Michael H. Kershaw

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.

Redirecting migration of T cells to chemokine secreted from tumors by genetic modification with CXCR2.

T-cell-based immunotherapies provide a promising means of cancer treatment although durable antitumor responses are infrequent. A potential reason for these shortcomings may lie in the observed lack of trafficking of specific T cells to tumor. Our increasing knowledge of the process of trafficking involving adhesion molecules and chemokines affords us the opportunity to intervene and correct deficiencies in this process. Chemokines can be expressed by a range of tumors and may serve as suitable targets for directing specific T cells toward tumor. We initially sought to identify which chemokines were produced by a range of human tumor cell lines, and which chemokines and chemokine receptors were expressed by cultured T cells. We identified two chemokines: Growth-Regulated Oncogene-alpha (Gro-alpha; CXCL1) and Regulated on Activation Normal T Cell-Expressed and Secreted (RANTES; CCL5), to be secreted by several human tumor cell lines. Expression was also detected in fine-needle aspirates of melanoma from patients. In addition, we determined the expression of several chemokine receptors on cultured human T cells including CCR1, CCR2, CCR4, CCR5, CXCR3, and CXCR4. Cultured, activated human T cells expressed the chemokines lymphotactin (XCL1), RANTES, macrophage inflammatory protein-1 alpha (MIP-1 alpha; CCL3) and MIP-1 beta (CCL4), but no appreciable Gro-alpha. In a strategy to direct T cells toward chemokines expressed by tumors we chose Gro-alpha as the target chemokine because it was produced by tumor and not by T cells themselves. However, T cells did not express the receptor for Gro-alpha, CXCR2, and therefore, T cells were transduced with a retroviral vector encoding CXCR2. Calcium ion mobilization, an important first step in chemokine receptor signaling, was subsequently demonstrated in transduced T cells in response to Gro-alpha. In addition, Gro-alpha was chemotactic for T cells expressing CXCR2 in vitro toward both recombinant protein and tumor-derived chemokine. Interestingly we demonstrate, for the first time, that Gro-alpha was able to induce interferon-gamma (IFN-gamma) secretion from transduced T cells, thereby extending our knowledge of other potential functions of CXCR2. This study demonstrates the feasibility of redirecting the migration properties of T cells toward chemokines secreted by tumors.

Dual-specific T cells combine proliferation and antitumor activity

An effective immune response against cancer requires the activation and expansion of specific T cells. Tumor antigens, however, are generally poor immunogens. To achieve expansion of tumor-reactive T cells in vivo, we used a strategy of generating dual-specific T cells that could respond to a powerful immunogen while also possessing tumor reactivity. We generated dual-specific T cells by genetic modification of alloreactive T cells with a chimeric receptor recognizing folate-binding protein, an ovarian cancer–associated antigen. Mouse dual-specific T cells responded in vitro to both allogeneic antigen and tumor cells expressing folate-binding protein, and expanded in number in vivo in response to immunization with allogeneic cells. Most importantly, the combination of dual-specific T cells and immunization had an antitumor effect in vivo. We also generated human dual-specific T cells and characterized the dual-specific nature of individual clones. Assigning the tasks of expansion and tumor reactivity to different receptors within the same lymphocyte may help to overcome the problem of poor immunogenicity of tumor antigens.

Recognition of human colon cancer by T cells transduced with a chimeric receptor gene.

Transduction with chimeric T-cell receptor genes can be used to redirect primary T lymphocytes to recognize specific antigens (Ags), including ovarian and breast cancer Ags. To extend this approach to colon cancer we report here redirection of T cells using a chimeric receptor recognizing the colon cancer-associated Ag EGP40. Chimeric T cell receptors were constructed by ligating single-chain genes of either of two EGP40-specific monoclonal antibodies (CO17.1A, GA733) to the Fc receptor γ-signaling chain. Retroviral vectors incorporating these constructs were used to transduce a murine T-cell line and human peripheral blood lymphocytes. These modified T cells were analyzed for specific recognition of colon cancer lines by measuring cytokine release and lytic activity against tumor targets. Murine lymphocytes transduced with the chimeric receptor based on GA733, but not CO17.1A, released cytokine specifically in response to EGP40-expressing colon cancer cell lines. Recognition of colon cancer targets by murine lymphocytes was blocked by the addition of GA733 antibody or soluble EGP40 Ag, confirming that colon cancer recognition is based on specific chimeric receptor-Ag interaction. Human lymphocytes transduced with chimeric GA733 specifically recognized colon carcinoma cells in cytokine release assays and lysed EGP40-expressing tumor cells. Genetic modification of T cells can be used to redirect T cells against EGP40-expressing tumor cells. The expression of chimeric GA733 in the autologous lymphocytes of patients may provide a source of tumor-reactive cells with therapeutic application against colon cancer.

Generation of gene-modified T cells reactive against the angiogenic kinase insert domain-containing receptor (KDR) found on tumor vasculature.

The destruction of newly forming tumor vasculature is a promising approach to inhibit tumor growth. The goal of the present study was to investigate whether human lymphocytes gene modified to express a chimeric receptor specific for the angiogenic endothelial cell receptor, KDR, could react against KDR(+) cells. Gene-modified lymphocytes specifically lysed KDR(+) cells and secreted cytokines in response to KDR(+) target cells including human umbilical vein endothelial cells (HUVECs). Anti-KDR lymphocytes induced HUVECs to secrete the chemokine interleukin 8 and upregulate the adhesion molecules VCAM and E-selectin, which may be important in the recruitment of further immune effector cells to tumor. These KDR-specific lymphocytes may be useful in the adoptive immunotherapy of a broad range of cancers by inducing immune-mediated destruction of tumor neovasculature.

MHC class I and class II presentation of tumor antigen in retrovirally and adenovirally transduced dendritic cells

The unique antigen-presenting capabilities of dendritic cells (DCs) make them an attractive means with which to initiate an antitumor immune response. Using DCs transduced with tumor antigens for immunotherapy has several theoretical advantages over peptide-pulsed DCs including the possibility that transduced DCs are capable of presenting epitopes on both class I and class II MHC molecules. To test this theory, we inserted the human tumor antigen gp100 into mouse DCs transgenic for HLA-DRβ1*0401 using either adenoviral vector or a VSV-G pseudotyped retroviral vector. DCs transduced with tumor antigen were able to be recognized by both a murine CD8+ T-cell clone and a murine CD4+ T-cell line in a cytokine release assay, thereby demonstrating presentation of both MHC class I and class II gp100 epitopes. This study describes the simultaneous presentation of a tumor-associated antigen to both CD4+ and CD8+ T cells and lends support to the use of gene-modified DCs as a means to initiate both CD4+ and CD8+ antitumor responses.

Increased functional expression of transgene in primary human lymphocytes using retroviral vectors modified with IRES and splicing motifs

Genetic modification of human lymphocytes is being employed in strategies to correct enzyme deficiencies, encode cytokines and to redirect lymphocytes to antigenic targets other than those encoded by their endogenous T cell receptor. However, expression of transgenes in primary lymphocytes is generally low. Reasoning that vector modification may lead to increased transgene expression and subsequent increases in function, we have performed two retroviral vector modifications and report their effect on the functional expression in primary lymphocytes. A chimeric receptor specific for the colon carcinoma-associated antigen, EGP40, was initially incorporated into the retroviral vector LXSN. In this vector, receptor expression is driven by the Moloney murine leukemia virus LTR, and neomycin phosphotransferase expression driven by the SV40 promoter. Replacement of SV40 with an internal ribosomal entry site (IRES) increased the transgene activity of a mouse T cell line and human PBL as judged by increased cytokine release in response to antigen positive target cells. A further increase in transgene function was generated by the additional incorporation of a splice acceptor motif into the construct. Human PBL transduced with vector incorporating both IRES and intron were consistently more effective at lysing antigen positive colorectal carcinoma cells.