Joachim Kopp

Vaccination with autologous non‐irradiated dendritic cells in patients with bcr/abl+ chronic myeloid leukaemia

In chronic myeloid leukaemia (CML), dendritic cells (DC) and leukaemic cells share a common progeny, leading to constitutive expression of putative tumour antigens, such as bcr/abl, in DC. In this phase‐I/II study, autologous DC were used as a vaccine in patients with chronic phase bcr/abl+ CML, who had not achieved an adequate cytogenetic response after treatment with α‐interferon or imatinib. Ten patients were enrolled, DC were generated from peripheral blood monocytes and vaccination consisted of four subcutaneous injections of increasing numbers of DC (1–50 × 106 cells per injection) on days 1, 2, 8 and 21. Vaccination was feasible and safe. Improvement of the cytogenetic/molecular response, as detected by fluorescence in situ hybridization of peripheral blood mononuclear cells (PBMC), was possibly related to vaccination in four of 10 patients. In three of these patients, T cells recognizing leukaemia‐associated antigens became detectable. The proliferative capacity of PBMC in response to autologous DC increased after vaccination in all evaluable patients. We conclude that vaccination with autologous, non‐irradiated ‘leukaemic’ DC is feasible, safe and induces anti‐leukaemic T‐cell responses in some CML patients. DC vaccination might be useful in CML as postremission therapy, i.e. after treatment with tyrosine kinase inhibitors.

Cryopreservation of mature monocyte-derived human dendritic cells for vaccination: influence on phenotype and functional properties.

Abstract. In the past decade there has been increasing evidence that tumor antigen-loaded dendritic cells (DC) are able to elicit anti-tumor T-cell responses. Initial clinical data for different tumor entities are encouraging, with objective tumor regressions being observed in some patients. Since GMP production of DC for clinical vaccination protocols is a time- and cost-intensive procedure, cryopreservation of DC in aliquots ready for clinical use would significantly facilitate DC-based vaccination in the clinic. We asked whether freezing and thawing alters the phenotype or functional properties of DC. DC from healthy volunteers and from patients with chronic myeloid leukemia (CML) were analyzed after freezing and thawing for their viability, morphology, immunophenotype (FACS profile), T-cell stimulatory capacity (mixed lymphocyte reaction) and mobility (time-lapse cinemicroscopy). Our results demonstrate that cryopreservation does not cause significant changes in the phenotype or function of DC, neither in DC from healthy volunteers nor in those from CML patients. Our data indicate that cryopreserved aliquots of DC are suitable for clinical application in DC-based immunotherapy protocols.

Influence of CD80, Interleukin-2, and Interleukin-7 Expression in Human Renal Cell Carcinoma on the Expansion, Function, and Survival of Tumor-Specific CTLs

Purpose: A renal cell carcinoma (RCC) line, RCC-26, has been identified as a suitable candidate for development of an allogeneic tumor cell vaccine based on its expression of a variety of tumor-associated antigens (TAA). To improve immunogenicity, RCC-26 cells were genetically engineered to express CD80 alone or in combination with interleukin (IL)-2 or IL-7. The effect of these modifications on proliferation, function, and survival of autologous and allogeneic tumor-specific CTLs was assessed. Experimental Design: RCC-26 sublines expressing different transgenes were tested for their capacity to reactivate cytokine secretion and cytotoxicity in autologous tumor-infiltrating lymphocytes, to improve proliferation and survival of tumor-associated T cells present in autologous peripheral blood, and to induce tumor-associated responses in naive allogeneic lymphocytes. The expression of several common TAA was quantitated in the RCC-26 sublines using reverse transcription-PCR to identify surrogate markers for immune monitoring in clinical trials. Results: Gene-modified RCC-26 cells showed enhanced immunogenicity. CD80 expression was necessary to induce RCC-associated CTL in blood of healthy allogeneic donors. It also improved proliferation of autologous effector-memory T cells. Further enhancement was achieved with IL-2 through induction of the antiapoptosis protein Bcl-xL. The candidate vaccine lines overexpressed several common TAA that are suitable markers for immune monitoring. Conclusions: RCC-26 cells coexpressing CD80 and cytokine transgenes display improved immunogenic characteristics, supporting their use as allogeneic tumor cell vaccines for HLA-A2-matched patients with metastatic RCC.

Phase 1 Trial of Allogeneic Gene-Modified Tumor Cell Vaccine RCC-26/CD80/IL-2 in Patients with Metastatic Renal Cell Carcinoma

Preclinical studies showed that the allogeneic tumor cell line RCC-26 displayed natural immunogenic potential that was enhanced through expression of CD80 costimulatory molecules and secretion of interleukin-2. Here we report the study of RCC-26/CD80/IL-2 cells in a phase 1 vaccine trial of renal cell carcinoma patients with metastatic disease (mRCC). Fifteen patients of the HLA-A*0201 allotype, with at least one metastatic lesion, were included. Irradiated vaccine cells were applied in increasing doses of 2.5, 10, and 40 x 10(6) cells over 22 weeks. Primary study parameters included safety and toxicity. Sequential blood samples were analyzed by interferon-gamma enzyme-linked immunospot assays to detect tumor antigen-associated (TAA) effector cells. The vaccine was well tolerated and the designated vaccination course was completed in 9 of 15 patients. Neither vaccine-induced autoimmunity nor systemic side effects were observed. Delayed-type hypersensitivity skin reactions were detected in 11 of 12 evaluated patients and were particularly strong in patients with prolonged survival. In parallel, vaccine-induced immune responses against vaccine or overexpressed TAA were detected in 9 of 12 evaluated patients. No tumor regressions occurred according to RECIST (Response Evaluation Criteria in Solid Tumors) criteria; however, median time to progression was 5.3 months and median survival was 15.6 months, indicating substantial disease stabilization. We conclude that vaccine use was safe and feasible in mRCC. Clinical benefits were limited in these patients with advanced disease; however, immune monitoring revealed vaccine-induced responses against multiple TAAs in the majority of study participants. These results suggest that this vaccine could be useful in combination therapies and/or minimal residual disease.

High immune response rates and decreased frequencies of regulatory T cells in metastatic renal cell carcinoma patients after tumor cell vaccination.

Our previously reported phase I clinical trial with the allogeneic gene-modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (TH1/TH2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies.

Allogeneic gene-modified tumor cells (RCC-26/IL-7/CD80) as a vaccine in patients with metastatic renal cell cancer: a clinical phase-I study

Despite novel targeted agents, prognosis of metastatic renal cell cancer (RCC) remains poor, and experimental therapeutic strategies are warranted. Transfection of tumor cells with co-stimulatory molecules and/or cytokines is able to increase immunogenicity. Therefore, in our clinical study, 10 human leukocyte antigen (HLA)-A*0201+ patients with histologically-confirmed progressive metastatic clear cell RCC were immunized repetitively over 22 weeks with 2.5–40 × 106 interleukin (IL)-7/CD80 cotransfected allogeneic HLA-A*0201+ tumor cells (RCC26/IL-7/CD80). Endpoints of the study were feasibility, safety, immunological and clinical responses. Vaccination was feasible and safe. In all, 50% of the patients showed stable disease throughout the study; the median time to progression was 18 weeks. However, vaccination with allogeneic RCC26/IL-7/CD80 tumor cells was not able to induce TH1-polarized immune responses. A TH2 cytokine profile with increasing amounts of antigen-specific IL-10 secretion was observed in most of the responding patients. Interferon-γ secretion by patient lymphocytes upon antigen-specific and non-specific stimulation was substantially impaired, both before and during vaccination, as compared with healthy controls. This is possibly due to profound tumor-induced immunosuppression, which may prevent induction of antitumor immune responses by the gene-modified vaccine. Vaccination in minimal residual disease with concurrent depletion of regulatory cells might be one strategy to overcome this limitation.

Allogeneic partially HLA-matched dendritic cells pulsed with autologous tumor cell lysate as a vaccine in metastatic renal cell cancer: A clinical phase I/II study

Multi-kinase inhibitors have been established for the treatment of advanced renal cell cancer, but long-term results are still disappointing and immunotherapeutic approaches remain an interesting experimental option particularly in patients with a low tumor burden. DC are crucial for antigen-specific MHC-restricted T cell immunity. Furthermore, allogeneic HLA-molecules pose a strong immunogenic signal and may help to induce tumor-specific T cell responses. In this phase I/II trial, 7 patients with histologically confirmed progressive metastatic RCC were immunized repetitively with 1 × 107 allogeneic partially HLA-matched DC pulsed with autologous tumor lysate following a schedule of 8 vaccinations over 20 weeks. Patients also received 3 Mio IE IL-2 s.c. once daily starting in week 4. Primary endpoints of the study were feasibility and safety. Secondary endpoints were immunological and clinical responses. Vaccination was feasible and safe with no severe toxicity being observed. No objective response could be documented. However, while all patients had documented progress at study entry, 29% of the patients showed SD throughout the study with a mean TTP of 24.6 weeks (range 5 to 96 weeks). In 3/7 patients, TH1-polarized immune responses against RCC-associated antigens were observed. In one patient showing a minimal clinical response and a TTP of 96 weeks, clonally proliferated T cells against yet undefined antigens were induced by the vaccine. Vaccination with tumor antigen loaded DC remains an interesting experimental approach, but should rather be applied in the situation of minimal residual disease after systemic therapy. Additional depletion of regulatory cells might be a promising strategy.

Gene expression profiling of peripheral blood mononuclear cells during treatment with a gene-modified allogeneic tumor cell vaccine in advanced renal cell cancer: tumor-induced immunosuppression and a possible role for NF-κB.

Tumor‐induced immunosuppression remains a major challenge for immunotherapy of cancer patients. To further elucidate why an allogeneic gene‐modified [interleukin‐7 (IL‐7)/CD80‐cotransfected] renal cell cancer (RCC) vaccine failed to induce clinically relevant TH‐1‐polarized immune responses, peripheral blood mononuclear cells from enrolled study patients were analyzed by gene expression profiling (GEP) both prior and after vaccination. At baseline before vaccination, a profound downregulation of gene signatures associated with antigen presentation, immune response/T cells, cytokines/chemokines and signaling/transcription factors was observed in RCC patients as compared to healthy controls. Vaccination led to a partial reversion of preexisting immunosuppression, however, GEP indicated that an appropriate TH‐1 polarization could not be achieved. Most interestingly, our results suggest that the nuclear factor‐kappa B signaling pathway might be involved in the impairment of immunological responsiveness and the observed TH‐2 deviation. In summary, our data suggest that GEP might be a powerful tool for the prediction of immunosuppression and the monitoring of immune responses within immunotherapy trials.

DC generation from peripheral blood mononuclear cells in patients with chronic myeloid leukemia: Influence of interferons on DC yield and functional properties.

ABSTRACT In Chronic Myeloid Leukemia (CML), standard treatment consists of modern tyrosine-kinase inhibitors (TKI). Nevertheless, there is evidence that immune responses against leukemia-associated antigens (LAA) may play an important role in disease control. Dendritic cell (DC)- based immunotherapy is able to induce T cell responses against LAA and might therefore pose an interesting therapeutic option in CML, especially in the setting of minimal residual disease (MRD). GMP production of DC for clinical vaccination remains a time- and cost- intensive procedure and standardized DC generation is warranted. We asked whether maturation-induction with IFN-γ and IFN-α has an influence on functional properties of DC derived from peripheral blood mononuclear cells (PBMC) in CML patients. Monocyte-derived DC from healthy donors and from patients with CML were analyzed after maturation-induction with our TNF-α-containing standard cytokine cocktail with or without addition of IFN-α and/or IFN-γ. Our results confirm that the addition of IFN-γ leads to enhanced IL-12 secretion in healthy donors. In contrast, in CML patients, IFN-γ was not able to increase IL-12 secretion, possibly due to a higher degree of cell adherence and lower cell yield during the cell culture. Our data suggest, that- in contrast to healthy donors-, additional interferons are not beneficial for maturation induction during large-scale DC production in patients with CML.