Metastatic colorectal cancer cells from patients previously treated with chemotherapy are sensitive to T-cell killing mediated by CEA/CD3-bispecific T-cell-engaging BiTE antibody.

BACKGROUND: Novel technologies to redirect T-cell killing against cancer cells are emerging. We hypothesised that metastatic human colorectal cancer (CRC) previously treated with conventional chemotherapy would be sensitive to T-cell killing mediated by carcinoembryonic antigen (CEA)/CD3-bispecific T-cell-engaging BiTE antibody (MEDI-565). METHODS: We analysed proliferation and lysis of CEA-positive (CEA+) CRC specimens that had survived previous systemic chemotherapy and biologic therapy to determine whether they could be killed by patient T cells engaged by MEDI-565 in vitro. RESULTS: At low concentrations (0.1-1 ng ml(-1)), MEDI-565+ T cells caused reduced proliferation and enhanced apoptosis of CEA+ human CRC specimens. High levels of soluble CEA did not impair killing by redirected T cells and there was no increase in resistance to T-cell killing despite multiple rounds of exposure. CONCLUSIONS: This study shows for the first time that metastatic CRC specimens derived from patients previously treated with conventional chemotherapy can be lysed by patient T cells. Clinical testing of cancer immunotherapies, such as MEDI-565 that result in exposure of tumours to large numbers of T cells, is warranted.

Optimization of vaccine responses with an E1, E2b and E3-deleted Ad5 vector circumvents pre-existing anti-vector immunity.

Recombinant serotype 5 adenovirus (Ad5) vectors lacking E1 expression induce robust immune responses against encoded transgenes in pre-clinical models, but have muted responses in human trials because of widespread pre-existing anti-adenovirus immunity. Attempts to circumvent Ad5-specific immunity by using alternative serotypes or modifying capsid components have not yielded profound clinical improvement. To address this issue, we explored a novel alternative strategy, specifically reducing the expression of structural Ad5 genes by creating E1 and E2b deleted recombinant Ad5 vectors. Our data show that [E1-, E2b-]vectors retaining the Ad5 serotype are potent immunogens in pre-clinical models despite the presence of significant Ad5-specific immunity, in contrast to [E1-] vectors. These pre-clinical studies with E1 and E2b-deleted recombinant Ad5 vectors suggest that anti-Ad immunity will no longer be a limiting factor, and that clinical trials to evaluate their performance are warranted.

Surrogate markers of response to cancer immunotherapy.

Clinically effective cancer immunotherapy has been sought for more than 100 years and has been recently applied most successfully in strategies that passively deliver immune effectors such as monoclonal antibodies (anti-CD20 for lymphoma and anti-HER2/neu for breast cancer), donor lymphocyte infusions in chronic myelongenous leukemia and non-myeloablative allogeneic peripheral blood progenitor transplants for renal cell carcinoma. There is mounting enthusiasm for strategies employing active stimulation of antitumour immune responses. These include vaccines based on tumour antigen proteins and peptides, autologous, allogeneic or gene-modified tumour cells, dendritic cells and antigen-encoding viral vector constructs. Indeed, randomised Phase III clinical trials of autologous tumour cell vaccines for colorectal cancer demonstrated an improvement in disease free survival and a trend toward improved overall survival [1]. Despite these preliminary successes, it is clear that the many strategies under development cannot all be evaluated for survival benefit in large clinical trials that require many years, patients and resources to complete. This highlights the need to develop intermediate markers to help prioritise which agents to test in prospective randomised Phase III trials.

Quantitating therapeutically relevant T-cell responses to cancer vaccines.

Successful application of active immunotherapy to the treatment of cancer will require stimulation of potent antigen-specific T-cell responses. It is not known how numerous or how potent these T cells must be in order to abrogate tumors, but the levels of immunity needed to control chronic viral infections may provide estimates for comparison. Evaluation of the efficacy of a vaccine strategy in attaining these levels of immunity will depend on the use of assays that create a picture of T-cell number and function that correlates with clinical outcomes. We discuss the currently available in vivo and in vitro T-cell assays and their relevance for detecting therapeutic levels of T-cell activity. We also propose a strategy for efficiently evaluating the immunologic efficacy of cancer vaccines so that the most promising candidates can be brought more rapidly into definitive clinical trials.

Assays for monitoring cellular immune responses to active immunotherapy of cancer.

Numerous cancer immunotherapy strategies are currently being tested in clinical trials. Although clinical efficacy will be the final test of these approaches, the long and complicated developmental pathway for these agents necessitates evaluating immunological responses as intermediate markers of the most likely candidates for success. This has emphasized the need for assays that accurately detect and quantitate T cell-mediated, antigen-specific immune responses. This review evaluates the currently used in vivo and in vitro methods of assessing T-cell number and function, including delayed-type hypersensitivity, tetramer analysis, ELISPOT, flow cytometry-based analysis of cytokine expression, and PCR-based detection of T-cell receptor gene usage or cytokine production. We provide examples of how each has been used to monitor recent clinical trials and a discussion of how well each correlates with clinical outcome.

Monitoring cellular immune responses to cancer immunotherapy.

Many clinical trials are testing the feasibility of stimulating the immune system to treat cancer. Although the efficacy of this approach will ultimately be determined by clinically relevant endpoints, detection of the magnitude and activity of the immune response is an important intermediate point in the development of these strategies. Assays that predict clinically relevant endpoints are particularly desirable for helping to determine which strategies should ultimately be tested in larger randomized clinical trials. In this review, we will discuss these cellular immunological assays and the current status of their role in clinical trials of immunotherapy.

Direct detection of cellular immune responses to cancer vaccines.

The evaluation of cancer immunotherapy is predicated on the hypothesis that markers of tumor antigen-specific T-cell immunity will cone-late with clinical efficacy. Establishing which candidate vaccines should enter large-scale clinical trials will necessitate optimal application of immunologic monitoring assays. Evidence suggests that available techniques are adequate for the direct detection of clinically significant antigen-specific T-cell responses from tissue specimens. To achieve this goal, it is important to have an understanding of individual methods and their limitations, to include appropriate control antigens in the monitoring strategy, and to incorporate statistical considerations into the design and analysis of such studies.

A subset of human monocyte-derived dendritic cells expresses high levels of interleukin-12 in response to combined CD40 ligand and interferon-gamma treatment.

Dendritic cells (DCs) may arise from multiple lineages and progress through a series of intermediate stages until fully mature, at which time they are capable of optimal antigen presentation and T-cell activation. High cell surface expression of CD83 is presumed to correlate with full maturation of DCs, and a number of agents have been shown to increase CD83 expression on DCs. We hypothesized that interleukin 12 (IL-12) expression would be a more accurate marker of functionally mature DCs capable of activating antigen-specific T cells. We used combinations of signaling through CD40, using CD40 ligand trimer (CD40L), and interferon gamma to demonstrate that CD83 expression is necessary but not sufficient for optimal production of IL-12 by DCs. Phenotypically mature DCs could be induced to produce high levels of IL-12 p70 only when provided 2 simultaneous stimulatory signals. By intracellular cytokine detection, we determined that only a subset of cells that express high levels of CD80 and CD83 generate large amounts of IL-12. DCs matured with both signals are superior to DCs stimulated with the individual agents in activating antigen-specific T cell in vitro. These findings have important implications regarding the identification, characterization, and clinical application of functionally mature DCs.

Quantitation of T-cell receptor frequencies by competitive polymerase chain reaction: dynamics of T-cell clonotype frequencies in an expanding tumor-infiltrating lymphocyte culture.

The use of T-cell receptor (TCR) genes as markers for antigen-reactive T cells is dependent on the ability of the TCR genes to rapidly identify antigen-reactive T-cell clonotypes in patient samples. We recently reported a competitive reverse-transcriptase polymerase chain reaction (cRT-PCR) method that can measure the frequency of individual TCRBV subfamilies and clonotypes in mixed lymphocyte populations more accurately than other semiquantitative PCR assays. However, it is impractical to measure changes in the absolute frequency of each TCRBV subfamily to identify those T cells with increasing frequency after antigen stimulation in vivo or in vitro. Therefore, we have modified our cRT-PCR method to more rapidly identify expanding T-cell populations by combining all of the TCRBV subfamily-specific competitors into a single sample to determine the relative abundance of each TCRBV subfamily. Using an expanding TIL 620 culture, we identified four TCRBV (BV2, BV12, BV17, and BV23) subfamilies that expanded over a 23-day period. These subfamilies accounted for 23% of the T cells in the day 35 culture and increased to 57%, 92%, and 80% of the days 44, 51, and 58 cultures respectively. Analysis of DNA sequences demonstrated that the observed expansion was caused primarily by a single clonotype within each subfamily. T cells expressing BV17 and BV23 recognized gp100 and MART-1 respectively. Therefore, this cRT-PCR method can detect expanding T-cell populations based solely on their TCRBV subfamily expression. Furthermore, T-cell expansion in a mixed TIL population was a good predictor of antigen reactivity.

Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity.

The tumor-associated-Ag MART-1 is expressed by most human melanomas. The genes encoding an alphabeta TCR from a MART-1-specific, HLA-A2-restricted, human T cell clone have been efficiently transferred and expressed in human PBL. These retrovirally transduced PBL cultures were MART-1 peptide reactive, and most cultures recognized HLA-A2+ melanoma lines. Limiting dilution clones were generated from three bulk transduced PBL cultures to investigate the function of individual clones within the transduced cultures. Twenty-nine of 29 CD8+ clones specifically secreted IFN-gamma in response to T2 cells pulsed with MART-1(27-35) peptide, and 23 of 29 specifically secreted IFN-gamma in response to HLA-A2+ melanoma lines. Additionally, 23 of 29 CD8+ clones lysed T2 cells pulsed with the MART-1(27-35) peptide and 15 of 29 lysed the HLA-A2+ melanoma line 888. CD4+ clones specifically secreted IFN-gamma in response to T2 cells pulsed with the MART-1(27-35) peptide. TCR gene transfer to patient PBL can produce CTL with anti-tumor reactivity in vitro and could potentially offer a treatment for patients with metastatic melanoma. This approach could also be applied to the treatment of other tumors and viral infections. Additionally, TCR gene transfer offers unique opportunities to study the fate of adoptively transferred T cells in vivo.

Quantitation of T-cell receptor frequencies by competitive PCR: generation and evaluation of novel TCR subfamily and clone specific competitors.

T cell receptor (TCR) V gene usage has been used to characterize the immune response to bacteria, viruses, allografts, self antigens, tumor antigens, and superantigens. Sensitive methods to detect changes in the frequency of TCR subfamilies or clonotypes might be useful in evaluating the efficacy of vaccines against infectious agents, immunotherapy treatments for cancer patients, or the status of autoimmune diseases. Two HLA-A2 restricted CTL clones expressing BV17 were isolated from a tumor infiltrating lymphocytes (TIL) culture of a patient with metastatic melanoma. One clone recognized the MART-1(27-35) peptide and the other clone recognized the gp100(209-217) peptide. The frequency of each of these CTL clones in an expanding TIL culture was measured using a novel competitive RT-PCR (cRT-PCR) strategy. cRT-PCR uses a single primer pair to amplify template cDNA simultaneously with a modified DNA competitor molecule. A rapid two-step PCR technique followed by a single cloning step was used to generate a TCR BV17 subfamily specific competitor or competitors specific for the MART-1(27-35) reactive CTL clone (CO-41) and the gp100(209-217) reactive CTL clone (CO-4). Each competitor contained a segment of the TCR BC region that served as an internal reference standard. Using the BV17 competitor we were able to accurately and reproducibly measure cDNA templates at a frequency as low as 1/100,000 using cDNA samples of known TCRBV subfamily composition. This competitor was used to monitor the frequency of BV17 expressing T cells in the TIL and PMBC of a patient with metastatic melanoma. We determined that the frequency of BV17 expressing T cells increased from 4.5% of the culture on day 35 to 60.7% of the culture on day 58. Expansion of the BV17 subfamily was due predominantly to the expansion of the CO-4 clone. This method can be used to meaningfully quantify the precursor frequency of T cell mRNA in prepared samples via TCR subfamily or TCR sequence specific primers.

Potential use of T cell receptor genes to modify hematopoietic stem cells for the gene therapy of cancer.

The purpose of this review is to illustrate some of the technical and biological hurdles that need to be addressed when developing new gene therapy based clinical trials. Gene transfer approaches can be used to "mark" cells to monitor their persistence in vivo in patients, to protect cells from toxic chemotherapeutic agents, correct a genetic defect within the target cell, or to confer a novel function on the target cell. Selection of the most suitable vector for gene transfer depends upon a number of factors such as the target cell itself and whether gene expression needs to be sustained or transient. The TCR gene transfer approach described here represents one innovative strategy being pursued as a potential therapy for metastatic melanoma. Tumor reactive T cells can be isolated from the tumor infiltrating lymphocytes (TIL) of melanoma patients. A retroviral vector has been constructed containing the T cell receptor (TCR) alpha and beta chain genes from a MART-1-specific T cell clone (TIL 5). Jurkat cells transduced with this virus specifically release cytokine in response to MART-1 peptide pulsed T2 cells, showing that the virus can mediate expression of a functional TCR. HLA-A2 transgenic mice are being used to examine whether transduced bone marrow progenitor cells will differentiate in vivo into mature CD8+ T cells expressing the MART-1-specific TCR. Expression of the human TCR alpha and beta chain genes has been detected by RT-PCR in the peripheral blood of HLA-A2 transgenic mice reconstituted with transduced mouse bone marrow. Expression of the TIL 5 TCR genes in the peripheral blood of these mice was maintained for greater than 40 weeks after bone marrow reconstitution. TIL 5 TCR gene expression was also maintained following transfer of bone marrow from mice previously reconstituted with transduced bone marrow to secondary mouse recipients, suggesting that a pluripotent progenitor or lymphocyte progenitor cell has been transduced.

Changes in the fine specificity of gp100(209-217)-reactive T cells in patients following vaccination with a peptide modified at an HLA-A2.1 anchor residue.

In a recent clinical trial, HLA-A2+ melanoma patients were vaccinated with a peptide derived from the melanoma Ag gp100, which had been modified at the second position (g9-209 2M) to enhance MHC binding affinity. Vaccination led to a significant increase in lymphocyte precursors in 10 of 11 patients but did not result in objective cancer responses. We observed that some postvaccination PBMC cultures were less reactive with tumor cells than they were with g9-209 peptide-pulsed T2 cells. In contrast, g9-209-reactive tumor-infiltrating lymphocyte cultures generally reacted equally with tumor cells and g9-209 peptide-pulsed T2 cells. To investigate this difference in T cell reactivity, T cell cloids derived from the PBMC of three patients vaccinated with g9-209 2M were compared with T cell cloids isolated from g9-209-reactive TIL cultures. All of the T cell cloids obtained from TIL reacted with HLA-A2+, gp100+ melanoma cell lines as well as with g9-209 and g9-209 2M peptide-pulsed targets. In contrast, only 3 of 20 PBMC-derived T cell cloids reacted with melanoma cell lines in addition to g9-209 and to g9-209 2M peptide-pulsed targets. Twelve of twenty PBMC-derived cloids reacted with g9-209 and g9-209 2M peptide-pulsed targets but not with melanoma cell lines. And 5 of 20 PBMC-derived cloids recognized only the g9-209 2M-modified peptide-pulsed targets. These results suggest that immunizing patients with the modified peptide affected the T cell repertoire by expanding an array of T cells with different fine specificities, only some of which recognized melanoma cells.

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 form 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 alpha/beta rearrangements. Furthermore, there were no conserved amino acids in the CDR3 regions of these TcRs, indicating that multiple TcR clonotypes 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.

Identification of epitope mimics recognized by CTL reactive to the melanoma/melanocyte-derived peptide MART-1(27-35).

CTL reactivity to the epitope MART-1(27-35), of the melanoma (self) antigen MART-1/melan A is frequently observed in tumor-infiltrating lymphocytes and may be readily elicited from the peripheral blood of melanoma patients that express HLA-A*0201. Available data suggest that these observations contrast with those made for other HLA-A*0201-presented melanoma self antigens regarding the regularity of observed CTL responses. Based on preliminary findings, we hypothesized that the CTL response to MART-1 might be augmented in part by T cell encounters with peptides derived from sources other than MART-1, which show sequence similarity to MART-1(27-35). To test this idea, a protein database search for potential MART-1 epitope mimics was done using criteria developed from analyses of effector recognition of singly-substituted peptide analogues of MART-1(27-35). Synthetic peptides were made for a portion of the sequences retrieved; 12/40 peptides tested were able to sensitize target cells for lysis by one or more anti-MART-1 effectors. The peptides recognized correspond to sequences occurring in a variety of proteins of viral, bacterial, and human (self) origin. One peptide derives from glycoprotein C of the common pathogen HSV-1; cells infected with recombinant vaccinia virus encoding native glycoprotein C were lysed by anti-MART-1 effectors. Our results overall indicate that sequences conforming to the A2.1 binding motif and possessing features essential to recognition by anti-MART-1 CTL occur frequently in proteins. These findings further suggest that T cells might encounter a variety of such sequences in vivo, and that epitope mimicry may play a role in modulating the CTL response to MART-1(27-35).

Combined PCR-heteroduplex and PCR-SSCP analysis for matching of HLA-A, -B and -C allotypes in marrow transplantation.

We describe a method for rapid matching of HLA-A, -B and -C allotypes using simultaneous polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and heteroduplex analysis. Electrophoresis is performed at ambient temperature without requirements for buffer cooling. SSCP and heteroduplexes are revealed as discrete spatially separated band clusters. Using HLA-A, -B and -C locus-specific PCR primers, matching for alleles at these loci can be performed in 5 h. We tested 17 serologically matched patient-unrelated donor pairs and found considerable microheterogeneity at the DNA level. We propose that this technology has several advantages over conventional low-resolution typing methods and represents a potentially valuable screening method in unrelated donor selection.

HLA-DQB1 and DQA1 matching by ambient temperature PCR-SSCP.

We have developed a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) protocol for rapid matching of DQA1 and DQB1 alleles. Electrophoresis can be performed at ambient temperature within the range 18-28 degrees C without continuous gel cooling. The method has been tested on 27 patient-potential bone marrow donor pairs for DQB1 and 31 pairs for DQA1. Bone marrow pairs were chosen to represent a broad range of common alleles based upon previous restriction fragment length polymorphism (RFLP) analysis type assignments. Samples were re-typed by PCR with sequence-specific primers (PCR-SSP) and the results compared to matching by PCR-SSCP analysis. There was a 100% correlation between PCR-SSP and PCR-SSCP analysis for DQB1, and a 97% correlation for DQA1 matching.