Slavoljub Milosevic

MHC-restricted fratricide of human lymphocytes expressing survivin-specific transgenic T cell receptors

The apoptosis inhibitor protein survivin is overexpressed in many tumors, making it a candidate target molecule for various forms of immunotherapy. To explore survivin as a target antigen for adoptive T cell therapy using lymphocytes expressing survivin-specific transgenic T cell receptors (Tg-TCRs), we isolated HLA-A2-allorestricted survivin-specific T cells with high functional avidity. Lymphocytes expressing Tg-TCRs were derived from these T cells and specifically recognized HLA-A2+ survivin+ tumor cells. Surprisingly, HLA-A2+ but not HLA-A2- lymphocytes expressing Tg-TCRs underwent extensive apoptosis over time. This demise was caused by HLA-A2-restricted fratricide that occurred due to survivin expression in lymphocytes, which created ligands for Tg-TCR recognition. Therefore, survivin-specific TCR gene therapy would be limited to application in HLA-A2-mismatched stem cell transplantation. We also noted that lymphocytes that expressed survivin-specific Tg-TCRs killed T cell clones of various specificities derived from HLA-A2+ but not HLA-A2- donors. These results raise a general question regarding the development of cancer vaccines that target proteins that are also expressed in activated lymphocytes, since induction of high-avidity T cells that expand in lymph nodes following vaccination or later accumulate at tumor sites might limit themselves by self-MHC-restricted fratricide while at the same time inadvertently eliminating neighboring T cells of other specificities.

Dendritic cells pulsed with RNA encoding allogeneic MHC and antigen induce T cells with superior antitumor activity and higher TCR functional avidity.

Adoptive transfer of T cells expressing transgenic T-cell receptors (TCRs) with antitumor function is a hopeful new therapy for patients with advanced tumors; however, there is a critical bottleneck in identifying high-affinity TCR specificities needed to treat different malignancies. We have developed a strategy using autologous dendritic cells cotransfected with RNA encoding an allogeneic major histocompatibility complex molecule and a tumor-associated antigen to obtain allo-restricted peptide-specific T cells having superior capacity to recognize tumor cells and higher functional avidity. This approach provides maximum flexibility because any major histocompatibility complex molecule and any tumor-associated antigen can be combined in the dendritic cells used for priming of autologous T cells. TCRs of allo-restricted T cells, when expressed as transgenes in activated peripheral blood lymphocytes, transferred superior function compared with self-restricted TCR. This approach allows high-avidity T cells and TCR specific for tumor-associated self-peptides to be easily obtained for direct adoptive T-cell therapy or for isolation of therapeutic transgenic TCR sequences.

Allorestricted T cells with specificity for the FMNL1-derived peptide PP2 have potent antitumor activity against hematologic and other malignancies

Cell-based immunotherapy in settings of allogeneic stem cell transplantation or donor leukocyte infusion has curative potential, especially in hematologic malignancies. However, this approach is severely restricted due to graft-versus-host disease (GvHD). This limitation may be overcome if target antigens are molecularly defined and effector cells are specifically selected. We chose formin-related protein in leukocytes 1 (FMNL1) as a target antigen after intensive investigation of its expression profile at the mRNA and protein levels. Here, we confirm restricted expression in peripheral blood mononuclear cells (PBMCs) from healthy donors but also observe overexpression in different leukemias and aberrant expression in transformed cell lines derived from solid tumors. We isolated allorestricted T-cell clones expressing a single defined TCR recognizing a particular HLA-A2-presented peptide derived from FMNL1. This T-cell clone showed potent antitumor activity against lymphoma and renal cell carcinoma cell lines, Epstein-Barr virus (EBV)-transformed B cells, and primary tumor samples derived from patients with chronic lymphocytic leukemia (CLL), whereas nontransformed cells with the exception of activated B cells were only marginally recognized. Allorestricted TCRs with specificity for naturally presented FMNL1-derived epitopes may represent promising reagents for the development of adoptive therapies in lymphoma and other malignant diseases.

Generation of allo-restricted peptide-specific T cells using RNA-pulsed dendritic cells: A three phase experimental procedure

Designer T cells expressing transgenic T cell receptors (TCR) with anti-tumor specificity offer new treatment options for cancer patients. We developed a three phase procedure to identify T cells of high avidity based on the fact that T cells recognizing peptides presented by allogeneic MHC efficiently kill tumor cells. Autologous dendritic cells (DC) are co-transfected with ivt-RNA encoding an allogeneic MHC molecule and a selected antigen to allow them to express allogeneic MHC-peptide complexes that activate allo-restricted peptide-specific T cells. This approach provides great flexibility for obtaining high-avidity T cells as potential sources of TCR for adoptive T cell therapy.

CD4+ and CD8+T-cell reactions against leukemia-associated- or minor-histocompatibility-antigens in AML-patients after allogeneic SCT☆

T-cells play an important role in the remission-maintenance in AML-patients (pts) after SCT, however the role of LAA- (WT1, PR1, PRAME) or minor-histocompatibility (mHag, HA1) antigen-specific CD4(+) and CD8(+)T-cells is not defined. A LAA/HA1-peptide/protein stimulation, cloning and monitoring strategy for specific CD8(+)/CD4(+)T-cells in AML-pts after SCT is given. Our results show that (1) LAA-peptide-specific CD8+T-cells are detectable in every AML-pt after SCT. CD8(+)T-cells, recognizing two different antigens detectable in 5 of 7 cases correlate with long-lasting remissions. Clonal TCR-Vβ-restriction exemplarily proven by spectratyping in PRAME-specific CD8(+)T-cells; high PRAME-peptide-reactivity was CD4(+)-associated, as shown by IFN-γ-release. (2) Two types of antigen-presenting cells (APCs) were tested for presentation of LAA/HA1-proteins to CD4(+)T-cells: miniEBV-transduced lymphoblastoid cells (B-cell-source) and CD4-depleted MNC (source for B-cell/monocyte/DC). We provide a refined cloning-system for proliferating, CD40L(+)CD4(+)T-cells after LAA/HA1-stimulation. CD4(+)T-cells produced cytokines (GM-CSF, IFN-γ) upon exposure to LAA/HA1-stimulation until after at least 7 restimulations and demonstrated cytotoxic activity against naive blasts, but not fibroblasts. Antileukemic activity of unstimulated, stimulated or cloned CD4(+)T-cells correlated with defined T-cell-subtypes and the clinical course of the disease. In conclusion we provide immunological tools to enrich and monitor LAA/HA1-CD4(+)- and CD8(+)T-cells in AML-pts after SCT and generate data with relevant prognostic value. We were able to demonstrate the presence of LAA-peptide-specific CD8(+)T-cell clones in AML-pts after SCT. In addition, we were also able to enrich specific antileukemic reactive CD4(+)T-cells without GvH-reactivity upon repeated LAA/HA1-protein stimulation and limiting dilution cloning.

Generation of tumor antigen-specific CD4+ and CD8+ T cells by simultaneous MHC-I and -II epitope presentation in vitro and in vivo

In recent years, activation of the patients immune system to defend against tumors was demonstrated to be a promising alternative strategy to classical cancer treatments. Dendritic cells (DCs) can present antigens on MHC-II and -I leading to the activation of CD4+ or CD8+ T cells, respectively. Stimulated CD4+ T cells act as helper cells for cytotoxic CD8+ T cells to kill tumor cells by mediating strong proliferation and licensing DCs, increasing their presentation capacity. Tumors bearing MHC-II molecules can also be directly destroyed by cytotoxic CD4+ T cells. As an efficient anti-tumor response strongly depends on the interplay of DCs, CD4+ and CD8+ T cells, these three cell types are considered to be essential for successful immunotherapy design. In clinical trials, DCs were either endogenously or exogenously loaded with tumor antigens for the presentation on MHC-I rather MHC-II. As the antigen presentation pathways differ, a simultaneous loading of MHC-I and -II was suboptimal. To overcome this obstacle, we used a signaling sequence (CrossTAg) to force MHC-II cross-presentation of tumor-associated antigens (TAAs) encoded by in vitro transcribed (ivt) RNA. Subsequently, peripheral blood lymphocytes were primed by DCs expressing TAAs on MHC-I and -II enabling activation and interaction of CD4+ and CD8+ T cells. By this approach, we were able to induce and identify TAA-specific CD4+ and CD8+ T cells in the same experiment. In addition, superior induction efficiency of DCs loaded with CrossTAg-TAA-ivtRNA compared to conventional TAA-ivtRNA was shown in the humanized NOD/scid IL2Rgnull (NSG) mouse model. NSG mice were engrafted with human peripheral blood mononuclear cells (PBMC) and vaccinated twice with DCs either transfected with CrossTAg-TAA-ivtRNA or conventional TAA-ivtRNA. Reisolated PBMC of mice vaccinated with CrossTAg-TAA-ivtRNA transfected DCs showed higher numbers of antigen-specific CD8+ T cells and stronger activation/cytotoxic activity against tumor cells. These in vitro and in vivo data illustrate the benefits of loading DCs with CrossTAg-linked target antigens as CD8+ and CD4+ T cells can be induced side by side allowing interactions and T cell help.

Induction and isolation of tumor antigen-specific CD4+ T lymphocytes using sorting signal directed MHC class-II expression

The adoptive transfer of tumor-antigen specific CD8+ T lymphocytes has shown highly promising but somewhat limited benefit in tumor immunotherapy. The poor objective clinical efficacy observed in early studies has been overcome partly by integration of patient preconditioning and improved technologies for T cell receptor gene therapy. Nevertheless, in accordance with results from peptide vaccine studies, we hypothesize that further improvement in therapeutic efficacy can be achieved through addition of tumor-antigen specific CD4+ T lymphocytes. CD4+ T cells provide pivotal help for activated cytotoxic T cells, especially by reducing exhaustion upon chronic antigen stimulation, and by supporting tumor infiltration capacity. In addition, CD4+ T lymphocytes are critical for the initiation of long lasting CD8+ T cell memory and can also exert direct antitumor effector functions, even in the absence of MHC class II expression. To support a systematic evaluation of potentially beneficial effects of CD4+ T cells in the adoptive T cell transfer setting, we developed an efficient method for the activation and isolation of tumor-antigen specific CD4+ T lymphocytes. Using selected tumor/testis antigens fused to cell internal sorting signals, we were able to utililze dendritic cells transfected with in vitro transcribed RNA for the efficient induction of tumor-antigen specific CD4+ T cells. Via antibody-mediated staining of CD40-ligand (CD40L) on the surface of specifically reactivated peripheral blood lymphocytes (PBL), we successfully isolated multiple tumor-antigen specific CD4+ T cell clones with diverse MHC class II allotype restrictions. Moreover, a method for the direct identification of CD4+ T cell epitops (DEPI) enabled us to define novel MHC-II restricted epitopes within the targeted tumor/testis antigens. Currently, isolated tumor-antigen specific CD4+ T cell clones are assessed for direct and indirect effector mechanisms to determine the possible contributions of CD4+ T lymphocytes in the immunotherapy of cancer.

Isolation of antigen-specific CD8+ T lymphocytes in vitro and in vivo

Depending on the mechanisms by which cancer develops and the strategies tumors use to escape the immune system, cancer therapies are more or less successful. A combination of different therapeutic agents, such as dendritic cell (DC) vaccines loaded with tumor antigens and adoptive transfer of tumor-specific T-cells, may enhance the chances of effective cures. Thus, for the isolation of T-cell receptors (TCR) that can be transferred into patient-derived lymphocytes, enabling T-cells to recognize and eliminate tumor cells, we induced tumor-associated-antigen (TAA)-specific T-cells by loading DC with in vitro transcribed (ivt) RNA coding for 5 different cancer-testis-antigens (CTA, GAGE-1, MAGE-A4, NY-ESO-1, SSX4 and XAGE-1). As CTA are self-antigens with a limited expression in immune privileged tissues and in various tumor entities, we needed to bypass negative selection of highly avid T-cells specific for CTA restricted by self-MHC molecules. Therefore, allo-reactive T-cells, which are able to recognize antigens on foreign MHC molecules, offer the possibility to gain high-affinity TCR. Using DC and responding PBL from two donors that expressed different MHC allotypes resulted in the presentation of CTA on several different MHC molecules encoded by endogenous HLA genes. In addition, taking into account that CD4+ T cell help is considered to be essential for the activation and expansion of antigen-specific CD8+ T-cells, target antigens were fused to cell internal localization signals (targeting) which led to presentation by both MHC-I and -II molecules of antigen-presenting cells, allowing the simultaneous activation of CD4+ and CD8+ T-cells. By this means, several CTA-specific T-cell clones were identified. Furthermore, we were able to demonstrate a clear benefit of using DC transfected with targeting-linked Melan-A-ivt-mRNA compared with DC transfected with standard Melan-A-ivt-mRNA in a humanized in vivo DC vaccination model. NOD/scid IL2Rγnull (NSG) mice are deficient for T-, B- and NK-cells, providing a niche for engraftment of human peripheral blood mononuclear cells (PBMC). Vaccination of reconstituted mice with DC loaded either with targeting-Melan-A-ivt-mRNA or with Melan-A-ivt-mRNA resulted in a superior induction-capacity of targeting-Melan-A-ivt-mRNA-transfected DC, demonstrated by significantly greater activation and proliferation of Melan-A-specific CD8+ T-cells. In conclusion, we were able to induce tumor-antigen specific CD8+ T-cells in vitro and in vivo by using targeting-linked antigens provided to DC. Since adoptive T-cell transfer and DC vaccination hitherto have failed to be optimally efficacious in clinical trials, the combination of these two approaches may provide greater clinical benefit to more patients.

In vitro and in vivo priming of T cells using dendritic cells loaded with internal cell localization signals fused cancer testis antigens

Cancer represents a unique cell deregulation disease in patients, therefore it has become clear in recent years that successful treatment will depend on personalized cancer therapy. The immune response against cancer is activated when antigen presenting cells (APC) pick up tumor antigens and present them to CD4 helper cells on MHC II molecules and to cytotoxic T lymphocytes (CTL) on MHC I molecules, respectively. Successful design of adoptive immunotherapies should thereby encompass three types of immune cells: APC, CD4 helper lymphocytes and CD8 CTL. We employed internal cell localization signals (ILS) to target endogenous antigens to both MHC I and MHC II molecules. We loaded dendritic cells (DC) with in vitro transcribed (ivt) mRNA coding for 5 different cancer-testis-antigens (CTAs) (GAGE-1, MAGE-A4, NY-ESO-1, SSX4 and XAGE-1) fused to ILS signals. These DCs which presented CTAs at high levels on MHC I and II were used for in vitro priming of unseparated PBL allowing activation and co-interactions of CD4 and CD8 T cells with concordant selection for immunodominat CTA. Expression of CD154 on CD4 T cells and CD137 on CD8 T cells were used for specific cell sorting, enabling isolation of CD4 T cell clones specific for 4 of 5 antigens to be obtained in one experiment. These clones used multiple TCR for CTA recognition as well as different MHC II allotypes for antigen presentation. Multiple CD8 T cell clones recognizing 5 of 5 CTA were also isolated. These clones used different TCRs and epitopes were presented by different MHC I allotypes. Therefore, this methodology enabled high throughput isolation of many different cancer specific CD4 and CD8 T cells. T cell priming using CTA fused to ILS signals was also assessed for priming efficiency in an in vivo DC vaccination model. We used NOD/scid IL2Rγnull (NSG) mice deficient for T-, B- and NK-cells to engraft with human peripheral blood mononuclear cells. Autologous DC were transfected with ivt-mRNA of melan-A fused to ILS signals or with melan-A ivt-mRNA without these signals. Upon second vaccination mice reconstituted with human PBL, were sacrificed and specific cytokine secretion by human CD8 T cells was measured upon co-cultivation with melanoma cell lines. Superior activation of melanoma specific CD8 T cells was seen when DC with melan-A fused to ILS signals was used for vaccination. Taken together, these data show that physiological ILS signals could be used to enable efficient priming of CD4 and CD8 T cells in vitro as well as in vivo. The isolated TCR provide potential reagents for use in adoptive transfer of genetically engineered T cells. Furthermore, DC loaded with ILS-fused CTA could be used as efficient DC vaccines for priming of both CD4 and CD8 T cells in vivo.