Specific Cytotoxic T Lymphocytes Research Articles

Boosting In Vivo CAR-Redirected Virus-Specific CTLs With Universal-Artificial Antigen Presenting Cells

Virus-specific cytotoxic T lymphocytes (CTLs) targeting EBV and/or CMV and engrafted with chimeric antigen receptors (CARs) can receive appropriate costimulation from professional antigen presenting cells (APCs) that process latent viral antigens and can then target tumor cells through their CAR. In a clinical trial in neuroblastoma patients, CAR-redirected virus specific CTLs persisted for over 6 weeks after infusion and produced complete tumor responses in 3/11 patients. To improve the in vivo expansion and persistence of CAR-redirected virus specific CTLs, we determined whether K562 cells engineered to express viral antigens and either CD40L or OX40L can act as universal artificial APCs (aAPCs) to boost CAR-modified virus-specific CTLs in vivo through a cross-presentation mechanism. Using CMV as the viral antigen model, we found that aAPCs/pp65 produce superior in vitro activation of CMV-specific CTLs from seropositive healthy donors (292±56 IFNγ spot forming cells (SFC)/105 cells) than control non modified K562 (83±25 IFNγ SFC/105 cells). The frequency of CMV-CTLs was further enhanced if the aAPC coexpressed CD40L (aAPCs/pp65/CD40L) (502±104 SFC/105 cells; p<0.001) but not by incorporation of OX40L (aAPCs/pp65/OX40L) (357±40 SFC/105 cells) or the combination CD40L/OX40L (477±91 SFC/105 cells). We then assessed whether these aAPCs can boost in vivo CMV-specific CTL responses in NSG mice engrafted with human cells from CMV-seropositive healthy donors. Spleens isolated from mice that had been vaccinated with either aAPCs/pp65 or aAPCs/pp65/CD40L or aAPCs/pp65/OX40L showed an increased frequency of CMV-specific precursors (57±24 vs. 41±14 vs. 35±17 IFNγ-SFC/105 cells) as compared to mice infused with control aAPCs (27±6) (p=0.005). In contrast to the in vitro experiments, the combination in vivo of aAPCs/CD40L/pp65 and aAPCs/OX40L/pp65 elicited the highest number of CMV-specific precursors (101±21 SFC/105 cells). We then tested if CMV-CTLs retained this response to the aAPC vaccine when the CTLs were grafted with a CAR. As a model we used a CAR targeting the neuroblastoma associated antigen GD2. We found that in vitro stimulation with aAPCs/CD40L/OX40L/pp65 promoted the greatest increase of CMV-specific (925±201 IFNγ SFC/105 cells) and CAR-GD2 responses (2725±585 IFNγ SFC/105 cells). The effector function of CAR+ CMV-CTLs was maintained through both their native TCR and their CAR as indicated by cytotoxicity against both GD2+ target cells (CHLA-255=63±14% at a 20:1 E:T ratio) and pp65-infected cells (59±3%); there was no killing of GD2- targets (Raji = 18%±8%) or of CMV uninfected cells (3%±1%). Sustained persistence of CAR+ CMV-CTLs in response to the aAPCs was also maintained in vivo. Thus, vaccination of NSG mice with aAPCs/CD40L/OX40L/pp65 enhanced the frequency of CAR+ CTLs (range 2.4% - 6.25%) as compared to mice vaccinated with control aAPCs (0.98% - 1.08%). IFNγ ELISpot assays confirmed increased functional frequency of both CAR and pp65 mediated recognition of T cells isolated from mice vaccinated with aAPCs/CD40L/OX40L/pp65 against GD2+ targets (71±24 SFC/105 cells) and pp65-infected targets (85±16) as compared to control mice (23±8 and 41±11, respectively) (p=0.048 and p=0.035). Importantly, vaccination increased the antitumor activity of CAR-CTLs in a xenograft model of neuroblastoma since 48% of the mice vaccinated with aAPCs/CD40L/OX40L/pp65 were tumor free by day 40, while mice vaccinated with control aAPCs uniformly succumbed to the tumor (p≤0.037). In conclusion, we have identified a broadly applicable strategy to stimulate CAR redirected virus-specific CTLs in vivo through their native TCR that lengthens their persistence and thereby increases their antitumor activity. Disclosures:Savoldo:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding.

Frequent T cell responses against immunogenic targets in lung cancer patients for targeted immunotherapy

To date, lung cancer is one of the leading causes of cancer mortality with short overall survival despite adequate therapy. New immunotherapeutic strategies using peptides derived from tumor-associated antigens (TAAs) can induce a specific cytotoxic T cell (CTL) response leading to a targeted tumor cell death. In the present study, we addressed whether there are further significant immunogenic candidate targets that may induce strong immune reactions with a high frequency in lung cancer patients eligible for cellular immunotherapeutic approaches, such as in a polyvalent vaccination approach. In this study, we investigated specific CTL responses of 14 HLA-A*0201-positive patients (of 33 screened patients) with non-small cell lung cancer (NSCLC; n=12) or small cell lung cancer (SCLC; n=2) against several known and novel TAA-derived peptides from lung cancer and/or other tumor entities, by measuring granzyme B (GrB) and/or interferon γ (IFNγ) secretion using enzyme-linked immunospot (ELISpot) analysis. Specific T cell responses could be detected for hTERT (4/13), two MAGE-A3-derived peptides (4/13 and 3/13, respectively), RHAMM (4/14), PRAME (8/14), G250 (7/12), survivin (3/13), HER2 (5/10) and WT1 (2/14), but also novel epitopes derived from Aurora kinase A (4/13) and B (5/13). Additionally, simultaneous CTL responses against the different peptides were examined and specific T cell responses against at least one of these TAAs could be detected in 13/14 (93%) patients. It could be shown that all patients with immune reactions against RHAMM and hTERT showed also immune responses against PRAME. Furthermore, patients with CTL responses against the Aurora kinase A peptide (Aura A1) also demonstrated a response against the Aurora kinase B peptide (Aura B1). Taken together, we showed that these TAA-derived peptides induce frequent specific T cell responses in patients with metastatic lung cancer and are, therefore, novel candidates for targeted immunotherapies and polyvalent approaches.

A constructed HLA-A2-restricted pMAGE-A1278–286tetramer detects specific cytotoxic T lymphocytes in tumour tissuesin situ

To construct a human leucocyte antigen (HLA)-A2-restricted peptide 278-286 of melanoma-associated antigen family A, 1 (pMAGE-A1(278-286)) tetramer to analyse the distribution of cytotoxic T lymphocytes (CTLs) in tumour tissue and tumour-adjacent normal tissue. A HLA-A2-pMAGE-A1(278-286) tetramer was constructed. The distribution of pMAGE-A1(278-286)-specific CTLs was investigated in tumour tissues and tumour-adjacent normal tissues from patients with hepatocellular carcinoma using in situ HLA-A2-pMAGE-A1(278-286) tetramer staining. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis indicated that HLA-A2 heavy and light chain proteins were successfully obtained. The successful construction of the HLA-A2-pMAGE-A1(278-286) monomer was confirmed with Western blot analysis using W6/32 antibody. Flow cytometry confirmed the specific binding of HLA-A2-pMAGE-A1(278-286) tetramer to pMAGE-A1(278-286)-specific CTLs. In situ HLA-A2-pMAGE-A1(278-286) tetramer staining demonstrated that the number of pMAGE-A1(278-286)-specific CTLs in tumour tissues was significantly higher than in tumour-adjacent normal tissues. The HLA-A2-pMAGE-A1(278-286) tetramer was useful for the detection of pMAGE-A1(278-286)-specific CTLs in both tumour tissues and tumour-adjacent normal tissues. In situ tetramer staining is a powerful tool for investigating the distribution of pMAGE-A1278-286-specific CTLs in the tumour microenvironment.

Identification of human leucocyte antigen (HLA)‐A*0201‐restricted cytotoxic T lymphocyte epitopes derived from HLA‐DOβ as a novel target for multiple myeloma

Despite the recent development of effective therapeutic agents against multiple myeloma (MM), new therapeutic approaches, including immunotherapies, remain to be developed. Here we identified novel human leucocyte antigen (HLA)-A*0201 (HLA-A2)-restricted cytotoxic T lymphocyte (CTL) epitopes from a B cell specific molecule HLA-DOβ (DOB) as a potential target for MM. By DNA microarray analysis, the HLA-DOB expression in MM cells was significantly higher than that in normal plasma cells. Twenty-five peptides were predicted to bind to HLA-A2 from the amino acid sequence of HLA-DOB. When screened for the immunogenicity in HLA-A2-transgenic mice immunized with HLA-DOB cDNA, 4 peptides were substantially immunogenic. By mass spectrometry analysis of peptides eluted from HLA-A2-immunoprecipitates of MM cell lines, only two epitopes, HLA-DOB232-240 (FLLGLIFLL) and HLA-DOB185-193 (VMLEMTPEL), were confirmed for their physical presence on cell surface. When healthy donor blood was repeatedly stimulated in vitro with these two peptides and assessed by antigen-specific γ-interferon secretion, HLA-DOB232-240 was more immunogenic than HLA-DOB185-193 . Additionally, the HLA-DOB232-240 -specific CTLs, but not the HLA-DOB185-193 -specific CTLs, displayed an major histocompatibility complex class I-restricted reactivity against MM cell lines expressing both HLA-A2 and HLA-DOB. Taken together, based on the physical presence on tumour cell surface and high immunogenicity, HLA-DOB232-240 might be useful for developing a novel immunotherapy against MM.

Mutations and phylogenetic analysis of the equine influenza virus (H3N8) nucleoprotein isolated in Morocco

The equine influenza (EI) is a highly contagious respiratory viral disease of equines. The aim of the present study was to determine the amino acid mutation sequences of the partial nucleoprotein (NP), which includes four epitopes of the equine leucocyte antigen (ELA) of A/equine/Nador/1/1997 (H3N8). These epitopes are critical for their binding to major histocompatibility molecule complex (MHC) class I and recognition by specific Cytotoxic T Lymphocytes (CTLs). The isolate was subjected to RT-PCR amplifications followed by sequencing analysis. Phylogenetic analysis showed that Moroccan isolate belongs to equine host-specific lineage and more closely related to Italian strains A/equine/Rome/5/1991 and A/equine/Italy/1062/1991. Amino acid sequence comparison of the NP showed that the strain A/equine/Nador/1/1997 has twelve substitutions at the residues T/284/A, A/286/T, R/293/K, I/299/V, V/312/I, N/319/K, S/344/L, V/353/I, M/374/I, C/377/N, N/397/S and R/452/K. All substitutions concerned both the interaction domains NP–NP and NP–PB2. However, the mutation N319 K enhances the NP–PB2 interaction and polymerase activity in mammalian infected cells. S/344/L mutation was located on the FEDLRVSNFI epitope (aa 338–347), this substitution is likely to help the virus to overcome the barrier of cell-mediated immunity of the host. The identified mutations were grouped into two groups, one included residues that facilitate the adaptation and evolution of influenza viruses within the equine lineage such as A/286/T, R/293/K, S/344/L, V/353/I and R/452/K, while the second contained the substitutions which enhance the virulence as polymerase activity (N319K) and mutations that affect CTL epitopes, resulting in an escape from immune surveillance by specific CTLs (S/344/L).

Relationship between effect of lamivudine in the treatment of cirrhotic patients with uncompensated hepatitis B with HBV genotypes and cytotoxic T lymphocyte

To explore relationship between effect of Lamivudine in the treatment of cirrhotic patients with uncompensated hepatitis B with hepatitis B virus (HBV)genotypes and HBV specific cytotoxic T lymphocytes (CTL). 80 cases of uncompensated cirrhotic hepatitis B (40 cases with genotype B and 40 with genotype C), HBV DNA positive, HBeAg positive and human leukocyte antigen (HLA)-A2 positive,were treated with Lamivudine 100 mg/d, one year later, its effect and relationship with HBV genotypes and HBV specific CTL were observed. HBV DNA turned negative:40 cases with genotype B turned negative (100%). In the 9th and 10th month of treatment, there was one case with genotype C had YMDD variation respectively and Adefovir dipivoxil was used for treatment, of the rest 38 cases, HBV DNA of 26 cases (68.42%) turned negative,HBV DNA negative rate of patients with genotype is lower than that of patients with genotype B, chi2 = 14.91, P < 0.01. HBeAg turned negative: 18 cases with genotype B (45%) turned negative, more than that of patients with genotype C (7 cases, 18.42%), chi2 = 6.32, P < 0.05. Peripheral blood HBV specific CTL level: before treatment, it was (0.33 +/- 0.03)% of patients with genotype B,higher than that of patients with genotype C [(0.11 +/- 0.02)%], t = 8.12, P < 0.001. 1 year after treatment: it was (0.44 +/- 0.04)% of patients with genotype B, higher than that before treatment, t = 4.01, P < 0.001, it was also higher than that of patients with genotype C 1 year after treatment [(0.23 +/- 0.03)%], t = 5.63, P < 0.01, alanine amino-transferase (ALT) returned to normal: 38 cases with genotype B (95%) returned to normal, more than that of patients with genotype C (28 cases, 73.68%), X2 = 6.79, P < 0.01. Effect of Lamivudinein the treatment of cirrhotic patients with uncompensated hepatitis B is better in patients with genotype B than patients with genotype C, its mechanism may be related to lower level of HBV specific CTL in patients with genotype C than patients with genotype B.

Identification of HLA ligands and T-cell epitopes for immunotherapy of lung cancer.

Lung cancer is the most common cancer worldwide. Every year, as many people die of lung cancer as of breast, colon and rectum cancers combined. Because most patients are being diagnosed in advanced, not resectable stages and therefore have a poor prognosis, there is an urgent need for alternative therapies. Since it has been demonstrated that a high number of tumor- and stromal-infiltrating cytotoxic T cells (CTLs) is associated with an increased disease-specific survival in lung cancer patients, it can be assumed that immunotherapy, e.g. peptide vaccines that are able to induce a CTL response against the tumor, might be a promising approach. We analyzed surgically resected lung cancer tissues with respect to HLA class I- and II-presented peptides and gene expression profiles, aiming at the identification of (novel) tumor antigens. In addition, we tested the ability of HLA ligands derived from such antigens to generate a CTL response in healthy donors. Among 170 HLA ligands characterized, we were able to identify several potential targets for specific CTL recognition and to generate CD8+ T cells which were specific for peptides derived from cyclin D1 or protein-kinase, DNA-activated, catalytic polypeptide and lysed tumor cells loaded with peptide. This is the first molecular analysis of HLA class I and II ligands ex vivo from human lung cancer tissues which reveals known and novel tumor antigens able to elicit a CTL response.

Nicotine Inhibits Memory CTL Programming

Nicotine is the main tobacco component responsible for tobacco addiction and is used extensively in smoking and smoking cessation therapies. However, little is known about its effects on the immune system. We confirmed that multiple nicotinic receptors are expressed on mouse and human cytotoxic T lymphocytes (CTLs) and demonstrated that nicotinic receptors on mouse CTLs are regulated during activation. Acute nicotine presence during activation increases primary CTL expansion in vitro, but impairs in vivo expansion after transfer and subsequent memory CTL differentiation, which reduces protection against subsequent pathogen challenges. Furthermore, nicotine abolishes the regulatory effect of rapamycin on memory CTL programming, which can be attributed to the fact that rapamycin enhances expression of nicotinic receptors. Interestingly, naïve CTLs from chronic nicotine-treated mice have normal memory programming, which is impaired by nicotine during activation in vitro. In conclusion, simultaneous exposure to nicotine and antigen during CTL activation negatively affects memory development.

Preliminary observation on antitumor effect of HPV58 composite DNA vaccine

To initially observe the antitumor immune of PVAX1-HPV58mE6E7FcGB composite DNA vaccine. Before detecting immune effect of the vaccine, the B16-HPV58E6E7 tumor cell line was built which could steadily express HPV58E6E7 fusion gene. Then, HPV58E6E7-GST fusion protein as an antigen was expressed and purified. Before or after immunized with the vaccine, the C57BL/6 mice were challenged by B16-HPV58E6E7 cells. Anti-tumor transplantation and tumor growth inhibition experiment were performed to observe prevention and treatment effects on the vaccine. Specific humoral and cellular immune responses in the immunized mice were detected by ELISA, enzyme linked immunospot assay (ELISPOT) and cytotoxic T lymphocyte (CTL) method. In the anti-tumor transplantation experiment, tumor formation rate was only 9/15 in the mice which were immunized by PVAX1-HPV58mE6E7FcGB vaccine, time before tumor formation was the longest [(13.6 ± 1.7) days] and tumor growth was the slowest in the vaccine group. In tumor growth inhibition experiment, inhibition rate reached 81.4% in the vaccine group. Except tumor formation rate, all data in the vaccine group was superior to the pure antigen PVAX1-HPV58mE6E7Fc group (P < 0.05). Humoral immune effect showed that both the vaccine and the pure antigen could induce specific antibody in the immunized mice, and the highest titer were 1: 25 600 and 1: 12 800, respectively. Although there was not significant difference of antibody titer between the vaccine and the pure antigen group (P > 0.05), the number of activated T cells in the vaccine group was almost four times as that in the pure antigen group [(219 ± 34)/4×10(5) spleen lymphocytes versus (55 ± 25)/4×10(5) spleen lymphocytes, P < 0.05], and the highest specific CTL that vaccine induced was significantly higher than that of pure antigen (43.3% versus 31.3%, P < 0.05). Humoral and cellular immune response could be effectively stimulated by PVAX1-HPV58mE6E7FcGB composite DNA vaccine. Growth of B16-HPV58E6E7 cells was significantly inhibited in the immunized mice. The cellular immune effect on the vaccine was superior to the pure antigen. Therefore, PVAX1-HPV58mE6E7FcGB could be used as a candidate vaccine for immune therapy to the HPV58 positive tumors and precancerous lesions.

Adoptive immunotherapy with autologous cytotoxic T lymphocytes (CTLs) pulsed ex vivo with patient-derived tumor cells in heavily pretreated, advanced renal cell carcinoma (aRCC) patients: A feasibility study.

4575 Background: Immunotherapy still remains the only treatment which proved able to induce long-lasting complete responses in aRCC. The aim of the present study was to evaluate the feasibility and safety of adoptive immunotherapy with ex vivo-generated autologous CTLs pulsed with patient-derived tumor cells in heavily pre-treated subjects with aRCC. Methods: This study was performed in accordance with local regulations on the production of cell derivatives; local Ethical Committee approval was obtained on a named patient basis, after obtaining each patient’s informed consent. CTLs were generated stimulating CD8-enriched lymphocytes with dendritic cells (DCs) pulsed with fresh autologous tumor cells in the presence of IL-12 and IL-7; these specific anti-tumor CTLs were expanded and then reinfused after a lymphodepleting chemotherapy with CTX and fludarabine, followed by s.c. administration of low-dose IL-2. Disease status was assessed after every two re-infusions. Eight patients were enrolled, 6 with a clear cell RCC, 1 with a papillary RCC, and 1 with a mixed, papillary and collecting ducts, RCC. All patients have been previously treated with a median of 5 prior treatment lines; the median time between diagnosis of aRCC and tumor biopsy was 55 months (15-185). Results: Six of the 8 enrolled patients have produced suitable tumor cell lines, and 5 of them had been already re-infused with tumor-specific CTLs. All patients received at least 2 CTL infusions (range: 2-5+); mean of totally infused CTLs was 10.7 x 109(range: 4-26.6). All CTLs infusions were well tolerated, with no evidence of autoimmune reactions. In terms of antitumor activity, one patient achieved a partial response, 3 had a slowly progressing disease, and one patient is still not evaluable; furthermore, in 3 patients disease-related symptoms improved. Conclusions: Our preliminary results suggest that immunotherapy with autologous CTLs pulsed ex-vivo with patient-derived tumor cells is feasible and safe in heavily pre-treated aRCC patients. Signs of activity were recorded, suggesting the need to further expand this cohort of patients.

A phase I/II study of multiple peptides cocktail vaccine for advanced/recurrent ovarian cancer.

TPS3116 Background: Despite improvement in chemotherapy including the molecular targeting agents, advanced or recurrent ovarian cancer (A/ROC) are still incurable. Some tumor associated proteins, hypoxia-inducible protein 2 (HIG2), and tumor related vascular endothelial growth factor receptor 1,2 (VEGFR1,VEGFR2) were found to be candidates as new targets, and their epitope peptides have been shown to have the ability to induce specific cytotoxic T lymphocyte ( CTL) responses. We conducted a phase I/II study of these peptides cocktail vaccine (PCV) in patients with A/ROC in order to evaluate toxicity, immunological response, and tumor response. Methods: 23 patients positive for human leukocyte antigen (HLA)-A2402 (A24) and 15 patients with HLA-A0201(A02) were enrolled in this study. Enrollment is still on going up to 30 evaluable patients in each group. PCV for A24 comprises FOXM1(Forkhead Box M1), MELK(maternal embryonic leucine zipper kinase), HJURP(Holliday junction recognition protein), VEGFR1 and VEGFR2. As for A02- PCV comprises HIG2, VEGFR1and VEGFR2. Cocktails were made at a dose of 1mg of each peptide with GMP grade-adjuvant, MONTANIDE ISA51. Vaccination schedule included weekly subcutaneous administration for first 12 weeks, then bi-weekly administration for next 16 weeks, then further vaccination was done monthly for 8 times and 3-6 months as a maintenance step according to patient’s need. Pre- and post-vaccination blood samples were obtained from the patients for toxicity assessment and immunological evaluation. Clinical responses were evaluated every three months by RECIST v 1.1. Results: PCV were generally well tolerated with no major adverse events, and most of the patients developed specific CTL responses. One patient showed complete response, two showed partial response and 10 showed stable disease in 22 evaluable patients. At the time of the analysis, median overall survival was 5 months (from 30 to 623 days) and 9 months(from 54 to 921 days),in A24 and A02, respectively. 11 patients remained alive with median follow up of 9 months. Conclusions: These findings suggest these peptides cocktail vaccines are safe and applicable for advanced/recurrent ovarian cancer. Clinical trial information: UMIN000003862, UMIN000003860.

Introduction of Exogenous T-cell Receptors Into Human Hematopoietic Progenitors Results in Exclusion of Endogenous T-cell Receptor Expression

Current tumor immunotherapy approaches include the genetic modification of peripheral T cells to express tumor antigen-specific T-cell receptors (TCRs). The approach, tested in melanoma, has led to some limited success of tumor regression in patients. Yet, the introduction of exogenous TCRs into mature T cells entails an underlying risk; the generation of autoreactive clones due to potential TCR mispairing, and the lack of effective negative selection, as these peripheral cells do not undergo thymic selection following introduction of the exogenous TCR. We have successfully generated MART-1-specific CD8 T cells from genetically modified human hematopoietic stem cells (hHSC) in a humanized mouse model. The advantages of this approach include a long-term source of antigen specific T cells and proper T-cell selection due to thymopoiesis following expression of the TCR. In this report, we examine the molecular processes occurring on endogenous TCR expression and demonstrate that this approach results in exclusive cell surface expression of the newly introduced TCR, and the exclusion of endogenous TCR cell surface expression. This suggests that this stem cell based approach can provide a potentially safer approach for anticancer immunotherapy due to the involvement of thymic selection.

From viral genome to specific peptide epitopes: methods for identifying porcine T cell epitopes based on in silico predictions, in vitro identification and ex vivo verification (P6098)

Abstract The affinity with which major histocompatibility complex (MHC) class I molecules bind peptides is instrumental to presentation of viral epitopes to cytotoxic T lymphocytes (CTLs). We analyzed three swine leukocyte antigen (SLA) molecules for complete nonamer peptide-based binding matrices in order to predict likely candidates for peptide-SLA binding. These results were combined with binding predictions generated by the algorithm, NetMHCpan (http://www.cbs.dtu.dk/services/NetMHCpan/) in order to select peptide candidates for in vitro analysis. The correlation between high affinity and high stability was determined using luminescence oxygen channeling (LOCI) and scintillation proximity assay (SPA) for peptides bound by the commonly occurring SLA molecules, SLA-1*0401, SLA-2*0401, and SLA-3*0401. Using these tools, peptides bound by SLA with high affinity and stability were identified from a library of 10.000 peptides. T cell epitopes were identified using peptide-SLA complexes assembled into fluorescent tetramers to stain swine influenza specific CTLs derived from immunized animals and MHC-defined pigs vaccinated against foot-and-mouth disease virus. These results demonstrate the broad applicability of methods originally developed for analysis of human leukocyte antigen (HLA) presentation of peptides. The methods presented provide a timely and cost-effective approach to CTL epitope discovery that can be applied to diseases of swine and of other mammalian species of interest.

Antibody–peptide–MHC fusion conjugates target non-cognate T cells to kill tumour cells

Attempts to generate robust anti-tumour cytotoxic T lymphocyte (CTL) responses using immunotherapy are frequently thwarted by exhaustion and anergy of CTL recruited to tumour. One strategy to overcome this is to retarget a population of virus-specific CTL to kill tumour cells. Here, we describe a proof-of-principle study using a bispecific conjugate designed to retarget ovalbumin (OVA)-specific CTL to kill tumour cells via CD20. A single-chain trimer (SCT) consisting of MHCI H-2K(b)/SIINFEKL peptide/beta 2 microglobulin/BirA was expressed in bacteria, refolded and chemically conjugated to one (1:1; F2) or two (2:1; F3) anti-hCD20 Fab' fragments. In vitro, the [SCT×Fab'] (F2 and F3) redirected SIINFEKL-specific OT-I CTL to kill CD20(+) target cells, and in the presence of CD20(+) target cells to provide crosslinking, they were also able to induce proliferation of OT-I cells. In vivo, activated OT-I CTL could be retargeted to kill [SCT×Fab']-coated B cells from hCD20 transgenic (hCD20 Tg) mice and also EL4 and B16 mouse tumour cells expressing human CD20 (hCD20). Importantly, in a hCD20 Tg mouse model, [SCT×Fab'] administered systemically were able to retarget activated OT-I cells to deplete normal B cells, and their performance matched that of a bispecific antibody (BsAb) comprising anti-CD3 and anti-CD20. [SCT×Fab'] were also active therapeutically in an EL4 tumour model. Furthermore, measurement of serum cytokine levels suggests that [SCT×Fab'] are associated with a lower level of inflammatory cytokine release than the BsAb and so may be advantageous clinically in terms of reduced toxicity.

Abstract LB-321: Combination dendritic cell and conventional chemoradiation therapy improves survival for rectal cancer in a murine model.

Abstract The goal of neoadjuvant therapy in rectal cancer is to downstage or preferably eradicate the tumor prior to resection. Achieving complete pathologic response (CPR) using neoadjuvant chemoradiation is associated with improved overall and disease free survival. The aim of this study was to achieve improved complete pathologic response rates and survival by combining intra-tumoral injection of dendritic cells with conventional therapy. A syngeneic rectal cancer cell line (CT26) was subcutaneously injected into the thigh of immunocompetent Balb/c mice. Mice either received no treatment, conventional chemoradiation (CXRT) (5-fluorouracil + XRT) for 3 weeks, or CXRT plus tumor-lysate pulsed dendritic cells (CXRT/DC). Specific anti-CT26 cytotoxic T cell (CTL) activity was determined using standard chromium release assay. Flow cytometry for immune cell subsets was performed on cells isolated from spleens and tumors. Animals in the control group showed predictable uninhibited growth and all were euthanized due to tumor size. CXRT animals showed improved survival but none achieved complete response. Thirty three percent of the animals in CXRT/DC group exhibited CPR. Median survival for the control, CXRT, and CXRT/DC treated animals was 24, 46.5, 67 days, (p&amp;lt;0.0001). Splenocytes from CXRT/DC treated mice exhibited higher specific anti-CT26 CTL activity (28%) when compared to CXRT (17%), untreated control (11%) and naïve mice (&amp;lt;1%). Flow cytometry demonstrated an increase in regulatory T cells (Tregs), macrophages, granulocytic myeloid derived suppressor cells (MDSCs) and monocytic MDSCs in tumor bearing animals, (p&amp;lt;0.05). Dendritic cell activity stimulates anti-tumor CTL responses, but may also decrease MDSC and Treg mediated immunosuppression. Addition of dendritic cells to conventional chemoradiation therapy for rectal cancer increases complete pathologic response and thereby survival via immune modulation. Further analysis will allow subset comparisons between treatment groups. Citation Format: Swapnil D. Kachare, Jennifer M. Cayer, F. Melinda Carver, Zachary P. Englert, Kathryn M. Verbanac, Emmanuel E. Zervos, Nasreen A. Vohra. Combination dendritic cell and conventional chemoradiation therapy improves survival for rectal cancer in a murine model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-321. doi:10.1158/1538-7445.AM2013-LB-321

Peptide FLNPDVLDI of heparanase is a novel HLA-A2-restricted CTL epitope and elicits potent immunological antitumor effects in vitro with an 8-branched design

The aim of this study was to predict and characterize a novel HLA-A2-restricted T-cell epitope of the human heparanase (HPSE) protein, as well as to evaluate its antitumor immunological effects in vitro with an 8-branched multiple antigenic peptide (MAP) design. The amino acid sequence of HPSE was scanned, and the cytotoxic T lymphocyte (CTL) epitopes were predicted using HLA-A2-restricted epitope-predictive algorithms based on supermotif and quantitative motif methods. The affinity of candidate peptides to HLA-A2 was evaluated using peptide-binding assay, by virtue of the characteristics of T2 cells. The MAPs consisting of the selected peptides were synthesized using an 8-branched design. The specific CTL-inducing ability of the MAPs was assessed by LDH release assay, and the CTL activity was evaluated by INF-γ ELISPOT assay. Among the predicted nonapeptides, the FLNPDVLDI peptide of HPSE showed the highest affinity to the HLA-A2 molecule. The 8-branched MAP comprising FLNPDVLDI induced specific CTLs for human HPSE in vitro, which effectively secreted IFN-γ and potently lysed HCC97-H human hepatocarcinoma cells and SW-480 human colonic carcinoma cells. The nonapeptide FLNPDVLDI of human HPSE appears to be a novel HLA-A2-restricted CTL epitope, and its 8-branch designed MAP is capable of inducing a potent HPSE-specific CTL response against tumor cells in vitro. Our study provides theoretical evidence for the peptide-based antitumor immunotherapy.

The modification of Tapasin enhances cytotoxic T lymphocyte activity of intracellularly delivered CTL epitopes &amp;lt;italic&amp;gt;via&amp;lt;/italic&amp;gt; cytoplasmic transduction peptide

Previous studies have demonstrated that the therapeutic vaccine based on the enhancement of hepatitis B virus (HBV)-specific cytotoxic T lymphocyte (CTL) activity may lead to viral clearance in HBV-infected individuals. The endoplasmic reticulum (ER) chaperone Tapasin plays an important role in major histocompatibility complex (MHC) class I assembly and enhances specific MHC class I-restricted CTL activity by allowing more peptides to be translocated into the ER. Combining the specificity of hepatitis B core antigen (HBcAg) CTL epitope, the cell-penetrating property of cytoplasmic transduction peptide (CTP), and chaperone Tapasin may elicit robust specific HBV immune responses. In the present study, we confirmed the cytoplasmic localization preference of CTP-HBcAg(18-27)-Tapasin fusion protein in vitro and evaluated the effects on promoting bone marrow-derived dendritic cells (BMDCs) maturation and enhancing T cells response to generate specific CTLs. Our results showed that CTP-HBcAg(18-27)-Tapasin fusion protein could not only penetrate into the cytoplasm exactly and effectively to elevate Tapasin expression, but also increase the expression of surface molecules (CD80, CD83, CD86, and MHC-I) and secretion of cytokine (IL-12p70) of DCs. Moreover, DCs treated with the above fusion proteins increased significantly the cytokine secretion of proliferated T cells in vitro, the percentages of IFN-γ(+)CD8(+) T cells and specific CTL responses compared with control groups. In conclusion, the modification of Tapasin can enhance the presentation of targeting antigens via intracellular delivery to DCs and elicit specific CTL immune responses efficiently.

A new rat model of prostate cancer.

The current status of immunotherapy of cancer dictates that tumour cells should express appropriate antigenic peptides on the cell surface associated with MHC class I antigen. Therapies designed to activate immunity against cell surface expressed target peptides are required to induce antigen specific cytotoxic T-lymphocytes (CTL) capable of mediating tumour rejection and that such immune responses should produce immunological memory. The necessity for both MHC class I (CTL response) and class II (T-helper response) - restricted responses has been well documented and the nature of tumour antigens expressed on a wide variety of human cancers widely reported.

Association of Neisseria gonorrhoeae OpaCEA with Dendritic Cells Suppresses Their Ability to Elicit an HIV-1-Specific T Cell Memory Response

Infection with Neisseria gonorrhoeae (N. gonorrhoeae) can trigger an intense local inflammatory response at the site of infection, yet there is little specific immune response or development of immune memory. Gonococcal surface epitopes are known to undergo antigenic variation; however, this is unlikely to explain the weak immune response to infection since individuals can be re-infected by the same serotype. Previous studies have demonstrated that the colony opacity-associated (Opa) proteins on the N. gonorrhoeae surface can bind human carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on CD4+ T cells to suppress T cell activation and proliferation. Interesting in this regard, N. gonorrhoeae infection is associated with impaired HIV-1 (human immunodeficiency virus type 1)-specific cytotoxic T-lymphocyte (CTL) responses and with transient increases in plasma viremia in HIV-1-infected patients, suggesting that N. gonorrhoeae may also subvert immune responses to co-pathogens. Since dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a key role in the induction of an adaptive immune response, we investigated the effects of N. gonorrhoeae Opa proteins on human DC activation and function. While morphological changes reminiscent of DC maturation were evident upon N. gonorrhoeae infection, we observed a marked downregulation of DC maturation marker CD83 when the gonococci expressing CEACAM1-specific OpaCEA, but not other Opa variants. Consistent with a gonococcal-induced defect in maturation, OpaCEA binding to CEACAM1 reduced the DCs’ capacity to stimulate an allogeneic T cell proliferative response. Moreover, OpaCEA-expressing N. gonorrhoeae showed the potential to impair DC-dependent development of specific adaptive immunity, since infection with OpaCEA-positive gonococci suppressed the ability of DCs to stimulate HIV-1-specific memory CTL responses. These results reveal a novel mechanism to explain why infection of N. gonorrhoeae fails to trigger an effective specific immune response or develop immune memory, and may affect the potent synergy between gonorrhea and HIV-1 infection.

Trivalent Live Attenuated Influenza-Simian Immunodeficiency Virus Vaccines: Efficacy and Evolution of Cytotoxic T Lymphocyte Escape in Macaques

There is an urgent need for a human immunodeficiency virus (HIV) vaccine that induces robust mucosal immunity. CD8(+) cytotoxic T lymphocytes (CTLs) apply substantial antiviral pressure, but CTLs to individual epitopes select for immune escape variants in both HIV in humans and SIV in macaques. Inducing multiple simian immunodeficiency virus (SIV)-specific CTLs may assist in controlling viremia. We vaccinated 10 Mane-A1*08401(+) female pigtail macaques with recombinant influenza viruses expressing three Mane-A1*08401-restricted SIV-specific CTL epitopes and subsequently challenged the animals, along with five controls, intravaginally with SIV(mac251). Seroconversion to the influenza virus vector resulted and small, but detectable, SIV-specific CTL responses were induced. There was a boost in CTL responses after challenge but no protection from high-level viremia or CD4 depletion was observed. All three CTL epitopes underwent a coordinated pattern of immune escape during early SIV infection. CTL escape was more rapid in the vaccinees than in the controls at the more dominant CTL epitopes. Although CTL escape can incur a "fitness" cost to the virus, a putative compensatory mutation 20 amino acids upstream from an immunodominant Gag CTL epitope also evolved soon after the primary CTL escape mutation. We conclude that vaccines based only on CTL epitopes will likely be undermined by rapid evolution of both CTL escape and compensatory mutations. More potent and possibly broader immune responses may be required to protect pigtail macaques from SIV.