Autologous Cytotoxic T Lymphocytes Research Articles

Long-lasting responses with chemotherapy followed by T-cell therapy in recurrent or metastatic EBV-related nasopharyngeal carcinoma.

Refractory or metastatic nasopharyngeal carcinoma (NPC) patients have a poor prognosis due to the lack of effective salvage treatments and prolonged survival by means of combination chemotherapy being described only for a minority of younger patients with oligometastatic disease. Targeting the Epstein - Barr virus (EBV) proteins expressed in NPC cells has been shown to be a feasible strategy that could help control systemic disease. Between 2011 and 2014, 16 patients with recurrent/metastatic EBV-NPC received first-line chemotherapy (CT) followed by 2 doses of autologous cytotoxic EBV specific T-lymphocytes (15-25 x 107 total cells/dose, 2 weeks apart), based on our previous studies showing the feasibility and efficacy of this infusion regimen. Cumulative overall survival (OS) and median OS were analysed in the whole population and according to specific clinical and biological parameters. All patients received the planned T-cell therapy schedule, 9 after reaching partial (n=5) or complete (n=4) disease remission with CT, and 7 after failing to obtain benefit from chemotherapy. No severe adverse events were recorded. Patients who received cytotoxic T-lymphocytes (CTLs) had a cumulative 10-year OS of 44%, with a median OS of 60 months (95% CI 42-62). Patients responding to CT, with oligometastatic disease (<3 disease sites), and plasma EBV-DNA <1000 copies/mL had a better outcome. Autologous EBV-specific CTLs transplanted following conventional first-line CT demonstrated promising efficacy with several patients obtaining long-lasting disease control. The rationale provided by this study, with the crucial role likely played by the timing of CTL administration when trying to induce synergy with conventional treatment needs to be confirmed in a prospective controlled trial.

229-LB: Generation of Human CAR-Treg Cells for Inducing Immunological Tolerance against Diabetogenic T Cells in Human Pancreatic Islets

We have developed pancreatic beta-cell, antigen-specific, Chimeric Antigen Receptor (CAR) T regulatory cells (Tregs) and explored their therapeutic potential against type 1 diabetes (T1D)/Latent Autoimmune Diabetes of Adult (LADA) in human pancreatic tissues ex-vivo. Two human Glutamic Acid Decarboxylase 65 kD isoform (GAD65) B-cell paratopes known to bind to two immunodominant regions in the N-terminal (CAR-N) and Middle (CAR-M) regions of human GAD65 were selected for assembly onto T cell receptors of Tregs. We first show that GAD65-CAR Tregs had a superior proliferation index (3 times than naïve Tregs) and were able to suppress diabetogenic, T cells in-vitro and this suppression was significantly superior to naïve Tregs at 2:1 (Tresp vs Treg) ratio (p&amp;lt;0.004). Further, in ex vivo co-culture experimentation with pancreatic islets, confocal images demonstrated homing of GAD65-CAR Tregs to pancreatic islets as early as 24 hrs. With live immunofluorescence imaging and flow cytometry we demonstrated that GAD65-CAR Tregs from T1D/LADA patient were able to suppress diabetogenic cytotoxic T lymphocytes (CTLs) (p&amp;lt;0.04) in the immune cell microenvironment in presence of heterologous pancreatic islet ex vivo. These findings verify that GAD65-CAR Tregs recognize endogenous GAD65 on isolated islets in an MHC-independent manner, are proliferative robustly and suppress autologous CTLs. Thus, GAD65-CAR Tregs can induce tolerance against T1D diabetogenic T cells and the use of GAD65-CAR Tregs can prove a promising strategy for inducing tolerance in islet transplantation to replace the immunosuppressive drugs in a safe potent manner. Finally, homing of the GAD65-CAR Tregs to the islets, provided evidence for antigen-specific re-directionality. The robust proliferation and superior suppression of diabetogenic T cells by GAD65-CAR Tregs validate the functionality of GAD65-CAR Tregs in inducing tolerance in T1D patients and host vs graft islet rejections. Disclosure S. Imam: None. S. Rafiqi: None. J. C. Jaume: None. Funding University of Toledo

Autologous cytotoxic T-cell receptor T cell therapy (SCG101) against hepatitis B surface antigen phase I/II trial for patients with advanced hepatitis B-related hepatocellular carcinoma.

TPS4181 Background: The overall prognosis of patients with advanced Hepatitis B-related hepatocellular carcinoma (HCC) remains poor, and treatment options are limited. In an IIT study conducted in China (NCT05339321), SCG101 following lymphodepletion (cy-flu) as second-line setting or beyond in patients with Hep B-related HCC has shown acceptable safety profile, reduction in HBsAg levels (&gt;1 log) and tumor control. A phase I/II trial has been initiated to evaluate safety and RP2D of autologous SCG101 T cell therapy in Hepatitis B virus-related HCC (NCT05417932). Methods: This multicenter, open-label, 3+3 dose-escalation phase I trial is ongoing across 7 sites in the United States, Singapore and Hong Kong SAR, and a parallel study is done in mainland China across 8 sites. Eligible patients have a histologically confirmed HCC, not amenable to curative intent (surgery or locoregional therapy) and failed ≥2 standard line of therapies, match any of HLA-A*02:01, *02:02, *02:03, *02:04, *02:07, *02:09,*02:16, have positive serum (or tumor) HBsAg, HBV-DNA &lt;2000 IU/ml, BCLC B or C, CP≤7, with measurable disease at screening (mRECIST and RECIST v1.1), ECOG PS&lt;1. Patients with CNS metastasis, prior liver transplant, autoimmune disease or prior cell therapy are excluded. SCG101 T cells is manufactured in Singapore under cGMP and ship to all study sites across the U.S, Singapore and Hong Kong SAR, while SCG101 T cells for the China sites is manufactured in SCG Cell Therapy’s China facility. Patients will receive a single dose of SCG101 T cell therapy following lymphodepletion (cy-flu). Endpoints include safety, MTD and RP2D. Secondary endpoints include ORR, DOR, PFS, OS, changes in serum HBsAg. Safety is monitored together with Data Review Committee. The study is actively enrolling, and the phase II portion of the trial is expected to be initiated by H2 2023. Autologous SCG101 T cell therapy may provide a new treatment option for patients with advanced Hepatitis B virus-related HCC. Clinical trial information: NCT05417932 .

Use of B cell–bound HLA-A2 class I monomers to generate high-avidity, allo-restricted CTLs against the leukemia-associated protein Wilms tumor antigen

AbstractRecent studies have detected Wilms tumor antigen (WT1)-specific cytotoxic T lymphocytes (CTLs) in patients with acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) and demonstrated that most of these CTLs were low avidity. Although HLA-mismatched donors can mount high-avidity CTLs against HLA-A2-presented peptides of WT1, a dominant anti-alloimmune response usually obscures detection of peptide-specific CTLs. Here we explored the feasibility of using recombinant HLA-A2 monomers containing single peptide epitopes as immunogens to generate peptide-specific CTLs from allogeneic donors. We demonstrate that the coating of HLA-A2- B lymphocytes with A2/peptide monomers provides a strong stimulus for autologous peptide-specific CTLs. After 3 to 5 rounds of stimulation a population of CD8+ T cells binding A2/peptide tetramers is easily detectable by fluorescence-activated cell sorting analysis. Furthermore, sorted A2/WT1 tetramer-positive CTLs display strong cytotoxic activity against leukemia cells expressing WT1 endogenously but not against WT1- human tumor cells. Thus, HLA/peptide monomers may be useful to isolate peptide-specific donor lymphocytes for treatment of patients with leukemia after HLA-mismatched transplantation. (Blood. 2004;103:4613-4615)

TUMOR INFLATING LYMPHOCYTES. PURIFICATION, EXPANDING AND CYTOTOXICITY ANALISYS ON PRIMARY TUMOR CULTURES

Tumor Infiltrating Lymphocytes (TILs) is one of the most promising sources of autologous cytotoxic T-cells for adoptive immunotherapy, which has already shown high efficiency in the treatment of metastatic melanoma. In this study, we isolated TILs from surgical material obtained by resection of solid tumors (primary and metastatic adenocarcinomas of various localization, melanoma, glioblastoma), studied their population composition and developed protocols for the purification expanding, and activation of CD4 +, CD8 + cytotoxic antitumor lymphocytes. Cells were obtained from samples of resected tumors in 15 patients; in each case we obtain an autologous pair: the primary tumor culture and the TILs culture. We could isolate viable lymphocytes in 100% of cases. Isolated TILs were successfully expanded in our specialized medium using various combinations of IL-2 / IL-15 / IL-21 // IL-7, anti-CD3 and anti-CD28. Immunophenotyping showed that the obtained TILs are a heterogeneous mixture of CD4 +, CD8 + cells containing populations of CD3 +, CD4, CD8 cells, CD4 + CD25 + CD127 T-regulatory cells, CD45RA, CCR7 + memory T-cells, CD56 + CD16- NK cells. The initial cultures of TILs were also characterized by a high level of PD1 expression, indicating their low antitumor cytotoxicity. Using different protocols of isolation, expansion, and activation, we obtained a cell preparation containing 80% of CD8+ PD1 activated TILs in an amount sufficient for adoptive therapy (500106 or more). An in vitro study of the cytotoxicity of obtained TILs in primary cultures of homologous tumors using RTCA Icelligence showed high cytotoxicity, providing almost 100% tumor cell death. Our developed protocol for the production and activation of TILs can be recommended for the phase I-II clinical trials of adoptive immunotherapy of recurrent, highly metastatic solid tumors.

The efficacy and safety of Epstein-Barr virus-specific antigen peptide-activated cytotoxic T-cells treatment for refractory or recurrent angioimmunoblastic T-cell lymphoma: A prospective clinical observational study.

The efficacy and safety of Epstein-Barr virus (EBV)-specific antigen peptide-activated cytotoxic T lymphocytes (CTLs) in the treatment of refractory or recurrent angioimmunoblastic T-cell lymphoma (AITL) was determined in this prospective one-arm clinical study. Seven males and two females were enrolled with a median age of 70 years. The tumor stages were all stage III and IV. All patients had group B symptoms and IPI scores of 3 to 5 points. All patients received chemotherapy before CTLs infusion which the median chemotherapy cycle was three. The diseases states before CTLs included five cases of disease progression (PD), two cases of recurrence (R), and two cases with residual lesions after chemotherapy. Eight patients received HLA-haploidentical EBV-specific CTLs, and one patient chose autologous CTLs. The number of transfused cells was 1.67 to 2.38 × 1010 for one course of CTLs therapy. One patient was treated with three courses of CTLs, three patients were treated with two courses of CTLs, and five patients were treated with one course of CTLs. During the infusion, eight patients had fever, one patient had rash, and no graft-vs-host diseases were observed. The EBV-DNA decreased by more than two orders of magnitude in six patients, and the response rate was 66.7%. Two patients of PD status achieved complete remission (CR), one patient of PD status achieved partial remission, two patients with residual lesions after chemotherapy achieved CR, and four patients had no response. The objective remission rate was 55.6%. After the median follow-up of 14.5 months, five patients died, and three patients were completely relieved while one patient was lost during follow-up. The 3-year overall survival was 44.4% and 3-year progression-free survival was 33.3%. EBV-specific antigen peptide-activated CTLs showed positive effect in certain patients with refractory and recurrent AITL with high clinical safety.

The synergistic antitumor effect of arsenic trioxide combined with cytotoxic T cells in pulmonary metastasis model of colon cancer.

Adoptive T cell therapy, including cytotoxic T lymphocytes (CTLs), represents a promising non-toxic anticancer strategy. The effects of this therapy can be impaired by tumor-infiltrated regulatory T cells (Tregs). Autologous murine CTLs acquired using cryopreservation exhibited a cytotoxic effect equivalent to that of conventional CTLs. The killing activity of CTLs was enhanced significantly using arsenic trioxide (ATO), accompanied by reduction in Tregs in vitro. Results using a pulmonary metastasis model of colon cancer indicated that compared with the control group, ATO group, and CTLs group, metastatic node number decreased significantly (p<0.001, p<0.001, p<0.001, respectively) and survival time was prolonged (p<0.001, p=0.669, p=0.158, respectively) in the ATO plus CTLs group. The number of infiltrated Foxp3+ Tregs decreased in the tumor center, but increased in the peri-tumor tissue. Our results indicate that this approach represents a practical protocol for acquiring autologous CTLs and a feasible strategy that uses a synergistic combination of ATO plus CTLs to treat pulmonary metastases of colon cancer.

Role of HIV-specific CD8+ T cells in pediatric HIV cure strategies after widespread early viral escape.

Recent studies have suggested greater HIV cure potential among infected children than adults. A major obstacle to HIV eradication in adults is that the viral reservoir is largely comprised of HIV-specific cytotoxic T lymphocyte (CTL) escape variants. We here evaluate the potential for CTL in HIV-infected slow-progressor children to play an effective role in "shock-and-kill" cure strategies. Two distinct subgroups of children were identified on the basis of viral load. Unexpectedly, in both groups, as in adults, HIV-specific CTL drove the selection of escape variants across a range of epitopes within the first weeks of infection. However, in HIV-infected children, but not adults, de novo autologous variant-specific CTL responses were generated, enabling the pediatric immune system to "corner" the virus. Thus, even when escape variants are selected in early infection, the capacity in children to generate variant-specific anti-HIV CTL responses maintains the potential for CTL to contribute to effective shock-and-kill cure strategies in pediatric HIV infection.

Induction and Analysis of Cytotoxic T-Lymphocytes that Recognize Autologous Oral Squamous Cell Carcinoma.

It is essential that cytotoxic T-lymphocytes (CTLs) recognize tumor-associated antigens (TAAs) and exhibit antitumor effects in immunological responses for tumor rejection. However, only a few cases with oral squamous cell carcinoma (OSCC) have been studied for these antitumor mechanisms. We established a cancer cell line and autologous CTL pair for identifying TAAs in OSCC, despite difficulties in establishing such a pair. A cell line (OTM) from a primary lesion and a CTL line (TcOTM) were derived from OSCC in a 64-year-old female patient. CTL clones were generated by repetitive limiting dilutions. Accurate characterization was performed by in vitro analysis. The TcOTM clone showed specific cytotoxic activity against OTM cells in an human leukocyte antigen (HLA)-A24-restricted manner. Furthermore, it exerted cytotoxicity against the allogenic HLA-A24 cell lines. These data indicated that the TcOTM clone recognized a TAA presented by HLA-A24 cells. This unique method will allow for identification of unknown TAAs for OSCC in the future.

Immunogenicity of oncolytic vaccinia viruses JX-GFP and TG6002 in a human melanoma in vitro model: studying immunogenic cell death, dendritic cell maturation and interaction with cytotoxic T lymphocytes.

Oncolytic virotherapy is an emerging immunotherapeutic modality for cancer treatment. Oncolytic viruses with genetic modifications can further enhance the oncolytic effects on tumor cells and stimulate antitumor immunity. The oncolytic vaccinia viruses JX-594-GFP+/hGM-CSF (JX-GFP) and TG6002 are genetically modified by secreting granulocyte-macrophage colony-stimulating factor (GM-CSF) or transforming 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). We compared their properties to kill tumor cells and induce an immunogenic type of cell death in a human melanoma cell model using SK29-MEL melanoma cells. Their influence on human immune cells, specifically regarding the activation of dendritic cells (DCs) and the interaction with the autologous cytotoxic T lymphocyte (CTL) clone, was investigated. Melanoma cells were infected with either JX-GFP or TG6002 alone or in combination with 5-FC and 5-FU. The influence of viral infection on cell viability followed a time- and multiplicity of infection dependent manner. Combination of virus treatment with 5-FU resulted in stronger reduction of cell viability. TG6002 in combination with 5-FC did not significantly strengthen the reduction of cell viability in this setting. Expression of calreticulin and high mobility group 1 protein (HMGB1), markers of immunogenic cell death (ICD), could be detected after viral infection. Accordingly, DC maturation was noted after viral oncolysis. DCs presented stronger expression of activation and maturation markers. The autologous CTL clone IVSB expressed the activation marker CD69, but viral treatment failed to enhance cytotoxicity marker. In summary, vaccinia viruses JX-GFP and TG6002 lyse melanoma cells and induce additional immunostimulatory effects to promote antitumor immune response. Further investigation in vivo is needed to consolidate the data.

T-Cell Therapy Using Interleukin-21-Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression.

Purpose Peripheral blood-derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti-CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8+ T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses, highlighting the promise of this strategy.

CMRF-56+ blood dendritic cells loaded with mRNA induce effective antigen-specific cytotoxic T-lymphocyte responses

ABSTRACTThere are numerous transcriptional, proteomic and functional differences between monocyte-derived dendritic cells (Mo-DC) and primary blood dendritic cells (BDC). The CMRF-56 monoclonal antibody (mAb) recognizes a cell surface marker, which is upregulated on BDC following overnight culture. Given its unique ability to select a heterogeneous population of BDC, we engineered a human chimeric (h)CMRF-56 IgG4 mAb to isolate primary BDC for potential therapeutic vaccination. The ability to select multiple primary BDC subsets from patients and load them with in vitro transcribed (IVT) mRNA encoding tumor antigen might circumvent the issues limiting the efficacy of Mo-DC. After optimizing and validating the purification of hCMRF-56+ BDC, we showed that transfection of hCMRF-56+ BDC with mRNA resulted in efficient mRNA translation and antigen presentation by myeloid BDC subsets, while preserving superior DC functions compared to Mo-DC. Immune selected and transfected hCMRF-56+ BDC migrated very efficiently in vitro and as effectively as cytokine matured Mo-DC in vivo. Compared to Mo-DC, hCMRF-56+ BDC transfected with influenza matrix protein M1 displayed superior MHC peptide presentation and generated potent antigen specific CD8+ T-cell recall responses, while Wilms tumor 1 (WT1) transfected CMRF-56+ BDC generated effective primary autologous cytotoxic T-cell responses. The ability of the combined DC subsets within hCMRF-56+ BDC to present mRNA delivered tumor antigens merits phase I evaluation as a reproducible generic platform for the next generation of active DC immune therapies.

CADM1/TSLC1 Identifies HTLV-1-Infected Cells and Determines Their Susceptibility to CTL-Mediated Lysis.

Human T cell lymphotropic virus-1 (HTLV-1) primarily infects CD4+ T cells, causing inflammatory disorders or a T cell malignancy in 5% to 10% of carriers. The cytotoxic T lymphocyte (CTL) response is a key factor that controls the viral load and thus the risk of disease. The ability to detect the viral protein Tax in primary cells has made it possible to estimate the rate at which Tax-expressing infected cells are eliminated by CTLs in persistently infected people. However, most HTLV-1-infected cells are Tax–at a given time, and their immunophenotype is poorly defined. Here, we aimed to identify a cell-surface molecule expressed by both Tax+ and Tax–HTLV-1-infected cells and use it to analyse the CTL response in fresh peripheral blood mononuclear cells. Cell adhesion molecule 1 (CADM1/TSLC1) was the best single marker of HTLV-1 infection, identifying HTLV-1-infected cells with greater sensitivity and specificity than CD25, CCR4 or ICAM-1. CADM1+CD4+ T cells carried a median of 65% of proviral copies in peripheral blood. In a cohort of 23 individuals, we quantified the rate of CTL-mediated killing of Tax+ and Tax−CADM1+ cells. We show that CADM1 expression is associated with enhanced susceptibility of infected cells to CTL lysis: despite the immunodominance of Tax in the CTL response, Tax+CADM1– cells were inefficiently lysed by CTLs. Upregulation of the CADM1 ligand CRTAM on CD8+ T cells correlated with efficient lysis of infected cells. Tax–CADM1+ cells were lysed at a very low rate by autologous CTLs, however, were efficiently killed when loaded with exogenous peptide antigen. High expression of CADM1 on most HTLV-1-infected cells in the face of enhanced CTL counterselection implies that CADM1 confers a strong benefit on the virus.

Case report: long-lasting response in a patient with metastatic renal cell cancer receiving antitumor cytotoxic T lymphocytes.

Adoptive immunotherapy can be a therapeutic option to treat cancer patients with advanced-stage disease refractory to conventional therapies. We used T-cell therapy with autologous antitumor cytotoxic T lymphocytes (CTLs) in a patient with metastatic renal cell carcinoma. Autologous antitumor CTLs obtained by stimulating CD8-enriched cells with dendritic cells pulsed with irradiated apoptotic tumor cells and expanded in vitro were transfused on days +14 and +28 following chemotherapy and every 2 to 4 months thereafter. Treatment with high doses of antitumor CTLs proved to be safe and induced immunological responses and long-lasting clinical benefit in our patient.

Identification of Four-Jointed Box 1 (FJX1)-Specific Peptides for Immunotherapy of Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is highly prevalent in South East Asia and China. The poor outcome is due to late presentation, recurrence, distant metastasis and limited therapeutic options. For improved treatment outcome, immunotherapeutic approaches focusing on dendritic and autologous cytotoxic T-cell based therapies have been developed, but cost and infrastructure remain barriers for implementing these in low-resource settings. As our prior observations had found that four-jointed box 1 (FJX1), a tumor antigen, is overexpressed in NPCs, we investigated if short 9–20 amino acid sequence specific peptides matching to FJX1 requiring only intramuscular immunization to train host immune systems would be a better treatment option for this disease. Thus, we designed 8 FJX1-specific peptides and implemented an assay system to first, assess the binding of these peptides to HLA-A2 molecules on T2 cells. After, ELISPOT assays were used to determine the peptides immunogenicity and ability to induce potential cytotoxicity activity towards cancer cells. Also, T-cell proliferation assay was used to evaluate the potential of MHC class II peptides to stimulate the expansion of isolated T-cells. Our results demonstrate that these peptides are immunogenic and peptide stimulated T-cells were able to induce peptide-specific cytolytic activity specifically against FJX1-expressing cancer cells. In addition, we demonstrated that the MHC class II peptides were capable of inducing T-cell proliferation. Our results suggest that these peptides are capable of inducing specific cytotoxic cytokines secretion against FJX1-expressing cancer cells and serve as a potential vaccine-based therapy for NPC patients.

Lentivirus-induced ‘Smart’ dendritic cells: Pharmacodynamics and GMP-compliant production for immunotherapy against TRP2-positive melanoma

Monocyte-derived conventional dendritic cells (ConvDCs) loaded with melanoma antigens showed modest responses in clinical trials. Efficacy studies were hampered by difficulties in ConvDC manufacturing and low potency. Overcoming these issues, we demonstrated higher potency of lentiviral vector (LV)-programmed DCs. Monocytes were directly induced to self-differentiate into DCs (SmartDC-TRP2) upon transduction with a tricistronic LV encoding for cytokines (granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4)) and a melanoma antigen (tyrosinase-related protein 2 (TRP2)). Here, SmartDC-TRP2 generated with monocytes from five advanced melanoma patients were tested in autologous DC:T cell stimulation assays, validating the activation of functional TRP2-specific cytotoxic T lymphocytes (CTLs) for all patients. We described methods compliant to good manufacturing practices (GMP) to produce LV and SmartDC-TRP2. Feasibility of monocyte transduction in a bag system and cryopreservation following a 24-h standard operating procedure were achieved. After thawing, 50% of the initial monocyte input was recovered and SmartDC-TRP2 self-differentiated in vitro, showing uniform expression of DC markers, detectable LV copies and a polyclonal LV integration pattern not biased to oncogenic loci. GMP-grade SmartDC-TRP2 expanded TRP2-specific autologous CTLs in vitro. These results demonstrated a simpler GMP-compliant method of manufacturing an effective individualized DC vaccine. Such DC vaccine, when in combination with checkpoint inhibition therapies, might provide higher specificity against melanoma.

Biophysical Characterization of TCR Variants with Reengineered Specificity and Affinity

Autologous cytotoxic T‐cells, CD8+ T‐cells, recognize and target certain cancer cells through an interaction between T‐cell receptors (TCR) and antigen bound major histocompatibility complex (MHC complex) on the target cells. This phenomenon has led to advent of adoptive immunotherapy for melanoma. In this therapy, T‐cells from the patient are genetically engineered to express TCRs that respond specifically to melanoma associated antigens and then reintroduced into patients. The first set of clinical trials examining this approach in humans utilized DMF4 and DMF5 TCRs, and showed that DMF4 TCR led to 13% tumor regression, whereas DMF5 TCR led to 30% tumor regression. Upon biophysical characterization it was shown that the improvement in immunogenicity from DMF5 is due to its higher affinity towards the cognate melanoma antigen. This made higher affinity TCRs therapeutically relevant. Traditionally higher affinity TCRs have been generated via in vitro selection techniques. However, in vitro selection methods have inherent limitations. Most notably, gaining peptide independent TCR binding. Structure guided design (SGD) provides an alternative technique for developing therapeutically relevant TCRs. It is an in silico method that combines structural and biophysical information with a modeling software to generate high affinity TCR variants, allowing for highly controlled manipulation of affinity and specificity. While SGD of TCRs is promising, it is in early stages of development and requires further optimization. Structural and biophysical characterization of TCR variants that have high affinity or re‐engineered specificity will help improve TCR SGD.

Establishment and operation of a Good Manufacturing Practice-compliant allogeneic Epstein-Barr virus (EBV)-specific cytotoxic cell bank for the treatment of EBV-associated lymphoproliferative disease.

Epstein-Barr virus (EBV) is associated with several malignancies, including post-transplant lymphoproliferative disorder (PTLD). Conventional treatments for PTLD are often successful, but risk organ rejection and cause significant side effects. EBV-specific cytotoxic T lymphocytes (CTLs) generated in vitro from peripheral blood lymphocytes provide an alternative treatment modality with few side effects, but autologous CTLs are difficult to use in clinical practice. Here we report the establishment and operation of a bank of EBV-specific CTLs derived from 25 blood donors with human leucocyte antigen (HLA) types found at high frequency in European populations. Since licensure, there have been enquiries about 37 patients, who shared a median of three class I and two class II HLA types with these donors. Cells have been infused into ten patients with lymphoproliferative disease, eight of whom achieved complete remission. Neither patient with refractory disease was matched for HLA class II. Both cases of EBV-associated non-haematopoietic sarcoma receiving cells failed to achieve complete remission. Thirteen patients died before any cells could be issued, emphasizing that the bank should be contacted before patients become pre-terminal. Thus, this third party donor-derived EBV-specific CTL cell bank can supply most patients with appropriately matched cells and most recipients have good outcomes.

A Bacterial Flagellin in Combination With Proinflammatory Cytokines Activates Human Monocyte-derived Dendritic Cells to Generate Cytotoxic T Lymphocytes Having Increased Homing Signals to Cancer

Flagellin, the cognate ligand for toll-like receptor 5, has potent adjuvant activity in various vaccines. However, its efficacy in generating dendritic cells (DCs) remains contentious. This study assessed how efficaciously Vibrio vulnificus FlaB (v-FlaB) could be used in generating a potent DC to induce antigen-specific cytotoxic T lymphocytes (CTLs). Mature DCs (mDCs) induced by the combination of v-FlaB/TNFα/IFNα were significantly more potent in inducing specific anticancer immune responses compared with the standard DCs that were maturated by the conventional cytokine cocktail of TNFα/IL-1β/IL-6/PGE(2). The potent mDCs produced a higher level of interleukin (IL)-12p70 and polarized naive CD4(+) T cells more towards Th1-type cells, markedly increased antigen-specific CD8(+) T-cell number and significantly enhanced the induction of lytic enzymes in antigen-specific CD8(+) CTLs and sensitized CD3(+) T cells to produce higher number of interferon (IFN)γ-secreting cells. As a result, the mDCs produced more potent antigen-specific CTLs against the MART-1 and expressed higher levels of homing receptors CCR5 and CXCR3. More importantly, the v-FlaB/TNFα/IFNα-DCs generated from melanoma patients produced strong autologous CTLs with efficient cytotoxic activities. In conclusion, v-FlaB combined with tumor necrosis factor (TNF)α and IFNα can generate potent DCs which produce functionally active CTLs and that may have potential as a potent cancer vaccine.

Demethylating agent decitabine induces autologous cancer testis antigen specific cytotoxic T lymphocytes in vivo.

Cancer testis antigens (CTAs) are a novel group of tumor associated antigens. Demethylating agent decitabine was reported to be able to up-regulate CTAs through its hypomethylation mechanism, thus enhance the immunogenicity of leukemia cells. However, few researches have ever focused on the questions that whether this immunostimulatory effect of decitabine could induce autologous CTA specific cytotoxic T lymphocytes (CTLs) in vivo, and if so, whether this effect contributes to disease control. In this study, we aimed to show that decitabine could induce specific autologous CTLs against some mouse CTAs in leukemia cells in vitro and in vivo. Several mouse CTAs were screened by RT-PCR. CTL specific to one of the CTAs named P1A was detected and sorted by P1A specific dimer by flow cytometry. The activity of specific CTLs was measured by real time RT-PCR. We firstly screened expression of some CTAs in mouse leukemia cells before and after decitabine treatment and found that decitabine treatment did up-regulate expression of many CTAs. Then we measured the CTLs' activity specific to a mouse CTA P1A in vivo and showed that this activity increased after decitabine treatment. Finally, we sorted these in vivo induced P1A specific CTLs by flow cytometry and demonstrated their cytotoxicity against decitabine treated leukemia cells. Our study showed the autologous immune response induced by decitabine in vivo. And more importantly, we firstly proved that this response may contribute to disease control. We believe that this immunostimulatory effect is another anti-cancer mechanism of decitabine, and this special effect would inspire new applications of decitabine in the field of leukemia treatment in the future.