Cytotoxic Lymphocyte Precursors Research Articles

CD137 Expression Identifies Leukemia Specific CTL After In Vitro Priming Of Cord Blood T Cells

IntroductionWe have previously reported that cord blood T cells (CBT) can be expanded ∼ 2 log after 2 weeks in culture following stimulation with CD3/CD28 co-stimulatory beads and exogenous IL2 + IL7. The expanded progeny retained the starting naive phenotype typical of unmanipulated cord blood T cells (Cancer Research, Post-expansion, CD137, CD40L and perforin expression remains minimal and these cells demonstrate no cytotoxicity against an immunogenic lymphoblastoid cell line (IM9) or against transplant recipient PHA blasts. However, when primed against (IM9) or IFN-γ treated myeloid leukemia cell lines in the presence of IL12, IL7 and IL15, after 3 weeks of restimulation, lineage restricted cytotoxic activity was demonstrable (Cancer Research, ). Recently, we have demonstrated that IL15 alone can support the priming and expansion of CTL (abs 414, ASBMT 2013, manuscript in preparation) from CD3/CD28 expanded antigen inexperienced CB T cells. HypothesisCD137 (4-1BB) is a member of the TNFR- family and is expressed on activated T cells. 4-1BB ligation and signaling augments the survival, proliferation and lytic function of activated effectors. Expression of CD137 on activated T cells reflects recent activation. Therefore it may identify antigen-specific CTL. Adoptive T cell therapy with a restricted TCRVβ repertoire compared to bulk, unselected CTL pools is preferable to exclude potentially GVHD-causing clones in the HLA-mismatched cord blood transplant setting. Here we tested the hypothesis that de novo CD137 expression may identify leukemia-specific CTL precursors and that CD137 selection step could enrich for CTL clones in the priming culture. MethodsIM9-specific CTL was generated from pre-expanded, >98% pure T cells using weekly stimulations with irradiated targets and IL15. After 3 weeks, cytotoxicity was tested against fresh IM9 leukemia cells in a Europium labeling assay. CD137 expressing cells were sorted on FACS Aria II (BD Biosciences). CD137- and CD137+ cells were re-tested for cytotoxicity and TCRβ CDR3 spectratyping was done to identify their clonality. RT2 profilerTM PCR array human Th1 & Th2 responses (SABiosciences) was employed to contrast the sorted populations for differential gene expression ResultsAfter 3 weeks of priming/expansion, 2-3 % of the CTL were CD137+. However, after overnight re-stimulation, CD137 expression increased to a range of 12-39% of all T cells (Figure-1). The cytotoxicity of bulk cells in the culture segregated into the CD137+ cells after FACS sort (Figure-2). Amongst the 23 TCR Vβ families tested, distinct oligoclonal expansion of Vβ7, Vβ5.1 and Vβ21 were observed in the CD137+ population in contrast with CD137- T cells from the same culture, where the 3 Vβ families retained Gaussian-like, multiple clonal distributions (Figure-3). Notably, despite the different genetic background of starting CBT, the IM9 specific CTL activity appears to reside in the same CTL clones based on the virtually identical TCR Vβ clonality. This finding suggests that CTL targets have the greater influence on shaping CTL repertoir and not the genetic background (HLA, etc) of CBT cells. While post-sort RT qPCR analysis demonstrate significantly increased message for CCR4, EBI3, TNFRSF9 and TNFSF4 genes, CD40LG, IL2, IL6, IL7R, SFTPD and SPP1 expressions were significantly decreased in CD137+ cells. ConclusionIL15 without other exogenous cytokines supports the in vitro priming and expansion of polyclonal CTL against leukemia cell lines from expanded cord blood T cells. Cytotoxicity dominates in the CD137 positive subset, therefore CD137 positive selection can be used to isolate and enrich leukemia specific CTLs that retain a predictable TCRVβ repertoire shaped by the leukemic target. These findings will have great influence on attaining clinically applicable leukemia-specific CTL protocols.▪▪▪ Disclosures:No relevant conflicts of interest to declare.

Deletion of naïve T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance

Alloreactive T-cell responses directed against minor histocompatibility (H) antigens, which arise from diverse genetic disparities between donor and recipient outside the MHC, are an important cause of rejection of MHC-matched grafts. Because clinically significant responses appear to be directed at only a few antigens, the selective deletion of naïve T cells recognizing donor-specific, immunodominant minor H antigens in recipients before transplantation may be a useful tolerogenic strategy. We have previously demonstrated that peptide-MHC class I tetramers coupled to a toxin can efficiently eliminate specific TCR-transgenic T cells in vivo. Here, using the minor histocompatibility antigen HY as a model, we investigated whether toxic tetramers could inhibit the subsequent priming of the two H2-Db-restricted, immunodominant T-cell responses by deleting precursor CTL. Immunization of female mice with male bone marrow elicited robust CTL activity against the Uty and Smcy epitopes, with Uty constituting the major response. As hypothesized, toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo CTL assay, and reduced the frequency of corresponding T cells. However, tetramer-mediated decreases in either T-cell population magnified CTL responses against the non-targeted epitope, suggesting that Db-Uty+ and Db-Smcy+ T cells compete for a limited common resource during priming. Toxic tetramers conceivably could be used in combination to dissect manipulate CD8+ T-cell immunodominance hierarchies, and to prevent the induction of donor-specific, minor H antigen CTL responses in allotransplantation.

Constant drug dose in human immuno‐deficiency virus‐infected patients to induce long‐term non‐progressor status: bifurcation and controllability approach

The authors propose a therapy consisting of a constant dosage of reverse transcription inhibitor and protease inhibitor to achieve long-term non-progressor (LTNP) status in human immuno-deficiency virus (HIV) patients. Based on the authors analyses of cytotoxic T lymphocyte precursor (CTLp) concentration at several equilibrium points and the bifurcation of these equilibrium points, they find that administration of drugs with an efficacy lower than a certain level induces a higher CTLp concentration. As a result, drug doses of moderate efficacy result in more patients with LTNP status than doses with high efficacy. In analyses of controllability, they show that a treatment of moderate efficacy is more efficient than one of very high efficacy in terms of controlling the immune system. Using simulations, they demonstrate that their proposed method results in LTNP status in HIV patients.

An Immunological Basis for Chronic Histiocytic Intervillositis in Recurrent Fetal Loss

Chronic histiocytic intervillositis (CHIV) is a rare type of placental pathology that is associated with reproductive loss at all gestational ages. The aim of the study was to investigate the relationship between the severity of CHIV and the outcome of pregnancy and to compare the immune response between CHIV patients and controls to explore an immunological origin of CHIV. Microscopic slides were reviewed and scored according to a previously published grading system in 30 pregnancies of 22 CHIV patients. Partner-specific mixed lymphocyte reactions, cytotoxic T-lymphocyte precursor frequencies (CTLpf), and anti-HLA antibodies were determined in four patients and seven controls. Higher CHIV scores are associated with worse pregnancy outcome. CHIV patients demonstrated a higher CTLpf against their partner compared to non-complicated pregnancies (P=0.03). The CTLpf was extremely high in 75% of the patients. Antipaternal HLA antibodies were only present in 75% of the CHIV patients compared to none of the controls (P=0.02). CHIV scores seem to be associated with the severity of adverse pregnancy outcome. High antipaternal cellular (T-cell) and humoral (B-cell) response to partner-specific CTLpf and the presence of anti-HLA antibodies directed to the partner suggest an immunologic origin of CHIV.

IL‐27 enhances the survival of tumor antigen‐specific CD8+ T cells and programs them into IL‐10‐producing, memory precursor‐like effector cells

IL-27 is a member of the IL-12 family of cytokines that is comprised of an IL-12 p40-related protein subunit, EBV-induced gene 3, and a p35-related subunit, p28. IL-27 functions through IL-27R and has been shown to have potent antitumor activity via activation of a variety of cellular components, including antitumor CD8(+) T-cell responses. However, the exact mechanisms of how IL-27 enhances antitumor CD8(+) T-cell responses remain unclear. Here we show that IL-27 significantly enhances the survival of activated tumor antigen-specific CD8(+) T cells in vitro and in vivo, and programs tumor antigen-specific CD8(+) T cells into memory precursor-like effector cells, characterized by upregulation of Bcl-6, SOCS3, Sca-1, and IL-10. While STAT3 activation and the CTL survival-enhancing effects can be independent of CTL IL-10 production, we show here that IL-27-induced CTL IL-10 production contributes to memory precursor cell phenotype induction, CTL memory, and tumor rejection. Thus, IL-27 enhances antitumor CTL responses via programming tumor antigen-specific CD8(+) T cells into a unique memory precursor type of effector cells characterized by a greater survival advantage. Our results have important implications for designing immunotherapy against human cancer.

Abstract B70: Generation of MAGE-A4 230-239 peptide-specific cytotoxic T lymphocytes using an alloreactive approach.

Abstract The aim of this project was to generate cytotoxic T lymphocytes (CTL) specific for the weakly immunogenic MAGE-A4 antigen, by an alloreactive approach. CTL may recognize and eliminate tumor cells. However, to avoid generation of autoreactive CTL, most CTL specific for tumor-associated antigens (TAA) are eliminated by negative selection during their development in the thymus. Thus, compared to virus-specific CTL, TAA-specific CTL precursors are often rare and more challenging to expand from peripheral blood. One TAA that is difficult to raise specific CTL against is the MAGE-A4 antigen, a cancer-testis antigen expressed by many tumors but not normal tissues. A MAGE-A4 230-239 peptide is processed (Duffour et al. Eur J Immunol 1999;29;3329–37) and presented by the HLA-A2 complex (Hilling et al. J Mol Biol 2001;310:1167-76). Still, rarely have MAGE-A4 230-239 specific CTL been found after in vitro stimulation or in vivo vaccination. We speculated that the reason was elimination of basically all precursors by negative selection. To get around this, we transfected HLA-A2 into mature dendritic cells (moDC) generated from HLA-A2 negative donor monocytes. In contrast to previous studies using this approach for other TAA (Stronen et al. Scand J Immunol 2009;69:319-28), we used the HLA-A2 gene in a plasmid instead of the HLA-A2 mRNA for transfection. This approach excludes in vitro transcription and avoids RNA stability issues. The transfection efficiency with our method was between 25 to 50% after 24 hours. We pulsed the HLA-A2 transfected moDC with peptide and co-cultured them with autologous CD8 T cells. As these CD8 T cells have not encountered HLA-A2 during development, they should contain higher frequencies of precursors specific for peptides presented by this HLA allele. Thus, the setting is allogeneic only for peptides presented by HLA-A2, and will from hereon be referred to as the HLA-A2 allogenic setting. As a control, we used the optimized MART-1 26-35 (A27L) peptide, known to generate potent in vitro responses even in CD8 T cells from healthy HLA-A2 positive donors using an autologous setting. We managed to expand MART-1 specific CTL in vitro from 50% of healthy HLA-A2 positive donors (2/4), when using PBMC and in every donor when using moDC (3/3) as antigen-presenting cells (APC). Thus it was not surprising that we could also generate MART-1 specific cells from all donors with the HLA-A2 allogeneic setting (3/3). In the case of MAGE-A4, we have never succeeded to generate specific CTL from HLA-A2 positive donors, neither with peptide-pulsed PBMC (0/8) nor with moDC (0/3) as APC. Therefore, it is very promising that we were able to generate MAGE-A4 specific cells from almost half of the donors (2/5) with the HLA-A2 allogenic setting. More donors will be included to determine the efficiency of the method. The MAGE-A4 and MART-1 specific cells generated by the HLA-A2 allogeneic approach were cloned and functionally tested. The clones for both specificities were similarly capable of killing peptide-pulsed target cells, for some at a high efficiency (80% lysis). MART-1 clones generally required less peptide for lysis (picomolars) than MAGE-A4 clones (nanomolars). This may be due to superior binding of the optimized MART-1 26-35 (A27L) peptide to the HLA-A2. The clones will be further phenotyped and tested for other effector functions such as cytokine production and lysis of MAGE-A4 expressing tumor-cells. We conclude that functional MAGE-A4 230-239 specific CTL can be generated by the HLA-A2 allogeneic method. Citation Format: Tanja Lövgren, Yumeng Mao, Maxi-Lu Böschen, Eva B. Allerbring, Adnane Achour, Isabel Poschke, Rolf Kiessling. Generation of MAGE-A4 230-239 peptide-specific cytotoxic T lymphocytes using an alloreactive approach. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B70.

Human Telomerase Reverse Transcriptase–specific T-Cell Receptor Gene Transfer Redirects T Cells to Display an Effective Antitumor Reactivity Against Adult T-Cell Leukemia

AbstractAbstract 1902 Background and Purpose:Adult T-cell leukemia (ATL) is one of the most refractory forms of T-cell malignancy that preferentially affects aged population that constrains clinical efficacy of chemotherapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, objective responses in a small part of patients who received allo-HSCT evidently emphasize the therapeutic potential of immune-mediated approaches. While, some previous studies described that activation of human telomerase reverse transcriptase (hTERT), one of tumor antigens was detected in ATL tumor cells. Collectively, in this study, we examined the feasibility of redirected T-cell based immunotherapy targeting hTERT to explore a novel treatment strategy for ATL. Methods:Approval for this study was obtained from our institutional review board. Written informed consent was obtained from all patients, healthy volunteers, and parents of cord blood donors in accordance with the Declaration of Helsinki. HLA-A*2402-restricted and hTERT461–469 epitope (VYGFVRACL)-specific TCR-α/β genes were newly cloned into a novel retroviral vector encoding siRNAs for endogenous TCR-α/β genes (hTERT-siTCR vector). Using hTERT-siTCR vector, hTERT-siTCRR-α/β genes were transduced into normal peripheral CD8+ T cells (hTERT-siTCR/CD8+), employed as effectors. HLA-A*2402+ or HLA-A*2402− ATL cell lines, freshly isolated patients’ ATL cells, normal peripheral blood mononuclear cells (PBMCs) from healthy volunteers, and CD34+ cells isolated from cord blood mononuclear cells (CBMCs) were employed as target cells. Anti-tumor reactivity mediated by hTERT-siTCR/CD8+ was assessed using standard 51Cr release assay. Expressions of hTERT mRNA and hTERT protein in ATL cell lines, freshly isolated ATL cells and normal CD4+ T cells were assessed by quantitative real-time PCR (QRT-PCR) and Western blotting. Frequencies of hTERT461–469–specific cytotoxic T-lymphocyte (CTL) precursors in periphery of ATL patients or healthy donors were measured by hTERT461–469/HLA-A*2402 tetramer assay. Finally, NOD/scid/γcnull (NOG) mice subcutaneously inoculated with luciferase-gene-modified HLA-A*2402+ ATL cell line, ATN-1 cells were treated with intravenous infusion of hTERT-siTCR/CD8+or non-gene-modified (NGM) ones, respectively. Thereafter, these mice were serially monitored for tumor growth using bioluminescence imaging and survival. Results:QRT-PCR and Western blotting revealed that hTERT was markedly overexpressed in both ATL cell lines and freshly isolated ATL cells, while less than undetectable in normal cells. hTERT461–469-specific CTL precursors were variably detectable in periphery of HLA-A*2402+ ATL patients, but not HLA-A*2402− ATL patients or HLA-A*2402+ healthy individuals. hTERT-siTCR/CD8+ were 30–50% positive for hTERT461–469/HLA-A*2402 tetramer and displayed HLA-A*2402-restricted and hTERT461–469–specific cytocidal activity against peptide-loaded K562-A24 cells. Those hTERT-siTCR/CD8+ successfully displayed tumoricidal activity against HLA-A*2402+ ATL cell line cells and freshly isolated HLA-A*2402+ ATL cells, but not HLA-A*2402− ones or HLA-A*2402+ normal cells including CD34+ CBMCs. Moreover, hTERT-siTCR/CD8+ generated from HLA-A*2402+ ATL patients also lysed autologous ATL tumor cells. Finally, using xenografted NOG mouse models, therapeutically infused hTERT-siTCR/CD8+, but not NGM ones from the identical donor successfully suppressed growth of inoculated ATL cells in vivo. Furthermore, NOG mice bearing advanced ATL tumor masses were also successfully treated with hTERT-siTCR/CD8+ in combination with bortezomib. Summary:In this study, we newly demonstrated that hTERT is a promising therapeutic tumor antigen for ATL. Moreover, redirected CD8+ T cells to express hTERT-specific TCR displayed antitumor activity against ATL tumor cells both in vitro and in vivo. Furthermore, our preliminary data represented antitumor activity against advanced ATL, in combination with bortezomib. Collectively, redirected T cell-based immunotherapy targeting hTERT seems clinically feasible and promising for the treatment of almost all ATL patients who are ineligible for allo-HSCT. Disclosures:No relevant conflicts of interest to declare.

Third Party Donor Derived EBV Specific T Cells for the Treatment of Refractory EBV-Related Malignancies in Immunodeficient Recipients

AbstractAbstract ▪577▪This icon denotes a clinically relevant abstractAdoptive immunotherapy is an effective strategy for the treatment of EBV+ lymphoproliferative diseases (EBV-LPD) arising after an allogeneic hematopoietic stem cell (HSCT) or solid organ transplant (SOT). This approach is, however, often limited by an inability to generate donor derived in vitro expanded EBV-specific cytotoxic T-lymphocyte (EBV-CTL) lines in a timely manner and/or the fact that EBV CTL lines derived from HLA non-identical donors may be restricted by non-shared HLA alleles.To date, we have treated 25 consecutive patients with an EBV LPD (N=20) or EBV Leiomyosarcoma (LMS) (N=5) with in vitro expanded EBV-CTLs derived from a donor other than the patient or their transplant (HSCT or SOT) donor. EBV CTLS were selected from a bank of 345 lines generated under GMP conditions from normal HSCT donors. Each donor was specifically consented for use of their T cells in patients other than their designated transplant recipient. Patients were recipients of unmodified (n=4), T cell depleted (n=5) or unrelated cord blood (n=5) HSCT, a solid organ transplant (n=6), a combined SOT and HSCT (n=1), or were non-transplanted patients with a primary immunodeficiency disease (n=4).EBV disease in transplanted patients was of host origin in 5 of 10 evaluable HSCT recipients, in 3 of 4 evaluable solid organ recipients, and in the one patient who underwent a combined HSCT/solid organ transplant. Third party EBV-CTLs were selected on the basis of HLA matching at a minimum of 2/8 recipient alleles. Where possible EBV-CTLs were selected that were restricted through HLA alleles present on the EBV+ tumor. HLA restriction was evaluated in vitro in 20 EBV-CTL donor lines. The restriction was at a single HLA allele (n=12), at two alleles (n=6) and at >than two alleles (n=2).Patients received infusions of 3rd party EBV-CTLs after failing a median of 2 prior therapies including rituximab in all but one case of EBV LPD. Four patients failed prior infusions with EBV-CTLs which were autologous (n=1), derived from their original HSCT (n=2) or from their solid organ donor (n=1). In two patients who progressed after treatment with EBV CTLs generated from their HSCT or organ donor, it was demonstrated that the donor derived EBV CTLs were restricted by a non-shared HLA allele.Patients received a median of 5 infusions most at 1×106 EBV-CTL/kg/infusion. Four patients received EBV-CTLs from >1 3rd party donor. Nine patients achieved a completed response. Nine patients died of progressive disease, 6 shortly after the first infusion (17–29 days). Two patients with LMS achieved long term stable disease (46 and 8 months); 5 achieved partial remissions which have been sustained in 4 (11- 68 months), and 1 patient progressed after 10 months in a partial remission. Response to EBV CTL therapy did not correlate with the degree of HLA matching between donor and recipient or donor and tumor. Radiographic and clinical responses correlated with detectable increases in the frequency of CTL precursors in the blood. However durable EBV CTL engraftment was not seen.One patient developed mild skin GvHD after infusion with 3rd party EBV-CTLs, but tolerated subsequent infusion of EBV-CTLs from an alternate 3rd party donor. Although no SOT recipient developed anti-HLA antibodies, one developed and episode of steroid responsive renal transplant rejection more than 6 months after infusion of EBV CTLs without evidence of donor (by STR analysis) in biopsied tissue.This study demonstrates a high response rate among patients with otherwise refractory EBV malignancy treated with EBV specific 3rd party CTLs restricted by HLA alleles shared by the tumor. Treatment failures correlated with the use of EBV CTLs restricted by HLA alleles not shared by the tumor. In addition two patients with a primary immunodeficiency disease who were unable to mount an endogenous EBV T cell response had transient but not durable responses to 3rd party cells. EBV CTLs can be effective when selected based on restriction to shared alleles despite significant HLA disparity. The bank of EBV specific T cells can provide an immediate source of HLA partially matched appropriately restricted T cells for adoptive immunotherapy to treat EBV associated malignancy. This enables treatment early in the course of disease and the use of EBV-CTL lines previously prepared and characterized in terms of HLA restriction. This is anticipated to maximize the response rate. Disclosures:No relevant conflicts of interest to declare.

Expression of chondroitin‐glucuronate C5‐epimerase and cellular immune responses in patients with hepatocellular carcinoma

Chondroitin-glucuronate C5-epimerase is an enzyme that converts D-glucuronic acid to L-iduronic acid residues in dermatan sulphate biosynthesis. It is also identified to be a tumour-associated antigen recognized by cytotoxic T cells (CTLs) and its enhanced expression in many cancers has been reported. In the present study, we investigated the usefulness of this molecule as an immunotherapeutic target in hepatocellular carcinoma (HCC). The expression of chondroitin-glucuronate C5-epimerase in hepatoma cell lines and HCC tissues was confirmed by immunofluorescence and immunohistochemical analysis. CTL responses were investigated by several immunological techniques using peripheral blood mononuclear cells (PBMCs) or tumour-infiltrating lymphocytes. To determine the safety of immunotherapy using chondroitin-glucuronate C5-epimerase-derived peptide, 12 patients with HCC were administered s.c. vaccinations of the peptides and analysed. Chondroitin-glucuronate C5-epimerase was expressed in HCC cell lines and human tissues including alpha-foetoprotein (AFP)-negative individuals. Chondroitin-glucuronate C5-epimerase-specific CTLs could be generated by stimulating PBMCs of HCC patients with peptides and they showed cytotoxicity against HCC cells expressing the protein. The frequency of CTL precursors investigated by enzyme-linked immunospot (ELISPOT) assay was 0-34 cells/3 × 10(5) PBMCs and the infiltration of interferon-gamma-producing CTLs into the tumour site was confirmed. In the vaccination study, no severe adverse events were observed and the peptide-specific CTLs were induced in 4 of 12 patients tested. Chondroitin-glucuronate C5-epimerase is a potential candidate for tumour antigen with immunogenicity and the peptides derived from this antigen could be useful in HCC immunotherapy.

Immune responses against islet allografts during tapering of immunosuppression – a pilot study in 5 subjects

Transplantation of isolated islet of Langerhans cells has great potential as a cure for type 1 diabetes but continuous immune suppressive therapy often causes considerable side effects. Tapering of immunosuppression in successfully transplanted patients would lower patients' health risk. To identify immune biomarkers that may prove informative in monitoring tapering, we studied the effect of tapering on islet auto- and alloimmune reactivity in a pilot study in five transplant recipients in vitro. Cytokine responses to the graft were measured using Luminex technology. Avidity of alloreactive cytotoxic T Lymphocytes (CTL) was determined by CD8 blockade. The influence of immunosuppression was mimicked by in vitro replenishment of tacrolimus and MPA, the active metabolite of mycophenolate mofetil. Tapering of tacrolimus was generally followed by decreased C-peptide production. T-cell autoreactivity increased in four out of five patients during tapering. Overall alloreactive CTL precursor frequencies did not change, but their avidity to donor mismatches increased significantly after tapering (P = 0·035). In vitro addition of tacrolimus but not MPA strongly inhibited CTL alloreactivity during tapering and led to a significant shift to anti-inflammatory graft-specific cytokine production. Tapering of immunosuppression is characterized by diverse immune profiles that appear to relate inversely to plasma C-peptide levels. Highly avid allospecific CTLs that are known to associate with rejection increased during tapering, but could be countered by restoring immune suppression in vitro. Immune monitoring studies may help guiding tapering of immunosuppression after islet cell transplantation, even though we do not have formal prove yet that the observed changes reflect direct effects of immune suppression on immunity.

Immune-modulating Effects of the Newest Cetuximab-based Chemoimmunotherapy Regimen in Advanced Colorectal Cancer Patients

Cetuximab is a human-murine chimeric monoclonal antibody to the epidermal growth factor receptor, active for advanced colorectal cancer treatment in combination with chemotherapy. Cetuximab mainly acts by inhibiting epidermal growth factor receptor-mediated pathways in cancer cells; however, in the human host, its IgG1 backbone may offer additional antitumor activity that includes FcγRs-mediated antibody-dependent cell cytotoxicity, phagocytosis, cross priming, and tumor-specific T-cell-mediated immune response. These mechanisms are still under active investigation. At this purpose, we have performed an immunologic investigation in advanced colon cancer patients enrolled in an ongoing phase II trial aimed to test the toxicity and the biological and antitumor activity of a novel biochemotherapy regimen combining polychemotherapy with gemcitabine, irinotecan, levofolinic acid, and fluorouracil with cetuximab and with subcutaneous low-dose metronomic aldesleukin (GILFICet regimen). The peripheral blood mononuclear cells of the first 20 patients enrolled in the GILFICet trial were collected at baseline and after 6 treatment cycles and examined for immune-phenotype change by flow cytometry. Colon cancer-specific T-cell lines were also generated ex vivo from these samples and subsequently characterized for immune phenotype, functional activity, and antigen specificity. We found a treatment-related increase of circulating dendritic cells, natural killer cells, central memory T cells, and activated T cells with a T-helper 1 (Th1)-cytotoxic phenotype. In addition, the ex-vivo characterization of antigen-specific T cells derived from the treated patients revealed a significant increase in proliferating cytotoxic T-lymphocyte precursors specific for carcinoembryonic antigen and thymidylate synthase derivative epitope peptides. On these basis, we concluded that the GILFICet regimen exerts substantial immune-modulating activity that significantly affects tumor antigen-specific T-cell compartment with potential antitumor activity.

Phase Ib study of TSPP (thymidilate synthase poly-epitope peptide) vaccine in combination with GOLFIG chemoimmuno-burst in advanced colon cancer patients: Results from the TSPP/VAC-1/c trial.

e13098 Background: TSPP is a poly-epitope peptide anticancer vaccine able to generate a thymidylate syntase (TS) specific T cell response with antitumor activity in preclinical models, expecially when used in sequential combination with 5-FU. TS is in fact involved in DNA replication and is the major pharmacological target of fluoropyrimidines whose activity leads in turn to TS upregulation. On these basis we designed a phase Ib trial where we investigated the safety and the immunobiological activity of TSPP + GOLFIG (Gemcitabine + FOLFOX-4 + GM-CSF + IL-2) regimen. Methods: TSPP/VAC-1 (Eudract 2009-016897-33) is a monocentric dose finding Phase Ib trial. 12 pretreated metastatic colon cancer patients were enrolled between April and November 2011. All of them received biochemotherapy according to the GOLFIG regimen biweekly and TSPP (on day 7) diluted in motanide ISA720 (1:1) at the dosage of 100 (3 patients), 200 (3 patients) and 300 ug (6 patients). Results: No life-threatening adverse events were observed. Grade 3 leucopenia (2 cases), thrombocitopenia (3 cases), anemia (3 cases), nausea/vomiting (2 cases), and dyarrhea (4 cases) along with 3 cases of oxaliplatin reaction were recorded. Fever and flu-like symptoms were common along cytokine administration. An IFN-gamma ELISPOT assay recorded an increase in TS specific CTL precursors in 3/6 evaluable patients; it was also observed an increase in lymphocytes, eosinophils, terminal effector memory and central memory T cells and in both c-ANCA and p-ANCA (P= 0.02) in the peripheral blood and a significant reduction in inflammatory markers (LDH, CRP and ESR). Quality of life analysis revealed a general improvement in patients’health state along the treatment. 1 partial response, 6 disease stabilizations and 2 progessions of disease were recorded. TSPP Maximal Tolerated Dose was not reached, while the Optimal Biological Dose was identified at 300 ug. Conclusions: Our results suggest that the GOLFIG + TSPP vaccine combination is safe and immunologically active and deserves to be evaluated in a further phase II trial.

Abstract LB-229: Treatment of advanced cancer patients with a newest poly-epitope peptide vaccine to the thymidylate synthase(TSPP): a phase Ib (TSPP/VAC1) trial

Abstract Background TSPP is a poly-epitope peptide vaccine to the thymidylate syntase (TS) an enzyme commonly over-expressed in replicating cancer cells and recognized as a major target of fluoropyrimidines. TSPP showed immunological and anticancer activity in preclinical models, thus in the present study we investigated its safety and immunobiological activity in advanced cancer patients. This study was designed to identify TSPP Maximum Tolerated Dose (MTD) and Optimal Biological Dose (OBD) when used alone (arm A) or in combination with GM-CSF and IL-2 according to the IG-1 regimen (arm B). Patients and methods TSPP/VAC-1 (Eudract 2009-016897-33) is a monocentric phase Ib trial including 21 pretreated advanced cancer patients (12 in arm A and 9 in arm B) enrolled between April and October 2011 (12 with colorectal, 1 with breast, 1 with gastric, 1 with biliary tract and 6 with non small cell lung cancer). All patients presented a good performance status (ECOG ≤ 1). Each patient received every 3 weeks a sc administration of TSPP diluted in motanide ISA720 (1:1) at the dosage of 100 µg (6 patients), 200 µg (6 patients), and 300 µg (9 patients). Patients enrolled in arm B also received daily sc GM-CSF (100 µg) from day 1 to 5 and sc IL-2 (0.5 MIU) bid from day 6 to 15. Results No life-threatening adverse events were recorded; 1 case of grade 3 hypothyroidism (arm B), 4 cases of poly-articular arthropaty (arm A), and 4 cases of conjuntivitis (arm A) were observed. In arm B, cytokine-related flu-like syndrome, nausea, vomiting, and dyarrhea were commonly reported. An IFN-gamma ELISPOT assay revealed an increase in TS specific CTL precursors in 5 patients in arm A and 2 in arm B. It was also observed an increase in terminal effector memory and central memory T cells in the peripheral blood in both arms. Patients enrolled in arm A showed a significant decline in peripheral immune-regulatory (CD4+CD25hi+FoxP3+) T cells (baseline vs 3 vaccinations (B vs III): 3.8 vs 1.7 %; p= 0.041) and inhibitory myeloid cells (CD11c+CD15+) [Baseline (Bs) vs III vaccinations (Vc): 5.09 vs 1.02%; p= 0.05] associated to an increase in anti-proteinase III (Bs vs III Vs:0.48 vs 1.38; p= 0.04) and anti-myeloperoxidase auto-antibodies (Bs vs III Vc: 0.76 vs 1.50; p= 0.02) and reduced level of inflammatory markers (CRP, ESR, and LDH). A quality of life analysis did not reveal significant treatment-related changes in arm A, while a detrimental effect was observed in arm B. It was recorded 1 partial response (arm A);11 disease stabilitazions (8/12 patients arm A, and 3/9 arm B) and 8 disease progressions (3/12 patients arm A and 5/9 arm B). MTD was not reached, while OBD was defined as a TSPP dosage of 300 µg. Conclusion Our results suggest that the TSPP vaccine is safe and immunologically active. On these bases we believe that TSPP deserves to be evaluated in further phase II trials in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-229. doi:1538-7445.AM2012-LB-229

Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation

AbstractWe evaluated HLA-compatible donor leukocyte infusions (DLIs) and HLA-compatible or HLA-disparate EBV-specific T cells (EBV-CTLs) in 49 hematopoietic cell transplantation recipients with biopsy-proven EBV-lymphoproliferative disease (EBV-LPD). DLIs and EBV-CTLs each induced durable complete or partial remissions in 73% and 68% of treated patients including 74% and 72% of patients surviving ≥ 8 days after infusion, respectively. Reversible acute GVHD occurred in recipients of DLIs (17%) but not EBV-CTLs. The probability of complete response was significantly lower among patients with multiorgan involvement. In responders, DLIs and EBV-CTLs regularly induced exponential increases in EBV-specific CTL precursor (EBV-CTLp) frequencies within 7-14 days, with subsequent clearance of EBV viremia and resolution of disease. In nonresponders, EBV-CTLps did not increase and EBV viremia persisted. Treatment failures were correlated with impaired T-cell recognition of tumor targets. Either donor-derived EBV-CTLs that had been sensitized with autologous BLCLs transformed by EBV strain B95.8 could not lyse spontaneous donor-derived EBV-transformed BLCLs expanded from the patient's blood or biopsied tumor or they failed to lyse their targets because they were selectively restricted by HLA alleles not shared by the EBV-LPD. Therefore, either unselected DLIs or EBV-specific CTLs can eradicate both untreated and Rituxan-resistant lymphomatous EBV-LPD, with failures ascribable to impaired T-cell recognition of tumor-associated viral antigens or their presenting HLA alleles.

A Proposed Algorithm Predictive for Cytotoxic T Cell Alloreactivity

Previously, we showed that with an increasing number of amino acid differences in single HLA class I-mismatched molecules, the probability of T cell alloreactivity decreases. It is unlikely that every amino acid difference will affect T cell alloreactivity in a similar way; we hypothesized that the effect of an amino acid difference may be dependent on its position and/or physicochemical properties. We selected 131 patient/donor pairs with either a single HLA-A or -C mismatch in the graft-versus-host direction and that were compatible for HLA-B, -DRB1, and -DQB1. The alloreactive CTL precursor (CTLp) frequency was determined and associated with the amino acid differences between the single HLA class I mismatches. In the β sheet, only amino acids that are noncompatible in their physicochemical properties affect T cell alloreactivity, whereas in the α helices, both compatible and noncompatible amino acids affect CTLp outcome. Positions 62, 63, 73, 76, 77, 80, 99, 116, 138, 144, 147, and 163 were bivariately associated with CTLp outcome, irrespective of the total number of amino acid differences. In multivariate analysis, positions 62, 63, 73, 80, 116, 138, 144, and 163 were found to be most predictive for negative CTLp outcome. These results formed the basis for a weighted predictive mismatch score; pairs with the highest mismatch scores are estimated to be 13 times more likely to have a negative CTLp. This new algorithm may be a tool in donor selection for hematopoietic stem cell transplantation.

Reduction in Incidence of Severe Infections by Transplantation of High Doses of Haploidentical T Cells Selectively Depleted of Alloreactive Units

Abstract 3020▪FN2▪This icon denotes a clinically relevant abstract Background:The intensive T-cell depletion of the graft accompanying haploidentical stem cell transplantation (SCT) delays immune reconstitution and results in frequent and rapidly lethal infectious complications. The ability to accelerate immune reconstitution following HLA-haploidentical-SCT would extend safe transplantation to the large number of patients who do not have an HLA-matched donor. Methods:Twenty-seven adults with very high-risk malignancy entered a Phase I clinical trial of haplo-identical T-cell depleted allogeneic SCT followed by an immunotherapeutic strategy consisting of alloreactive T-lymphocyte depleted cells to accelerate immune reconstitution (ATIR) while preventing graft-versus-host disease (GVHD). Selective elimination of host-reactive T cells was achieved using a dibromorhodamine-based photodepletion approach. All stem cell grafts underwent in vitro immunomagnetic T cell depletion using CD34+ positive cell selection (Miltenyi). The myeloablative regimen consisted of TBI (1200 cGy), thiotepa (5 mg/kg), ATG (12.5 mg/kg) and fludarabine (200 mg/m2). No GVHD prophylaxis was administered. Results:Eight patients were enrolled and subsequently removed from the study because of leukemia relapse (n=4) or late identification of an unrelated donor (n=4). All 8 patients died. Nineteen patients (11 M, 8 F) with very high-risk hematologic malignancies (mostly refractory or relapsed acute myeloid leukemia (10) and myelodysplastic syndromes (4), and refractory biphenotypic leukemia (1), CLL (2), CML (1) and NHL (1)) proceeded with the trial. Median age was 54 years (range: 20–62). Patient and disease characteristics were similar between patient cohorts. Patients received incremental doses of ATIR cells, from 1×104 to 5×106 CD3 cells/kg at a median of 30 days (range: 28–39) after SCT. Greater than 90% of activated (CD25+CD44+) CD4 and CD8 T cells (p<0.004) and anti-host cytotoxic T lymphocyte precursors (CTLp) (p=0.0008) were depleted from the donor lymphocyte infusions (DLI). Interferon- γ responses against CMV, EBV and Influenza peptides were maintained post-photodepletion. Naive (TNaive) and central memory (TCM) T cells were also preserved, but mature effector memory populations (TEMRA) decreased after photodepletion. All patients showed complete donor chimerism and durable hematologic engraftment. No patients developed grade III-IV acute GVHD. Acute GVHD grade II developed in 4 patients at a median of 102 days post-transplant (range 45–125), 3 of these belonging to the 2 highest dose groups (2.0–5.0×106 CD3 cells/kg). Five patients developed de novo extensive chronic GVHD at a median of 4.8 months post-transplant. All patients responded rapidly to oral immunosuppression lasting for a median of 6.3 months, with only one patient treated for up to 17 months. NK (CD56) and B (CD19) cells were the first to recover, approximately 4 weeks post-transplant. Patients administered the highest T-cell doses (2.0–5.0×106 CD3 cells/kg) showed earlier reconstitution of CD3, CD4 and CD8 cells (all p≤0.02). The administration of increasing ATIR cell doses yielded gradually increasing proportions of TNaive of CD4 phenotype in the first 18 weeks, and of CD8 phenotype between weeks 18 and 36 post-ATIR. Time to the first infection was delayed in patients receiving high ATIR doses (p=0.015). During the first 6 months post-ATIR, the number of patients without infections was only 1 (14%) of the 7 low T-cell doses patients but increased to 8 (67%) in the high T-cell dose group (p=0.027). The overall survival was 47.4±22.4% (±95% confidence interval) and the event-free-survival 36.8±21.7% at 2 years, with a median follow-up of survivors of 4.0 years (range 3.1–5.1 years). Transplant-related mortality was decreased in the high (19.2%) versus the low ((65.7%) T-cell dose group, with a similar trend for improved survival in the high T-cell dose group (p=0.078). Conclusions:Post-transplant immunotherapy with photodepleted DLI decreased the incidence and severity of infections without inducing severe GVHD. These results document the benefits of administrating selectively depleted T-cells after haploidentical transplantation. Disclosures:Roy:Kiadis Pharma: Research Funding. Mielke:Kiadis Pharma: Research Funding. Egeler:Kiadis Pharma: Employment.

Human Telomerase Reverse Transcriptase-Specific T-Cell Receptor Gene Transfer Redirects T-Lymphocytes to Exert Effective Antitumor Reactivity Against Adult T-Cell Leukemia,

Abstract 4169 Background and PurposeCurrently adult T-cell leukemia (ATL) is one of the most chemotherapy-resistant T cell malignancies and the prognosis of ATL patients still remains very poor. Although several novel treatment options, for example anti-chemokine receptor 4 (CCR4) monoclonal antibody, look promise, development of other novel treatment options still remains warranted. Currently activated status of human telomerase reverse transcriptase (hTERT) in ATL cells has been underscored. Thus, in this study, in order to develop a novel T cell-based immunotherapy for the treatment of ATL, we investigated the feasibility of redirected T-cell using hTERT-specific T-cell receptor (TCR) gene transfer. MethodsApproval for this study was obtained from the Institutional Review Board of Ehime University Hospital. HLA-A*24:02-restricted and hTERT461–469epitope (residues: VYGFVRACL)-specific TCR -a/b genes (Tajima K et al. Int J Cancer 2004) cloned from our established hTERT-specific CTL clone were inserted into a novel GaLV-pseudotyped retroviral vector encoding built-in siRNAs for constant regions of endogenous TCR a /b genes (hTERT-siTCR vector). hTERT-siTCR vector was transfected into normal CD8+ T cells in RetroNectin (Takara Bio Inc.) coated-plates and these redirected CD8+ T cells (hTERT-siTCR CD8) were used as effector cells. HLA-A*24:02+ or A*24:02− ATL cell lines, HTLV-1 infected T-cell lines and freshly isolated ATL cells from patients were examined as target cells. All patients gave written informed consents in accordance with the declaration of Helsinki. Normal HLA-A*24:02+CD4+ T cells andHLA-A*24:02+cord blood CD34+ mononuclear cells (CB-CD34+) were similarly obtained from healthy volunteers. Expression of hTERT mRNA and hTERT protein in ATL cell lines, freshly isolated ATL cells, and HTLV-1 infected T cells were evaluated by quantitative real time PCR (QRT-PCR) using DCt method and Western blotting. Antileukemia reactivity mediated by hTERT-siTCR CD8 against ATL cells were examined by standard 51chromium release assay and flow-based CD107a assay. For the safety assessment, effector cells-mediated cytotoxicity against CB-CD34+ cells as normal hematopoietic progenitors was similarly examined. Peripheral blood mononuclear cells (PBMCs) from ATL patients were obtained on admission and in stable disease or remission after receiving treatments, and hTERT461–469 epitope responsive CD8+ T cells were detected using hTERT461–469peptide/ HLA-A*2402 tetramer or ELISPOT assay. ResultsQRT-PCR revealed that both freshly isolated ATL cells and ATL cell lines markedly overexpressed hTERT mRNA, while that in normal CD4+ cells and CB-CD34+ cells was less than undetectable. hTERT-siTCR CD8 cells were more than 40% positive for hTERT/HLA-A*2402 tetramer and successfully displayed HLA-A*2402-restricted and hTERT461–469-specific cytocidal effect against target-loaded C1R-A24 cells. Additionally, hTERT-siTCR CD8 cells successfully discriminated A*2402+ ATL cell lines (ATN-1, TL-Su) from A*2402− ones (TLO-m1, HUT-102); those all cell lines similarly overexpressed hTERT. Furthermore, those effector cells successfully killed freshly isolated HLA-A*2402+ ATL cells, but not A*2402− ones. Finally, hTERT461–469-specific cytotoxic T-lymphocyte precursors were detected at variable levels in PBMCs obtained from HLA-A*2402+ ATL patients, which shows hTERT overexpressed by ATL cells may be naturally processed in vivo. ConclusionAlthough much further invetigations are warranted, in our preliminary study, we have shown for the first time that hTERT could be a potential therapeutic target of redirected T cell-based immunotherapy for the treatment of ATL. We are now in detail examining efficacy and safety of those redirected T cells using xenograft mouse model. Disclosures:No relevant conflicts of interest to declare.

Cetuximab ± chemotherapy enhances dendritic cell‐mediated phagocytosis of colon cancer cells and ignites a highly efficient colon cancer antigen‐specific cytotoxic T‐cell response in vitro

Cetuximab is a human/mouse chimeric IgG1 monoclonal antibody (mAb) to epidermal growth factor receptor, approved for colorectal carcinoma treatment in combination with chemotherapy. The immune-mediated effects elicited by its human fraction of crystallization moiety might critically contribute to the overall anti-tumor effectiveness of the antibody. We therefore investigated cetuximab ability to promote colon cancer cell opsonization and phagocytosis by human dendritic cells (DCs) that are subsequently engaged in antigen-cross presentation to cytotoxic T-lymphocyte (CTL) precursors. Human colon cancer cell lines were evaluated for susceptibility to DC-mediated phagocytosis before and after treatment with chemotherapy ± cetuximab in vitro. Human DCs loaded with control or drug-treated cetuximab-coated colon cancer cells were used to in vitro generate cytotoxic T cell clones from peripheral blood mononuclear cells of human leucocyte antigen-A(*)02.01(+) donors. T-cell cultures were characterized for immune-phenotype and tumor-antigen specific CTL activity. The results confirmed that treatment of tumor cells with irinotecan + L-folinate + 5-flurouracil (ILF) or with gemcitabine + ILF increased tumor antigen expression. Moreover, malignant cells exposed to chemotherapy and cetuximab were highly susceptible to phagocytosis by human DCs and were able to promote their activation. The consequent DC-mediated cross-priming of antigens derived from mAb-covered/drug-treated cancer cells elicited a robust CTL anti-tumor response. On the basis of our data, we suggest a possible involvement of CTL-dependent immunity in cetuximab anti-cancer effects.

A MAGE-C2 antigenic peptide processed by the immunoproteasome is recognized by cytolytic T cells isolated from a melanoma patient after successful immunotherapy

We have pursued our analysis of a melanoma patient who showed almost complete tumor regression following vaccination with MAGE-A1 and MAGE-A3 antigens. We previously described high frequencies of tumor-specific CTL precursors in blood samples collected after but also before vaccination. A set of CTL clones were derived that recognized antigens different from those of the vaccine. Two of these antigens were peptides encoded by another MAGE gene, MAGE-C2. Here we describe the antigen recognized by another tumor-specific CTL clone. It proved to be a third antigenic peptide encoded by gene MAGE-C2, ASSTLYLVF. It is presented by HLA-B57 molecules and proteasome-dependent. Tumor cells exposed to interferon-gamma (IFN-γ) were better recognized by the anti-MAGE-C2(42-50) CTL clone. This mainly resulted from a better processing of the peptide by the immunoproteasome as compared to the standard proteasome. Mass spectrometric analyses showed that the latter destroyed the antigenic peptide by cleaving between two internal hydrophobic residues. Despite its higher "chymotryptic-like" (posthydrophobic) activity, the immunoproteasome did not cleave at this position, in line with the suggestion that hydrophobic residues immediately downstream from a cleavage site impair cleavage by the immunoproteasome. We previously reported that one of the other MAGE-C2 peptides recognized by CTL from this patient was also better processed by the immunoproteasome. Together, these results support the notion that the tumor regression of this patient was mediated by an antitumor response shaped by IFN-γ and dominated by CTL directed against peptides that are better produced by the immunoproteasome, such as the MAGE-C2 peptides.

Discontinuation of calcineurin inhibitors treatment allows the development of FOXP3+ regulatory T-cells in patients after kidney transplantation

This study investigated specific gene expression profiles in patients with donor-specific cytotoxic-hyporesponsiveness, reflected by cytotoxic T-lymphocyte precursor frequency (CTLpf). The effect of calcineurin inhibitor (CNI) withdrawal was studied on markers for cytotoxicity (perforin, granzyme B), apoptosis (Fas,FasL), Th1 and Th2 cytokines (IL-2, IL-10), Th1 and Th2 transcription factors (T-bet, GATA 3), Th17 transcription factor and cytokine (RORγt, IL-17), and for immune regulation/activation (CD25, FOXP3). Peripheral blood samples from renal allograft recipients (n = 18) more than two yr after transplantation with stable renal function were analyzed before and four months after CNI withdrawal. Additionally, systolic and diastolic blood pressure, cholesterol, serum creatinine and proteinuria were evaluated, and no significant differences were measured before and after CNI withdrawal. However, CNIs' discontinuation influenced peripheral gene expression profiles. After CNI withdrawal, the mRNA expression of Granzyme B, Perforin, Fas, FasL, T-bet, GATA3 and CD25 were significantly lower than during CNI treatment. After CNI discontinuation, donor-specific CTLpf decreased, while FOXP3 expression discriminated between detectable and non-detectable donor-specific cytolysis reactivity; FOXP3 transcript values were highest in absence of donor-specific cytotoxicity (p < 0.01). Our study shows CNI withdrawal in stable kidney transplant recipients two yr after transplantation is safe. Moreover, discontinuation of CNIs' treatment allows FOXP3+ regulatory T-cells development, resulting in a significant decrease of anti-donor immune reactivity.