Cor H. J. Lamers

Immune responses to transgene and retroviral vector in patients treated with ex vivo–engineered T cells

Adoptive transfer of immune effector cells that are gene modified by retroviral transduction to express tumor-specific receptors constitutes an attractive approach to treat cancer. In patients with metastatic renal cell carcinoma, we performed a study with autologous T cells genetically retargeted with a chimeric antibody receptor (CAR) directed toward carbonic anhydrase IX (CAIX), an antigen highly expressed in renal cell carcinoma. In the majority of patients, we observed distinct humoral and/or cellular anti-CAIX-CAR T-cell immune responses in combination with a limited peripheral persistence of transferred CAIX-CAR T cells in the majority of patients. Humoral immune responses were anti-idiotypic in nature and neutralized CAIX-CAR-mediated T-cell function. Cellular anti-CAIX-CAR immune responses were directed to the complementarity-determining and framework regions of the CAR variable domains. In addition, 2 patients developed immunity directed against presumed retroviral vector epitopes. Here, we document the novel feature that therapeutic cells, which were ex vivo engineered by means of transduction with a minimal γ-retroviral vector, do express immunogenic vector-encoded epitopes, which might compromise persistence of these cells. These observations may constitute a critical concern for clinical ex vivo γ-retroviral gene transduction in general and CAR-retargeted T-cell therapy in particular, and underscore the need to attenuate the immunogenicity of both transgene and vector.

Circulating endothelial cells in oncology: pitfalls and promises

Adequate blood supply is a prerequisite in the pathogenesis of solid malignancies. As a result, depriving a tumour from its oxygen and nutrients, either by preventing the formation of new vessels, or by disrupting vessels already present in the tumour, appears to be an effective treatment modality in oncology. Given the mechanism by which these agents exert their anti-tumour activity together with the crucial role of tumour vasculature in the pathogenesis of tumours, there is a great need for markers properly reflecting its impact. Circulating endothelial cells (CEC), which are thought to derive from damaged vasculature, may be such a marker. Appropriate enumeration of these cells appears to be a technical challenge. Nevertheless, first studies using validated CEC assays have shown that CEC numbers in patients with advanced malignancies are elevated compared to healthy controls making CEC a potential tool for among other establishing prognosis and therapy-induced effects. In this review, we will address the possible clinical applications of CEC detection in oncology, as well as the pitfalls encountered in this process.

TCR gene transfer: MAGE-C2/HLA-A2 and MAGE-A3/HLA-DP4 epitopes as melanoma-specific immune targets

Adoptive therapy with TCR gene-engineered T cells provides an attractive and feasible treatment option for cancer patients. Further development of TCR gene therapy requires the implementation of T-cell target epitopes that prevent “on-target” reactivity towards healthy tissues and at the same time direct a clinically effective response towards tumor tissues. Candidate epitopes that meet these criteria are MAGE-C2336-344/HLA-A2 (MC2/A2) and MAGE-A3243-258/HLA-DP4 (MA3/DP4). We molecularly characterized TCRαβ genes of an MC2/A2-specific CD8 and MA3/DP4-specific CD4 T-cell clone derived from melanoma patients who responded clinically to MAGE vaccination. We identified MC2/A2 and MA3/DP4-specific TCR-Vα3/Vβ28 and TCR-Vα38/Vβ2 chains and validated these TCRs in vitro upon gene transfer into primary human T cells. The MC2 and MA3 TCR were surface-expressed and mediated CD8 T-cell functions towards melanoma cell lines and CD4 T-cell functions towards dendritic cells, respectively. We intend to start testing these MAGE-specific TCRs in phase I clinical trial.

Process validation and clinical evaluation of a protocol to generate gene-modified T lymphocytes for imunogene therapy for metastatic renal cell carcinoma: GMP-controlled transduction and expansion of patient's T lymphocytes using a carboxy anhydrase IX-specific scFv transgene.

BACKGROUND Adoptive transfer of autologous T cells that are gene-transduced to express Ag-specific receptors represents an experimental strategy to provide tumor-specific immunity to cancer patients. We studied this concept in patients with metastatic renal cell cancer (RCC) using retroviral transduction of T cells with a single-chain Ab-G250 chimeric receptor [scFv(G250)]. We describe the validation of our clinical protocol for gene transduction and expansion of human T lymphocytes. METHODS A batch of scFv(G250) transgene-containing retrovirus was produced under conditions of good manufacturing practice (GMP). In addition to quality control and safety testing of the virus batch, extensive potency testing was performed, i.e. assessment of its functional transduction efficiency in primary human T cells. Subsequently, the clinical gene transduction and cell-expansion protocol was subjected to a series of process validations and a clinical evaluation using T cells obtained from healthy donors and three RCC patients. RESULTS The clinical batch of scFv(G250) transgene-containing retrovirus met the quality and safety control criteria. Small-scale transductions yielded 62-92% scFv(G250)+ T cells and, at a clinical scale, 50-84% transduction efficiencies were obtained. Patient and healthy donor T cells showed similar expansion potencies, and also yielded similar levels of scFv(G250)-mediated immune functions, i.e. specific cytolysis of G250-ligand expressing RCC cells and production of IFN-gamma upon stimulation with such cells. All T cell cultures were free of replication competent retroviruses. DISCUSSION We have shown that the validated batch of scFv(G250) transgene-containing retrovirus in combination with our GMP T-cell transduction and expansion protocol successfully generates clinically relevant numbers of functional scFv(G250) gene-modified T cells for patient treatment.

LOCAL BUT NO SYSTEMIC IMMUNOMODULATION BY INTRAPERITONEAL TREATMENT OF ADVANCED OVARIAN CANCER WITH AUTOLOGOUS T LYMPHOCYTES RE-TARGETED BY A BI-SPECIFIC MONOCLONAL ANTIBODY

We have reported a 27% overall anti‐tumor response using i.p. immunotherapy of advanced ovarian carcinoma with autologous, ex vivo expanded, T lymphocytes re‐targeted with bi‐specific monoclonal antibody OC/TR, combined with soluble OC/TR and low‐dose recombinant interleukin‐2 (IL‐2). This treatment had no effect on extraperitoneal disease. Therefore we studied in 13 patients whether this immunotherapeutic protocol resulted only in local or also in systemic immunomodulation. The phenotype of the ex vivo expanded lymphocytes was mainly CD3+, 4−, 8+, 16−, 56−. Their OC/TR‐re‐targeted cytolytic activity against Igrov‐1 ovarian‐carcinoma cells was approximately as high in responders as in non‐responders. Following most therapeutic cycles, the immunophenotype of lymphocytes recovered from the peritoneal fluid was similar to that of the infused T cells (i.e., mainly CD3+, 4−, 8+) and they were coated with OC/TR. However, cytolytic activity of the recovered lymphocytes against Igrov‐1 cells was low in direct assays, and only slightly increased after additional in vitro re‐targeting with OC/TR. Systemically, the i.p. immunotherapy resulted in a transient lymphopenia lasting for about 7 days, low (i.e., 5 to 13 ng/ml) serum concentrations of free, functional OC/TR, and very weak coating of circulating T lymphocytes with OC/TR. These peripheral‐blood T lymphocytes did not exert OC/TR‐re‐targeted cytolytic activity. Thus, locoregional OC/TR‐re‐targeted cellular immunotherapy resulted in substantial local immunomodulation and anti‐tumor effects but virtually no systemic immunomodulation. Int. J. Cancer 73:211–219, 1997.

Correlation between circulating endothelial cell counts and plasma thrombomodulin levels as markers for endothelial damage

Increased numbers of circulating endothelial cells (CEC) in peripheral blood have been observed in diseases with vascular involvement, and are considered a promising surrogate marker for vascular damage. It was the objective of this study to evaluate the correlation between putative soluble markers of endothelial injury, activation, and endothelial proliferation, and absolute numbers of CEC. CEC were evaluated in 125 healthy donors and 40 patients with metastatic carcinoma by automated CD146 driven immunomagnetic isolation. Plasma concentrations of E-selectin, endoglin, and thrombomodulin were assessed by ELISA in plasma obtained from 40 healthy donors and 40 patients. CEC numbers in blood were positively correlated with plasma thrombomodulin levels, but not with levels of E-selectin and endoglin. Multivariate analysis demonstrated a significant increase in CEC numbers with age. The levels of plasma biomarkers were not influenced by age. Higher levels of thrombomodulin and E-selectin were observed in males when compared to females. In conclusion, CEC numbers correlate positively with plasma levels of thrombomodulin.

High-dose regimen of interleukin-2 and interferon-alpha in combination with lymphokine-activated killer cells in patients with metastatic renal cell cancer

Seventy-two patients with metastatic renal cell cancer were treated with the combination of high-dose interleukin-2 (IL2), interferon-alpha (IFN alpha), and lymphokine-activated killer cells (LAK). Seventeen patients were entered in a feasibility part of the study (protocol 1) and 55 in an efficacy part (protocol 2). Protocol 2 differed from protocol 1 in the addition of IFN alpha to the first 5 days of IL2 infusion. Each patient was planned to receive two induction cycles. IL2, 18 MIU/m2/day, was administered continuously i.v. on days 1-5, and IFN alpha, 5 MIU/m2/day (protocol 2), was administered i.m. on days 1-5, followed by three daily lymphaphereses on days 7-9. On day 12, treatment was resumed with IL2 and IFN alpha on days 12-15 and LAK reinfusions on days 12-14. In protocol 1, three complete (CR) and one partial (PR) responses were achieved (response rate 24%). The median duration of response and the median survival were 18.1 and 13.9 months, respectively. The 3-year survival was 35%. Of the 51 evaluable patients in protocol 2, 6 achieved a CR and 13 a PR (response rate 37%). The median duration of response was 11.1 months. The median survival was 16.9 months. The 3-year survival was 35%. There were three treatment-related deaths. Other severe toxicities included hypotension, cardiotoxicity, pulmonary edema, renal toxicity, and infectious complications. In the two induction cycles, only 54 and 42% of the planned doses could be administered. We conclude that the use of high-dose regimens of IL2 and IFN alpha is not warranted, unless we can define more accurately which patients may experience long-term survival as a result of treatment.

Ten years after the first inspection of a candidate European centre, an EBMT registry analysis suggests that clinical outcome is improved when hematopoietic SCT is performed in a JACIE accredited program.

In 2010, JACIE, the Joint Accreditation Committee of ISCT (International Society for Cell Therapy) Europe and EBMT (European group for Blood and Marrow Transplantation) celebrated the tenth anniversary of the first inspection of a European hematopoietic SCT program. JACIE standards establish the criteria for a comprehensive quality management program that covers all three major domains of activity that are necessary for the delivery of HSCT: clinical, collection and processing, as well as their interactions with ancillary and supportive activities. Although more than 200 European programs have applied for JACIE accreditation, and more than 100 have been granted accreditation, a recent retrospective analysis of the large-size EBMT registry of autologous and allogenic hematopoietic HSCT demonstrates that one of the factors affecting the overall survival of recipients of allogenic transplantation is the status of the transplant program regarding JACIE accreditation. This provides one of the first demonstrations that introduction of a quality management system contributes to the overall survival of patients treated with a highly specific medical procedure, and represents a milestone in the implementation of JACIE.

Large-scale production of natural cytokines during activation and expansion of human T lymphocytes in hollow fiber bioreactor cultures

We studied the large-scale production of a variety of natural cytokines during the activation and expansion of human T lymphocytes in a hollow fiber bioreactor culture system. Peripheral blood mononuclear cells (PBMC) were activated using phytohemagglutinin plus recombinant interleukin-2 (IL-2). Phytohemagglutinin was either present in the hollow fiber bioreactor during the entire 15-16-day culture period or only during the 20-h preactivation of the PBMC in culture bags. The expanding T lymphocytes were mainly CD3+,8+ and exerted maximal natural, activated, bispecific monoclonal antibody-redirected and lectin-dependent cytolytic activities between days 9 and 13 of culture. IL-1 and IL-4 were only produced in low amounts. IL-8 and lymphotoxin were primarily produced during the first week of culture. Harvest of the hollow fiber bioreactor culture supernatant at the time of peak cytokine concentration would have yielded per 10(8) PBMC input between 3.7 and 4.9 micrograms of IL-8 (at days 2 or 3), and between 0.02 and 0.5 microgram of lymphotoxin (at days 6 or 7). Tumor necrosis factor-alpha and IL-6 were produced during the entire culture period of 15 or 16 days: per 10(8) PBMC input, between 0.1 and 0.4 microgram of tumor necrosis factor-alpha (at days 2 or 3) and between 0.03 and 0.5 microgram of IL-6 (at days 15 or 16). Production of interferon-gamma and granulocyte-macrophage colony-stimulating factor started from initiation of cultures onwards to reach peak levels at the end of the 15- or 16-day culture period, yielding at that time between 2.1 and 17.7 micrograms/ml of interferon-gamma and between 0.4 and 4.2 micrograms of granulocyte-macrophage colony-stimulating factor per 10(8) PBMC input. The production of tumor necrosis factor-alpha, IL-6, interferon-gamma, and granulocyte-macrophage colony-stimulating factor was proportional to the extent of lymphocyte multiplication. These results demonstrate the usefulness of hollow fiber bioreactor cultures to produce natural cytokines during the activation and expansion of predominantly CD3+,8+ T lymphocytes.

T cell receptor-engineered T cells to treat solid tumors: T cell processing toward optimal T cell fitness

Therapy with autologous T cells that have been gene-engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) provides a feasible and broadly applicable treatment for cancer patients. In a clinical study in advanced renal cell carcinoma (RCC) patients with CAR T cells specific for carbonic anhydrase IX (CAIX), we observed toxicities that (most likely) indicated in vivo function of CAR T cells as well as low T cell persistence and clinical response rates. The latter observations were confirmed by later clinical trials in other solid tumor types and other gene-modified T cells. To improve the efficacy of T cell therapy, we have redefined in vitro conditions to generate T cells with young phenotype, a key correlate with clinical outcome. For their impact on gene-modified T cell phenotype and function, we have tested various anti-CD3/CD28 mAb-based T cell activation and expansion conditions as well as several cytokines prior to and/or after gene transfer using two different receptors: CAIX CAR and MAGE-C2(ALK)/HLA-A2 TCR. In a total set of 16 healthy donors, we observed that T cell activation with soluble anti-CD3/CD28 mAbs in the presence of both IL15 and IL21 prior to TCR gene transfer resulted in enhanced proportions of gene-modified T cells with a preferred in vitro phenotype and better function. T cells generated according to these processing methods demonstrated enhanced binding of pMHC, and an enhanced proportion of CD8+, CD27+, CD62L+, CD45RA+T cells. These new conditions will be translated into a GMP protocol in preparation of a clinical adoptive therapy trial to treat patients with MAGE-C2-positive tumors.