with osteosarcoma (duration, 4 months). Most patients experienced transient fevers and the expected hematologic toxicities from lymphodepletion pretreatment. Two patients experienced transient grade 3 and 4 transaminase elevations. There were no treatment-related deaths. Conclusion These results demonstrate the safety and efficacy of administering autologous CD4+ T cells that are genetically engineered to express an MHC class II-restricted antitumor TCR that targets MAGE-A3. This clinical trial extends the reach of TCR gene therapy for patients with metastatic cancer.

T CELL-RECEPTOR V GENE USE BY CD4+ MELANOMA-REACTIVE CLONAL AND OLIGOCLONAL T-CELL LINES

Tumor-reactive CD4+ T cells can be isolated and expanded from the peripheral blood and tumor lesions of patients with melanoma. In contrast to CD8+ T cells, little is known about the antigens recognized by these CD4+ T cells. As a consequence, little is known about the diversity of the T-cell receptor (TcR) use by melanoma-reactive CD4+ T cells. To address these questions, a panel of clonal or highly oligoclonal CD4+ T-cell lines was established from a patient with metastatic melanoma. A CD4+ tumor-infiltrating lymphocyte (TIL) line was established that was highly oligoclonal and recognized only autologous melanoma cells but not allogeneic melanomas, suggesting the expression of a mutated or uniquely expressed antigen by this melanoma. The antigen recognized by the CD4+ TILs could be presented by intact melanoma cells or by autologous Epstein-Barr virus (EBV) B cells pulsed with melanoma cell lysates. A panel of CD4+ clonal and highly oligoclonal T-cell lines was isolated from peripheral blood mononuclear cells (PBMC) from this patient; these were also reactive with autologous melanoma cells or tumor extracts pulsed on autologous EBV B cells. Despite their reactivity with the autologous melanoma, we found no evidence of restricted TcR V gene use, because all six T-cell lines recognized antigen via different TcR α/β rearrangements. Furthermore, there were no conserved amino acids in the CDR3 regions of these TcRs, indicating that multiple TcR clono-types could mediate recognition of a single unique major histocompatibility (MHC) complex class II restricted melanoma antigen or that multiple MHC class II restricted melanoma antigens are expressed by the melanoma.

Isolation and Characterization of an HLA-DPB1*04: 01-restricted MAGE-A3 T-Cell Receptor for Cancer Immunotherapy.

Long-term tumor regressions have been observed in patients following the adoptive transfer of autologous tumor-infiltrating lymphocytes or genetically modified T cells expressing MHC class I-restricted T-cell receptors (TCRs), but clinical trials have not evaluated responses to genetically modified T cells expressing antitumor MHC class II-restricted TCRs. As studies carried out in a murine tumor model system have demonstrated that the adoptive transfer of CD4+ T cells could lead to the regression of established tumors, we plan to test the hypothesis that CD4+ T cells can also induce tumor regressions in cancer patients. In this study, 2 MAGE-A3-specific TCRs were isolated from a regulatory T-cell clone (6F9) and an effector clone (R12C9), generated from the peripheral blood of 2 melanoma patients after MAGE-A3 vaccination. The results indicated that T cells transduced with 6F9 TCR mediated stronger effector functions than R12C9 TCR. The 6F9 TCR specifically recognized MAGE-A3 and the closely related MAGE-A6 gene product, but not other members of the MAGE-A family in the context of HLA-DPB1*04:01. To test the feasibility of a potential clinical trial using this TCR, a clinical-scale procedure was developed to obtain a large number of purified CD4+ T cells transduced with 6F9 TCR. Because HLA-DPB1*04:01 is present in ∼60% of the Caucasian population and MAGE-A3 is frequently expressed in a variety of cancer types, this TCR immunotherapy could potentially be applicable for a significant portion of cancer patients.

A Phase I study of an HLA-DPB1*0401-restricted T cell receptor targeting MAGE-A3 for patients with metastatic cancers

Meeting abstracts Adoptive transfer of genetically-modified T cells is being explored as a salvage treatment for patients with selected metastatic cancers. Most of the current strategies utilize MHC class I-restricted T cell receptor (TCR) or chimeric antigen receptor (CAR) technologies to

Abstract 3357: Isolation of tumor cell subpopulations using semi-automated tissue dissociation and magnetic cell separation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The analysis of tumor resident cell and stem cell populations requires proper methods for tissue dissociation and cell purification. Over the past few years, there has been a particular focus on the study of tumor infiltrating lymphocytes (TIL) and cancer stem cells (CSC). TIL can be isolated and characterized using conventional lymphocyte markers whereas CSC-specific cell surface markers are currently established (1). Two important markers, namely CD24 and CD44, have been shown to define CSC in various neoplasms such as breast cancer (2) or colon cancer (3). We have established methods for the enzymatic and mechanical dissociation of solid tumors and optimized them according to the specific needs of a given tissue or cell type. Solid tumors are built up of a mixture of cell types, which are interconnected to each other and surrounded by an extracellular matrix composed of a variety of proteins and polysaccharides. The major goal was to disrupt the extracellular matrix and cell adhesion components without harming the integrity of the cell membrane. This was achieved by a combination of varying enzyme mixes, mechanical forces, incubation periods, and temperatures. The automation of all mechanical steps (gentleMACS™ dissociator) led to reproducible and moreover user independent results with reduced overall processing times. Viable tumor infiltrating lymphocytes (TIL) as well as tumor cells were obtained from melanoma metastases in high numbers. The isolated TIL expanded significantly faster than those from manually dissociated tumors and showed functionality in vitro and in vivo. As for cancer stem cells, we present novel tools for the immunomagnetic isolation of CD44+ and CD44+/CD24- cells. Using magnetic cell sorting (MACS®), a subpopulation of human teratocarcinoma cells could be isolated to a purity of 92% based on their expression of CD44. Furthermore, a combined depletion and positive selection approach allowed for the enrichment of a CD44+/CD24- human CML subpopulation from a starting frequency of 34% to final purity of 95%. The separation of pure tumor cell populations will be necessary for their further characterization and also targeted drug screening approaches. The methods presented here are broadly applicable as they can be easily adapted to other cancer tissues and cell types and should help to optimize and standardize the future basic and clinical cancer research. 1. Trumpp, A. and Wiestler, O. D. (2008); Nature Clin. Prac. Oncology.; Vol. 5 No. 6 2. Al-Hajj, M. et al. (2003); Proc. Natl. Acad. Sci. USA; Vol. 100 No. 7 3. Du, L. et al. (2008); Clin. Cancer Res.; 6751 14 (21) Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3357.

Abstract CT003: A phase I study of an HLA-DPB1*0401-restricted T-cell receptor targeting MAGE-A3 for patients with metastatic cancer

Background: Adoptive transfer of genetically-modified T cells is being explored as a salvage treatment for patients with selected metastatic cancers. Most of the current strategies utilize MHC class I-restricted T cell receptor (TCR) or chimeric antigen receptor (CAR) technologies to genetically modify CD8+ T cells or bulk T cells for patient treatment. Evidence indicates that CD4+ T cells can induce tumor regression, similar to CD8+ T cells. To test this hypothesis, an HLA-DPB1*0401-restricted TCR recognizing MAGE-A3 was isolated from a patient9s peripheral blood after MAGE-A3 peptide vaccination. Because HLA-DPB1*0401 is present in ∼60% of the Caucasian population and MAGE-A3 is expressed in up to one third of tumor specimens from a variety of cancer types, this TCR immunotherapy could potentially be applicable for a significant portion of cancer patients. Trial Design: Eligible patients were HLA-DPB1*0401 positive with MAGE-A3 positive tumor specimens, and had not responded or had recurred following at least one standard first line therapy for their disease. Patients received a lymphodepleting preparative regimen, followed by adoptive transfer of purified CD4+ T cells transduced with the HLA-DPB1*0401-restricted MAGE-A3 TCR plus systemic high-dose interleukin-2 (IL-2). A cell dose-escalation was conducted, treating 1 patient at each cohort (0.01, 0.03, 0.1, up to 30 billion cells), followed by 6 patients at the highest dose level (up to 100 billion cells). Clinical trial information: NCT02111850. Results: Fourteen patients were treated in this phase I study, including the last 6 patients at the highest dose level (78∼100 billion cells). Objective partial responses (RECIST) were observed in a patient with metastatic cervical cancer (ongoing at 11+ months), esophageal cancer (duration 3 months) and urothelial cancer (ongoing at 4+ months). High levels of IL-6 were detected in all patients’ serum samples after adoptive cell transfer. One month after the treatment, TCR-transduced T cells persisted at high levels in the peripheral blood of 6 patients who received the highest dose level, compared to patients who receive lower dose levels (0.01 ∼ 30 billion cells) (p = 0.0082). These results demonstrate the safety of administering autologous CD4+ T cells genetically-engineered to express an MHC class II-restricted anti-tumor TCR targeting MAGE-A3 and presents evidence for efficacy. We have started the phase II clinical trial to study the efficacy of this TCR therapy in different types of metastatic cancer. This clinical trial extends the reach of TCR gene therapy to patients with metastatic cancer. To our knowledge, this clinical trial represents the first genetically-modified CD4+ T cell immunotherapy against metastatic cancer. Citation Format: Yong-Chen Lu, Linda L. Parker, Tangying Lu, Zhili Zheng, Xin Yao, Paul F. Robbins, Steven A. Feldman, Pierre van der Bruggen, Christopher A. Klebanoff, Christian S. Hinrichs, Stephanie L. Goff, Richard M. Sherry, Udai S. Kammula, James C. Yang, Steven A. Rosenberg. A phase I study of an HLA-DPB1*0401-restricted T-cell receptor targeting MAGE-A3 for patients with metastatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT003.