S. H. van der Burg

Pre-clinical safety and efficacy of TA-CIN, a recombinant HPV16 L2E6E7 fusion protein vaccine, in homologous and heterologous prime-boost regimens

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical intraepithelial neoplasia (CIN) and cancer. A newly designed vaccine, comprising the HPV16 L2, E6 and E7 as a single fusion protein (TA-CIN), was shown to elicit HPV16-specific CTL, T-helper cells and antibodies in a pre-clinical mouse model. These immune responses effectively prevented outgrowth of HPV16-positive tumour cells in a prophylactic setting as well as in a minimal residual disease setting. CTL immunity was optimally induced when TA-CIN was employed in heterologous prime-boost regimens in combination with TA-HPV, a clinical grade vaccinia-based vaccine. These data provide a scientific basis for the use of TA-CIN, alone or in combination with TA-HPV in future human trials.

The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays

The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer (“CIMT”) in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the “CIMT monitoring panel”. A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8+ T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such “two-step” inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.

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.

Toward the harmonization of immune monitoring in clinical trials: Quo vadis?

A constantly increasing number of cancer immunotherapies are being investigated in clinical trials but no reliable biomarkers to predict clinical benefit currently exist. For some cancer types, biomarkers have proven to be meaningful predictors of patient outcomes (e.g., BCR-ABL in Chronic Myeloid Leukemia or PSA in prostate cancer) and were established as routine tools. As effects of cancer immunotherapy are mediated through the immune system, immune responses may act as natural biomarkers for clinical efficiency if the right factors can be reliably measured. Following this concept, cancer immunotherapy trials over the last decade have often included measures of tumor-specific cellular immune responses as endpoints to identify reliable surrogates for clinical benefit. Substantial efforts were invested throughout the immunotherapy field in setting up suitable cellular immune assays. However, to date, data from clinical trials do not consistently show that immune responses are correlated with clinical endpoints [1]. This lack of correlation is partially interpreted as a consequence of the high variability in assay results, due to the lack of assay standardization, validation and harmonization across laboratories. If harmonization of immune assays can be achieved, assays can be tested as surrogate endpoints in clinical trials, substantially accelerate the development of immunotherapeutic agents and, in addition, offer a rationale to pre-select groups of patients with high probability to benefit from subsequent immunotherapy. A plethora of different immunological assays to monitor antigen-specific T cell responses are available, and some are used by a majority of laboratories. Prime examples are the ELISPOT assay, intracellular cytokine staining (ICS), MHC-peptide multimer staining for detection of antigen-specific CD8+ and increasingly for CD4+ T cells and proliferation assays based on carboxyfluorescein succinimidyl ester (CFSE)-labelling. Additional functional assays that have been introduced more recently are based on CD154 up-regulation on activated antigen-specific CD4+ T cells or the detection of CD107a or cytotoxicity related molecules like perforin or granzyme in CD8+ T cells. For each of these assays, the use of different protocols that have evolved over time translates into a wide range of performances. Differences in the interpretation of the results obtained make comparisons of published data even more complex. This has led to lack of comparability as well as doubts about the integrity of the results that were obtained and published worldwide [2]. Recent developments clearly suggest that “proper” immunomonitoring will have to encompass the parallel use of several tests, which together assess the frequency, function and homing capacity of induced/stimulated T cells present in the circulation and preferentially also locally within the targeted tissue. In view of the influence of both natural and adaptive regulatory T cells on clinical outcome, immunomonitoring should also include screening for these cells. Only the combined use of several techniques may lead to a valid set of surrogate markers for specific immune interventions that allows clinical decisions or optimization of immunotherapeutic strategies. A related proposal was made by a community-wide consensus workshop on clinical trials with cancer vaccines, in which criteria for the use of immune assays were recommended including at least two validated assays to be used in parallel [3]. There is also a surprising disparity between the substantial financial and logistic efforts invested in reagents and man hours to develop immune monitoring techniques and the comparatively minor investment made to date in actually validating and standardizing the available techniques within and between laboratories. This has to be critically re-evaluated as efforts put in the adequate harmonization of assays will support the investments already made. They are also a pre-requisite to meet regulatory requirements on assay standards prior to them being considered as surrogate endpoints for clinical trials. This affects academic institutions and industry partners alike and may reach beyond cancer into the fields of autoimmune and infectious diseases.

Systemic and local human papillomavirus 16‐specific T‐cell immunity in patients with head and neck cancer

Squamous cell carcinomas of the head and neck (HNSCC), in particular those of the oropharynx, can be caused by human papilloma virus Type 16 (HPV16). Whereas these HPV‐induced oropharyngeal carcinomas may express the HPV16 E6 and E7 oncoproteins and are associated with better survival, the nonvirally induced HNSCC are associated with overexpression of p53. In this study we assessed the presence of systemic and local T cells reactive against these oncoproteins in HNSCC. An exploratory study on the presence, type and function of HPV16‐ and/or p53‐specific T cells in the blood, tumor and/or metastatic lymph node as measured by several immune assays was performed in an unselected group of 50 patients with HNSCC. Tumor tissue was tested for HPV DNA and the overexpression of p53 protein. Almost all HPV16+ tumors were located in the oropharynx. Circulating HPV16‐ and p53‐specific T cells were found in 17/47 and 7/45 tested patients. T cells were isolated from tumor cultures and/or lymph nodes of 20 patients. HPV16‐specific T cells were detected in six of eight HPV+ tumors, but in none of the 12 HPV‐tumors. Tumor‐infiltrating p53‐specific T cells were not detected. In depth analysis of the HPV16‐specific T‐cell response revealed that this response comprised a broad repertoire of CD4+ T‐helper Type 1 and 2 cells, CD4+ regulatory T cells and CD8+ T cells reactive to HPV16. The local presence of HPV16‐specific T‐cell immunity in HPV16‐induced HNSCC implicates a role in the antitumor response and support the development of immunotherapy for HNSCC.

Minimal information about T cell assays: the process of reaching the community of T cell immunologists in cancer and beyond

Many assays to evaluate the nature, breadth, and quality of antigen-specific T cell responses are currently applied in human medicine. In most cases, assay-related protocols are developed on an individual laboratory basis, resulting in a large number of different protocols being applied worldwide. Together with the inherent complexity of cellular assays, this leads to unnecessary limitations in the ability to compare results generated across institutions. Over the past few years a number of critical assay parameters have been identified which influence test performance irrespective of protocol, material, and reagents used. Describing these critical factors as an integral part of any published report will both facilitate the comparison of data generated across institutions and lead to improvements in the assays themselves. To this end, the Minimal Information About T Cell Assays (MIATA) project was initiated. The objective of MIATA is to achieve a broad consensus on which T cell assay parameters should be reported in scientific publications and to propose a mechanism for reporting these in a systematic manner. To add maximum value for the scientific community, a step-wise, open, and field-spanning approach has been taken to achieve technical precision, user-friendliness, adequate incorporation of concerns, and high acceptance among peers. Here, we describe the past, present, and future perspectives of the MIATA project. We suggest that the approach taken can be generically applied to projects in which a broad consensus has to be reached among scientists working in fragmented fields, such as immunology. An additional objective of this undertaking is to engage the broader scientific community to comment on MIATA and to become an active participant in the project.

Tumor-infiltrating CD14-positive myeloid cells and CD8-positive T-cells prolong survival in patients with cervical carcinoma.

One of the hallmarks of cancer is the influx of myeloid cells. In our study, we investigated the constitution of tumor‐infiltrating myeloid cells and their relationship to other tumor‐infiltrating immune cells, tumor characteristics and the disease‐specific survival of patients with cervical cancer (CxCa). Triple‐color immunofluorescence confocal microscopy was used to locate, identify and quantify macrophages (CD14), their maturation status (CD33) and their polarization (CD163) in a cohort of 86 patients with cervical carcinoma. Quantification of the numbers of myeloid cells revealed that a strong intraepithelial infiltration of CD14+ cells, and more specifically the population of CD14+CD33−CD163− matured M1 macrophages, is associated with a large influx of intraepithelial T lymphocytes (p = 0.008), improved disease‐specific survival (p = 0.007) and forms an independent prognostic factor for survival (p = 0.033). The intraepithelial CD8+ T‐cell and regulatory T‐cell (Treg) ratio also forms an independent prognostic factor (p = 0.010) and combination of these two factors reveals a further increased benefit in survival for patients whose tumor displays a dense infiltration with intraepithelial matured M1 macrophages and a high CD8 T‐cell/Treg ratio, indicating that both populations of immune cells simultaneously improve survival. Subsequently, we made a heatmap including all known immune parameters for these patients, whereby we were able to identify different immune signatures in CxCa. These results indicate that reinforcement and activation of the intratumoral M1 macrophages may form an attractive immunotherapeutic option in CxCa.

A phase I trial combining carboplatin/doxorubicin with tocilizumab, an anti-IL-6R monoclonal antibody, and interferon-α2b in patients with recurrent epithelial ovarian cancer

BACKGROUND The immune system is important in epithelial ovarian cancer (EOC). Interleukin-6 is associated with chemoresistance and an immune-suppressive tumor microenvironment. We investigated whether a combination of chemotherapeutics, blockade of interleukin 6 (IL-6) receptor (IL-6R; tocilizumab), and immune enhancer interferon-α (Peg-Intron) is feasible, safe, and able to enhance immunity in patients with recurrent EOC. PATIENTS AND METHODS In this dose-escalation study, patients received tocilizumab 1, 2, 4, or 8 mg/kg i.v., q4 weeks during the first three cycles of carboplatin (AUC5) plus doxorubicin [pegylated liposomal doxorubicin (PLD) 30 mg/m(2) or doxorubicin 50 mg/m(2) i.v., day 1, q4 weeks, for six cycles]. At the highest tocilizumab dose (8 mg/kg), Peg-Intron (1 µg/kg s.c.) was added. Peripheral blood mononuclear cells were collected for immunomonitoring at baseline, after three and six cycles. Dose-limiting toxicity (DLT), CA-125, and radiologic response were evaluated. RESULTS In the 23 patients enrolled, no DLT was established. The most frequent grade 3/4 adverse events (CTCAE v4.03) were neutropenia (23%), febrile neutropenia (19%), and ileus (19%). No treatment-related deaths occurred. Using CT evaluation, 11 of 21 assessable patients responded, 6 had stable disease and 3 progressive disease. Patients receiving highest dose tocilizumab showed a functional blockade of IL-6R with increased levels of serum IL-6 (P = 0.02) and soluble IL-6R (P = 0.008). Consequently, immune cells displayed decreased levels of pSTAT3, myeloid cells produced more IL-12 and IL-1β while T cells were more activated and secreted higher amounts of effector cytokines interferon-γ and tumor necrosis factor-α. An increase in sIL-6R was potentially associated with a survival benefit (P = 0.03). CONCLUSIONS Functional IL-6R blocking is feasible and safe in EOC patients treated with carboplatin/(pegylated liposomal)doxorubicin, using 8 mg/kg tocilizumab. This combination is recommended for phase II evaluation based on immune parameters. CLINICAL TRIAL REGISTER NCT01637532.

Induction of a primary human cytotoxic T-lymphocyte response against a novel conserved epitope in a functional sequence of HIV-1 reverse transcriptase

Objective: To identify novel major histocompatibility complex (MHC) class I‐restricted cytotoxic T‐lymphocyte (CTL) epitopes conserved in HIV‐1. Methods: Potential conserved CTL epitopes were selected using a predictive computer algorithm based on a human leukocyte antigen (HLA)‐A*0201 peptide‐binding motif and tested for actual binding to the human processing defective cell line 174.CEM T2 (T2). Hence, the amino‐acid sequences of 14 full‐length sequenced HIV‐1 strains were analysed. An in vitro primary peptide‐specific human CTL response was induced with responding lymphocytes of an HIV‐1‐seronegative donor. Responding T cells were cloned by limiting dilution and tested for their ability to recognize naturally processed antigen in a 51Cr‐release assay using recombinant vaccinia‐HIV protein‐infected B‐lymphoblastoid cells (B‐LCL) as target cells. Results: The analysis of peptides bearing the HLA‐A*0201 motif for conservation resulted in one peptide of Env, three of Gag and 12 of Pol. Only Gag340‐348, Pol83‐92, Pol267‐277 and Pol960‐968 showed binding properties to T2 comparable with those of known CTL epitopes Gag76‐84 SLYNTVATL and Pol468‐476 ILKEPVHGV. A successful primary MHC class I‐restricted CTL response was induced against Pol468‐476 and Pol267‐277 VLDVGDAYFSV, a peptide in a functional sequence of reverse transcriptase (RT). The resulting CD8+ CTL clones were peptide‐specific and able to specifically lyse recombinant vaccinia‐HIV‐1 RT‐infected HLA‐A*0201‐matched B‐LCL. Conclusion: The method used to screen proteins sequences for potential CTL epitopes, test selected peptides for binding to MHC class I and induction of an in vitro primary response against optimal binding peptides resulted in the identification of at least one novel conserved CTL epitope. The novel epitope is located in an area crucial for RT activity. This study demonstrates the feasibility of identifying highly conserved HIV‐1‐derived peptides capable of eliciting novel anti‐HIV‐1 CTL responses.

Performance of serum-supplemented and serum-free media in IFNγ Elispot Assays for human T cells

The choice of serum for supplementation of media for T cell assays and in particular, Elispot has been a major challenge for assay performance, standardization, optimization, and reproducibility. The Assay Working Group of the Cancer Vaccine Consortium (CVC-CRI) has recently identified the choice of serum to be the leading cause for variability and suboptimal performance in large international Elispot proficiency panels. Therefore, a serum task force was initiated to compare the performance of commercially available serum-free media to laboratories’ own medium/serum combinations. The objective of this project was to investigate whether a serum-free medium exists that performs as well as lab-own serum/media combinations with regard to antigen-specific responses and background reactivity in Elispot. In this way, a straightforward solution could be provided to address the serum challenge. Eleven laboratories tested peripheral blood mononuclear cells (PBMC) from four donors for their reactivity against two peptide pools, following their own Standard Operating Procedure (SOP). Each laboratory performed five simultaneous experiments with the same SOP, the only difference between the experiments was the medium used. The five media were lab-own serum-supplemented medium, AIM-V, CTL, Optmizer, and X-Vivo. The serum task force results demonstrate compellingly that serum-free media perform as well as qualified medium/serum combinations, independent of the applied SOP. Recovery and viability of cells are largely unaffected by serum-free conditions even after overnight resting. Furthermore, one serum-free medium was identified that appears to enhance antigen-specific IFNγ-secretion.