Sylvia Janetzki

Improved Endpoints for Cancer Immunotherapy Trials

Unlike chemotherapy, which acts directly on the tumor, cancer immunotherapies exert their effects on the immune system and demonstrate new kinetics that involve building a cellular immune response, followed by changes in tumor burden or patient survival. Thus, adequate design and evaluation of some immunotherapy clinical trials require a new development paradigm that includes reconsideration of established endpoints. Between 2004 and 2009, several initiatives facilitated by the Cancer Immunotherapy Consortium of the Cancer Research Institute and partner organizations systematically evaluated an immunotherapy-focused clinical development paradigm and created the principles for redefining trial endpoints. On this basis, a body of clinical and laboratory data was generated that supports three novel endpoint recommendations. First, cellular immune response assays generate highly variable results. Assay harmonization in multicenter trials may minimize variability and help to establish cellular immune response as a reproducible biomarker, thus allowing investigation of its relationship with clinical outcomes. Second, immunotherapy may induce novel patterns of antitumor response not captured by Response Evaluation Criteria in Solid Tumors or World Health Organization criteria. New immune-related response criteria were defined to more comprehensively capture all response patterns. Third, delayed separation of Kaplan–Meier curves in randomized immunotherapy trials can affect results. Altered statistical models describing hazard ratios as a function of time and recognizing differences before and after separation of curves may allow improved planning of phase III trials. These recommendations may improve our tools for cancer immunotherapy trials and may offer a more realistic and useful model for clinical investigation.

A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays.

Vaccines in general and HIV vaccines in particular are focusing ever more on the induction of cellular immunity specifically the generation of cytotoxic T cells (CTL). As progress is made towards developing a safe and effective HIV vaccine, there is a need for a robust, sensitive and reproducible assay to evaluate vaccine-induced cellular immunogenicity in Phase II/III trials. The enzyme-linked immunospot (ELISPOT) assay fits these criteria and is a technology that is readily transferable and amenable to high-through-put screening. There is a need for reagents that can be used as positive controls and for optimizing and standardizing the assay. We selected a panel of 23 8-11 mer Influenza virus (Flu), Cytomegalovirus (CMV) and Epstein Barr virus (EBV) epitopes recognized by CD8 positive T cells and presented by 11 class I HLA-A and HLA-B alleles whose cumulative frequencies represent >100% of Caucasian individuals. We examined interferon-gamma (IFN-gamma) secretion in peripheral blood mononuclear cells (PBMC) incubated with the peptides using a modified ELISPOT assay. IFN-gamma secretion was detected in 15/17 (88%) HIV-1 seronegative and 14/20 (70%) HIV-1 seropositive individuals. Release of IFN-gamma in response to the pool of peptides was CD8+ T cell mediated and HLA restricted. In vitro stimulation of PBMC with individual peptides or the pool of peptides led to the expansion of T cells capable of killing target cells expressing the appropriate viral antigen in a CTL assay. The size, shape and appearance of the spots produced using this peptide panel provided a standard for the establishment of acceptance criteria of spots for the evaluation of ELISPOT plates using an automated reader system.

IMMUNIZATION OF CANCER PATIENTS WITH AUTOLOGOUS CANCER-DERIVED HEAT SHOCK PROTEIN gp96 PREPARATIONS: A PILOT STUDY

Heat shock protein (HSP)–peptide complexes isolated from murine cancers elicit protective immunity and T lymphocytes specific for the cancer from which the HSPs are isolated. A pilot study was designed to test the feasibility, immunogenicity and toxicity of such treatment in cancer patients. Sixteen patients with assorted advanced malignancies, which had become refractory to established therapies, were recruited. The gp96 vaccine was prepared for each patient from tumor obtained from that patient. Anti‐tumor immune responses were evaluated using Elispot assays of T cells in peripheral blood after minimal in vitro stimulation. No unacceptable vaccine‐related toxicities or auto‐immune reactions were observed. Immunization with autologous gp96 elicited MHC I–restricted, tumor‐specific CD8+ T lymphocytes in 6/12 patients immunized. In addition, expansion of the NK cell population was seen in 8/13 of patients immunized. These observations are entirely consistent with the murine experience and form a firm basis for future trials with clinical end points, using autologous, patient‐specific HSP–peptide vaccines. Int. J. Cancer 88:232–238, 2000.

Heat shock protein vaccines against cancer

Vaccination of mice with heat shock proteins (HSPs) derived from a tumor makes the mice resistant to the tumor from which the HSP was obtained. This phenomenon has been demonstrated with three HSPs--gp96, hsp90, and hsp70. Vaccination with HSPs also elicits antigen-specific cytotoxic T lymphocytes (CTLs). The specific immunogenicity of HSPs derives apparently, not from the HSPs per se, but from the peptides bound to them. These observations provide the basis for a new generation of vaccines against cancer. The HSP-based cancer vaccines circumvent two of the most intractable hurdles to cancer immunotherapy. One of them is the possibility that human cancers, like cancers of experimental animals, are antigenically distinct. The prospect of identification of immunogenic antigens of individual cancers from patients is daunting to the extent of being impractical. The observation that HSPs chaperone antigenic peptides of the cells from which they are derived circumvents this extraordinary hurdle. Second, most current approaches to cancer immunotherapy focus on determining the CTL-recognized epitopes of cancer cell lines. This approach requires the availability of cell lines and CTLs against cancers. These reagents are unavailable for an overwhelming proportion of human cancers. In contrast, the HSP-based vaccines do not depend on the availability of cell lines or CTLs nor do they require definition of the antigenic epitopes of cancer cells. These advantages, among others, make HSPs attractive and novel immunogens against cancer.

Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI)

The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels.

“MIATA”—Minimal Information about T Cell Assays

Immunotherapy, especially therapeutic vaccination, has a great deal of potential in the treatment of cancer and certain infectious diseases such as HIV (Allison et al., 2006; Fauci et al., 2008; Feldmann and Steinman, 2005). Numerous vaccine candidates have been tested in patients with a variety of tumor types and chronic viral diseases. Often, the best way to assess the clinical potential of these vaccines is to monitor the induced T cell response, and yet there are currently no standards for reporting these results. This letter is an effort to address this problem.

Response definition criteria for ELISPOT assays revisited

No consensus has been reached on how to determine if an immune response has been detected based on raw data from an ELISPOT assay. The goal of this paper is to enable investigators to understand and readily implement currently available methods for response determination. We describe empirical and statistical approaches, identifying the strengths and limitations of each approach to allow readers to rationally select and apply a scientifically sound method appropriate to their specific laboratory setting. Five representative approaches were applied to data sets from the CIMT Immunoguiding Program and the response detection and false positive rates were compared. Simulation studies were also performed to compare empirical and statistical approaches. Based on these, we recommend the use of a non-parametric statistical test. Further, we recommend that six medium control wells or four wells each for both medium control and experimental conditions be performed to increase the sensitivity in detecting a response, that replicates with large variation in spot counts be filtered out, and that positive responses arising from experimental spot counts below the estimated limit of detection be interpreted with caution. Moreover, a web-based user interface was developed to allow easy access to the recommended statistical methods. This interface allows the user to upload data from an ELISPOT assay and obtain an output file of the binary responses.

Evaluation of CD8(+) T-cell frequencies by the Elispot assay in healthy individuals and in patients with metastatic melanoma immunized with tyrosinase peptide.

The lack of reproducible, quantitative assays for T‐cell responses has been a limitation in the development of cancer vaccines to elicit T‐cell immunity. We utilized the Elispot assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubations. CD8+ T‐cell reactivity was determined with an interferon (IFN)‐γElispot assay detecting T cells at the single cell level that secrete IFN‐γ. We studied both healthy individuals and patients with melanoma. Healthy HLA‐A*0201‐positive individuals showed a similar mean frequency of CD8+ cells recognizing a tyrosinase peptide, YMDGTMSQV, when compared with melanoma patients prior to immunization. The frequencies of CD8+ cells recognizing the tyrosinase peptide remained relatively constant over time in healthy individuals. Nine HLA‐A*0201‐positive patients with stage IV metastatic melanoma were immunized intradermally with the tyrosinase peptide together with the immune adjuvant QS‐21 in a peptide dose escalation study with 3 patients per dose group. Two patients demonstrated a significant increase in the frequency of CD8+ cells recognizing the tyrosinase peptide during the course of immunization, from approx. 1/16,000 CD8+ T cells to approx. 1/4,000 in the first patient and from approx. 1/14,000 to approx. 1/2,000 in the second patient. These results demonstrate that modest expansion of peptide‐specific CD8+ T cells can be generated in vivo by immunization with peptide plus QS‐21 in at least a subset of patients with melanoma. Int. J. Cancer 87:391–398, 2000.

Recommendations from the iSBTc-SITC/FDA/NCI Workshop on Immunotherapy Biomarkers

Purpose: To facilitate development of innovative immunotherapy approaches, especially for treatment concepts exploiting the potential benefits of personalized therapy, there is a need to develop and validate tools to identify patients who can benefit from immunotherapy. Despite substantial effort, we do not yet know which parameters of antitumor immunity to measure and which assays are optimal for those measurements. Experimental Design: The iSBTc-SITC (International Society for Biological Therapy of Cancer-Society for Immunotherapy of Cancer), FDA (Food and Drug Administration), and NCI (National Cancer Institute) partnered to address these issues for immunotherapy of cancer. Here, we review the major challenges, give examples of approaches and solutions, and present our recommendations. Results and Conclusions: Although specific immune parameters and assays are not yet validated, we recommend following standardized (accurate, precise, and reproducible) protocols and use of functional assays for the primary immunologic readouts of a trial; consideration of central laboratories for immune monitoring of large, multi-institutional trials; and standardized testing of several phenotypic and functional potential potency assays specific to any cellular product. When reporting results, the full QA (quality assessment)/QC (quality control) should be conducted and selected examples of truly representative raw data and assay performance characteristics should be included. Finally, to promote broader analysis of multiple aspects of immunity, and gather data on variability, we recommend that in addition to cells and serum, RNA and DNA samples be banked (under standardized conditions) for later testing. We also recommend that sufficient blood be drawn to allow for planned testing of the primary hypothesis being addressed in the trial, and that additional baseline and posttreatment blood is banked for testing novel hypotheses (or generating new hypotheses) that arise in the field. Clin Cancer Res; 17(10); 3064–76. ©2011 AACR.

Harmonization of Immune Biomarker Assays for Clinical Studies

Harmonizing immune assay use in clinical trials could reduce data variability and support immune biomarker development. Assays that measure a patient’s immune response play an increasingly important role in the development of immunotherapies. The inherent complexity of these assays and independent protocol development between laboratories result in high data variability and poor reproducibility. Quality control through harmonization—based on integration of laboratory-specific protocols with standard operating procedures and assay performance benchmarks—is one way to overcome these limitations. Harmonization guidelines can be widely implemented to address assay performance variables. This process enables objective interpretation and comparison of data across clinical trial sites and also facilitates the identification of relevant immune biomarkers, guiding the development of new therapies.