γ9 and δ2CDR3 domains regulate functional avidity of T cells harboring γ9δ2TCRs.

Immunotherapy with innate immune cells has recently evoked broad interest as a novel treatment option for cancer patients. γ9δ2T cells in particular are emerging as an innate cell population with high frequency and strong antitumor reactivity, which makes them and their receptors promising candidates for immune interventions. However, clinical trials have so far reported only limited tumor control by adoptively transferred γ9δ2T cells. As a potential explanation for this lack of efficacy, we found unexpectedly high variability in tumor recognition within the physiologic human γ9δ2T-cell repertoire, which is substantially regulated by the CDR3 domains of individual γ9δ2TCRs. In the present study, we demonstrate that the reported molecular requirements of CDR3 domains to interact with target cells shape the physiologic γ9δ2T-cell repertoire and, most likely, limit the protective and therapeutic antitumor efficacy of γ9δ2T cells. Based on these findings, we propose combinatorial-γδTCR-chain exchange as an efficient method for designing high-affinity γ9δ2TCRs that mediate improved antitumor responses when expressed in αßT cells both in vitro and in vivo in a humanized mouse model.

Pitfalls of vaccinations with WT1-, Proteinase3- and MUC1-derived peptides in combination with MontanideISA51 and CpG7909.

T cells with specificity for antigens derived from Wilms Tumor gene (WT1), Proteinase3 (Pr3), and mucin1 (MUC1) have been demonstrated to lyse acute myeloid leukemia (AML) blasts and multiple-myeloma (MM) cells, and strategies to enhance or induce such tumor-specific T cells by vaccination are currently being explored in multiple clinical trials. To test safety and immunogenicity of a vaccine composed of WT1-, Pr3-, and MUC1-derived Class I-restricted peptides and the pan HLA-DR T helper cell epitope (PADRE) or MUC1-helper epitopes in combination with CpG7909 and MontanideISA51, four patients with AML and five with MM were repetitively vaccinated. No clinical responses were observed. Neither pre-existing nor naive WT1-/Pr3-/MUC1-specific CD8+ T cells expanded in vivo by vaccination. In contrast, a significant decline in vaccine-specific CD8+ T cells was observed. An increase in PADRE-specific CD4+ T helper cells was observed after vaccination but these appeared unable to produce IL2, and CD4+ T cells with a regulatory phenotype increased. Taken into considerations that multiple clinical trials with identical antigens but different adjuvants induced vaccine-specific T cell responses, our data caution that a vaccination with leukemia-associated antigens can be detrimental when combined with MontanideISA51 and CpG7909. Reflecting the time-consuming efforts of clinical trials and the fact that 1/3 of ongoing peptide vaccination trails use CpG and/or Montanide, our data need to be taken into consideration.

HuMab-7D8, a monoclonal antibody directed against the membrane-proximal small loop epitope of CD20 can effectively eliminate CD20 low expressing tumor cells that resist rituximab-mediated lysis.

BACKGROUND: Incorporation of the chimeric CD20 monoclonal antibody rituximab in the treatment schedule of patients with non-Hodgkin's lymphoma has significantly improved outcome. Despite this success, about half of the patients do not respond to treatment or suffer from a relapse and additional therapy is required. A low CD20-expression level may in part be responsible for resistance against rituximab. We therefore investigated whether the CD20-expression level related resistance to rituximab could be overcome by a new group of CD20 mAbs (HuMab-7D8 and ofatumumab) targeting a unique membrane-proximal epitope on the CD20 molecule. DESIGN AND METHODS: By retroviral transduction of the CD20 gene into CD20-negative cells and clonal selection of transduced cells a system was developed in which the CD20-expression level is the only variable. These CD20 transduced cells were used to study the impact of rituximab and HuMab-7D8 mediated complement-dependent cytotoxicity. To study the in vivo efficacy of these mAbs an in vivo imaging system was generated by retroviral expression of the luciferase gene in the CD20-positive cells. RESULTS: We show that HuMab-7D8 efficiently killed CD20(low) cells that are not susceptible to rituximab-induced killing in vitro. In a mouse xenograft model, we observed a comparable increase in survival time between HuMab-7D8 and rituximab-treated mice. Most significantly, however, HuMab-7D8 eradicated all CD20-expressing cells both in the periphery as well as in the bone marrow whereas after rituximab treatment CD20(low) cells survived. CONCLUSIONS: Cells that are insensitive to in vitro and in vivo killing by rituximab as the result of their low CD20-expression profile may be efficiently killed by an antibody against the membrane-proximal epitope on CD20. Such antibodies should, therefore, be explored to overcome rituximab resistance in the clinic.

Complement-induced cell death by rituximab depends on CD20 expression level and acts complementary to antibody-dependent cellular cytotoxicity.

PURPOSE: The use of the CD20-specific antibody rituximab has greatly improved the response to treatment of CD20+ follicular lymphoma. Despite the success of rituximab, resistance has been reported and prognostic markers to predict individual response are lacking. The level of CD20 expression on tumors has been related to response, but results of several studies are contradictory and no clear relationship could be established. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are thought to be important effector mechanisms, but the exact mechanism of rituximab-mediated cell kill is still unknown. Importantly, no data have been reported on the combined contribution of CDC and ADCC. EXPERIMENTAL DESIGN: We have developed a system of clonally related CEM-CD20 cells by retroviral transfer of the human CD20 cDNA (n = 90). This set of cells, with the CD20 molecule as the only variable, was used to study the importance of CD20 expression level on rituximab-mediated CDC, ADCC, and the combination. RESULTS: We show a sigmoidal correlation of CD20 expression level and rituximab-mediated killing via CDC but not ADCC. On both high and low CD20-expressing cells, all CD20 molecules were translocated into lipid rafts after rituximab binding. Furthermore, CDC and ADCC act simultaneously and CDC-resistant cells are sensitive to ADCC and vice versa. CONCLUSIONS: These findings suggest that CDC depends on CD20 expression level and that both CDC and ADCC act complementary. These data give new insights into novel strategies to improve the efficacy of CD20-specific antibodies for the treatment of CD20+ tumors.

Identification of a 40S ribosomal protein S4-derived H-Y epitope able to elicit a lymphoblast-specific cytotoxic T lymphocyte response.

PURPOSE: The superior graft-versus-leukemia (GVL) effect of the female-to-male stem cell transplantation is partially independent from the concomitant graft-versus-host reactivity. However, the antigenic basis of this selective GVL response remains enigmatic, because no H-Y antigens with hematopoietic-restricted expression were identified. In this study, we report a novel H-Y epitope that is preferentially recognized on activated proliferating lymphocytes. EXPERIMENTAL DESIGN: We generated a CTL clone YKIII.8 that showed reactivity toward male B*5201+ CD40-activated B cells, EBV-lymphoblastoid cell lines, and phytohemagglutinin-activated T-cell blasts but little or no reactivity toward fibroblasts, CD14+ cells, or unstimulated B and T cells. The antigen recognized by YKIII.8 was identified by screening of a cDNA expression library, and its pattern of expression was investigated. RESULTS: cDNA of the male isoform of 40S ribosomal protein S4 was found to encode the antigenic peptide TIRYPDPVI, which was recognized by YKIII.8. Western blot analysis showed that rapidly proliferating cells overexpress the RPS4 protein in comparison with nonrecognized cell subsets. Retroviral transfer of YKIII.8 T-cell receptor resulted in preservation of the lymphoblast-specific reactivity pattern. CONCLUSION: Our findings suggest that CTL specific to certain epitopes of ubiquitously expressed H-Y antigens may specifically target lymphoblasts, contributing to the selective GVL effect of female-to-male stem cell transplantation.

Untouched GMP-Ready Purified Engineered Immune Cells to Treat Cancer.

PURPOSE: Engineering T cells with receptors to redirect the immune system against cancer has most recently been described as a scientific breakthrough. However, a main challenge remains the GMP-grade purification of immune cells selectively expressing the introduced receptor in order to reduce potential side effects due to poorly or nonengineered cells. EXPERIMENTAL DESIGN: In order to test a novel purification strategy, we took advantage of a model γδT cell receptor (TCR), naturally interfering with endogenous TCR expression and designed the optimal retroviral expression cassette to achieve maximal interference with endogenous TCR chains. Following retroviral transduction, nonengineered and poorly engineered immune cells characterized by a high endogenous αßTCR expression were efficiently depleted with GMP-grade anti-αßTCR beads. Next, the engineered immune cells were validated for TCR expression, function against a panel of tumor cell lines and primary tumors and potential allo-reactivity. Engineered immune cells were further validated in two humanized mouse tumor models. RESULTS: The untouched enrichment of engineered immune cells translated into highly purified receptor-engineered cells with strong antitumor reactivity both in vitro and in vivo. Importantly, this approach eliminated residual allo-reactivity of engineered immune cells. Our data demonstrate that even with long-term suboptimal interference with endogenous TCR chains such as in resting cells, allo-reactivity remained absent and tumor control preserved. CONCLUSIONS: We present a novel enrichment method for the production of untouched engineered immune cells, ready to be translated into a GMP-grade method and potentially applicable to all receptor-modified cells even if interference with endogenous TCR chains is far from complete.

Development and application of quantitative real time PCR and RT-PCR assays that discriminate between the full-length and truncated herpes simplex virus thymidine kinase gene.

Allogeneic donor T lymphocytes manipulated genetically to express the herpes simplex virus thymidine kinase (HSV-TK) gene have emerged as promising tools to alter the balance between graft versus host disease and graft versus leukemia after allogeneic stem cell transplantation, since they can be eliminated selectively in vivo with ganciclovir. Recently, it was reported that in SFCMM-3, an HSV-TK-encoding retroviral vector, two cryptic splice sites in the HSV-TK sequence led to the generation of an HSV-TK splice variant (deltaHSV-TK) that encodes a ganciclovir-resistant gene product. In order to quantify wtHSV-TK and deltaHSV-TK RNA levels we have developed two real time Taqman PCR assays. We demonstrate that the sensitivity of both PCR assays is 10(-4). It was found that the splice variant is generated in the packaging cell line and results in approximately 4.8+/-1.9% of virions that contain deltaHSV-TK RNA. After transduction of human T cells no significant increase in deltaHSV-TK RNA could be detected. Thus, at maximum 4.2+/-1.2% of T cells transduced with SFCMM-3 will be resistant to ganciclovir due to this mechanism only. Together, these assays provide a powerful method to monitor patients in future clinical trials.

UTY-specific TCR-transfer generates potential graft-versus-leukaemia effector T cells.

Immunotherapeutic approaches that target antigens that are differentially recognized on haematopoietic and non-haematopoietic cells may specifically enhance the graft-versus-leukaemia (GVL) effect of donor lymphocyte infusion. In this study, we have characterized a new HLA-B*5201-restricted epitope of the UTY gene. Unusually, presentation of this epitope was restricted to lymphoblasts. As a result, a T cell clone specific to this epitope recognized normal and malignant male B and T lymphoblasts, while showing little reactivity towards male HLA-B*5201+ fibroblasts. Transfer of its T cell receptor (TCR) into donor T cells led to the generation of large numbers of T cells, which acquired the specificity of the original clone, its avidity and the differential pattern of reactivity towards lymphoblasts and fibroblasts. Remarkably, the specific response of TCR-transferred T cells was significantly higher than that of the original clone. This is the first demonstration of the possibility to preserve the specific pattern of a T cell response to a differentially expressed antigen after TCR-transfer and to augment the amplitude of this response concomitantly. These results indicate that it may be feasible to enhance the GVL effect of donor lymphocyte infusions in lymphoproliferative malignancies by the transfer of TCRs specific to epitopes that are differentially recognized on lymphoblasts.

HLA-DRB1*16-restricted recognition of myeloid cells, including CD34+ CML progenitor cells.

The therapeutic effect of a human leucocyte antigen (HLA)-identical allogeneic stem cell transplantation (allo-SCT) for the treatment of haematological malignancies is mediated partly by the allogeneic T cells that are administered together with the stem cell graft. Chronic myeloid leukaemia (CML) is particularly sensitive to this graft-versus-leukaemia (GVL) effect. Several studies have shown that in allogeneic responses both CD4 and CD8 cells are capable of strong antigen-specific growth inhibition of leukaemic progenitor cells, but that CD4 cells mainly exert the GVL effect against CML. Efficient activation of allogeneic CD4 cells, as well as CD8 cells, may explain the sensitivity of CML cells to elimination by allogeneic T cells. Identification of the antigens recognized by CD4 cells is crucial in understanding the mechanism through which CML cells are so successful in activating allogeneic T cells. In the present report, we describe the characterization of an allogeneic CD4 T-cell clone, DDII.4.4. This clone was found to react against an antigen that is specifically expressed in myeloid cells, including CD34+ CML cells. The antigen recognition is restricted by HLA-DRB1*16. To our knowledge, this is only the second report on an allogeneic CD4 T-cell clone that reacts with early CD34+ myeloid progenitor cells.