Tumor-specific CTL Research Articles

An HLA-A2 Polyepitope Vaccine for Melanoma Immunotherapy

Epitope-based vaccination strategies designed to induce tumor-specific CD8 CTL are being widely considered for cancer immunotherapy. Here we describe a recombinant poxvirus vaccine that codes for ten HLA-A2-restricted epitopes derived from five melanoma Ags conjoined in an artificial polyepitope or polytope construct. Target cells infected with the melanoma polytope vaccinia were recognized by three different epitope-specific CTL lines derived from HLA-A2 melanoma patients, and CTL responses to seven of the epitopes were generated in at least one of six HLA-A2-transgenic mice immunized with the construct. CTL lines derived from vaccinated transgenic mice were also able to kill melanoma cells in vitro. Multiple epitopes within the polytope construct were therefore shown to be individually immunogenic, illustrating the feasibility of the polytope approach for melanoma immunotherapy. Tumor escape from CTL surveillance, through down regulation of individual tumor Ags and MHC alleles, might be overcome by polytope vaccines, which simultaneously target multiple cancer Ags.

In situ analysis of tumor-specific CTL effector and memory responses elicited by tumor vaccination.

In this study tumor-specific CTL effector and memory responses were analyzed in normal mice, in tumor bearing mice and in animals treated by active specific immunotherapy (ASI) with an autologous virus modified tumor vaccine. In the well defined DBA/2 mouse lymphoma model ESb, the tumor specific CTL response requires interactions of four different cell types and recognition of MHC class I and class II restricted tumor antigens either on host antigen presenting cells (APCs) or on the tumor cells. The most effective in situ activation of syngeneic tumor specific CTL can be generated within no more than 9 days by primary immunization in the ear pinna and restimulation in the peritoneal cavity. Here we describe modulatory effects of either cytokines or of virus infection of tumor stimulator cells during restimulation in the peritoneal cavity on the CTL memory response. The in situ peritoneal effector cell (PEC) response was augmented when Newcastle Disease Virus (NDV) was used to infect tumor cells which expressed the correct tumor associated antigen of the memory response but not when a third party tumor cell was infected and admixed. A response could also be augmented by co-administration of interferons-alpha, -beta or IL-2 and by pre-treatment with low dose cyclophosphamide. Therapeutic vaccination effects could be achieved in mice inoculated s.c. with this aggressive and metastatic tumor variant if the vaccine was given very early, i.e. 1-2 days after priming and when the vaccine was applied post-operatively, i.e. in situations of low tumor burden. A long-term CTL memory response could be demonstrated even two months after post-operative active specific immunotherapy with ESb-NDV vaccine.

Identification of New HER2/neu-Derived Peptide Epitopes That Can Elicit Specific CTL Against Autologous and Allogeneic Carcinomas and Melanomas

AbstractTwenty-two new HLA-A2.1-binding peptides derived from the protooncogene HER2/neu were identified and analyzed for their capacity to elicit peptide and tumor-specific CTL responses. We used peptide-pulsed autologous DC from the ascites of patients with ovarian carcinomas to induce CTL. Of the 22 tested new HER2/neu-derived epitopes that could bind HLA-A2 with high (IC50 < 50 nM) or intermediate (50 nM < IC50 < 500 nM) affinity, we report the recognition by CTL of at least four novel epitopes, including HER2(9435), HER2(9665), HER2(9689), and HER2(10952), and confirm that of the known HER2 (9369) epitope. These epitopes were able to elicit CTL that specifically killed peptide-sensitized target cells and, most importantly, a HER2/neu-transfected cell line and the autologous tumor cells. We also confirm that HER2/neu is overexpressed in several melanoma lines, and as a new finding, report that some of these lines are sensitive to CTL induced by the HER2 (9369), HER2(9435), and HER2(9689) epitopes. Finally, CTL clones specific for HER2 (9369), HER2(9435), and HER2(9689) epitopes were isolated from tumor-specific CTL lines, further demonstrating the immunodominance of these epitopes. These findings broaden the potential application of HER2/neu-based immunotherapy.

Depletion of IL-10- and TGF-β-Producing Regulatory γδ T Cells by Administering a Daunomycin-Conjugated Specific Monoclonal Antibody in Early Tumor Lesions Augments the Activity of CTLs and NK Cells

AbstractIt has been demonstrated that γδ T cells accumulating in early tumor lesions and those purified from spleen cells of tumor-bearing mice attenuate the activity of CTLs and NK cells. We, therefore, investigated whether depletion of γδ T cells from early lesions of tumors results in restoration of CTL and NK cell activities and subsequent regression of tumors. A daunomycin-conjugated anti-γδTCR mAb UC7-13D5 (Dau-UC7) was prepared to efficiently deplete γδ T cells. An in vitro study revealed that Dau-UC7 specifically lysed γδTCR+ cells and effectively inhibited splenic γδ T cells from tumor-bearing mice to produce cytotoxic cell-suppressive factors. Furthermore, intralesional injections of Dau-UC7 at an early stage of tumor development led to augmentation of tumor-specific CTL as well as NK cell activities and to the resultant regression or growth inhibition of the tumors. On analysis of cytokine profile, γδ T cells transcribed mRNAs for IL-10 and TGF-β, but not IL-4 or IFN-γ, suggesting the T regulatory 1-like phenotype. Finally, a blocking study with mAbs showed that the inhibitory action of γδ T cells on CTLs and NK cells was at least partly mediated by IL-10 and TGF-β. These results clearly demonstrated the novel mechanism by which T regulatory 1-like γδ T cells suppress anti-tumor CTL and NK activities by their regulatory cytokines in early tumor formation.

Cutting Edge: Rapid Cloning of Tumor-Specific CTL Suitable for Adoptive Immunotherapy of Melanoma

Adoptive immunotherapy using CTL has provided some clinical benefit to patients with metastatic melanoma. Use of cloned CTL of known specificity might improve clinical effect, but technical difficulties have limited exploration of this possibility. We have used fluorescence-driven cell sorting to clone tumor-specific CTL after staining with tetrameric MHC class I/peptide complexes. CTL specific for the melanoma Ags melan-A, tyrosinase, and MAGE3 were cloned from the peripheral blood, tumor-infiltrated lymph nodes, and skin metastases of five patients. Clones were isolated and characterized in as little as 6 weeks, much faster than is possible with previous techniques. We show that these CTL clones express markers compatible with immunotherapeutic use in melanoma, including the cutaneous lymphocyte Ag, which is associated with homing to skin.

Cytotoxic T lymphocyte response against non-immunoselected tumor antigens predicts the outcome of gene therapy with IL-12-transduced tumor cell vaccine.

The colon adenocarcinoma C26, carrying two endogenous tumor-associated antigens (TAA) recognized by CTL, has been transduced with the gene coding for the human folate receptor alpha (FR alpha) as an additional antigen in order to study the efficacy of vaccination against a tumor expressing multiple antigens. A dicistronic vector was used to transduce the IL-12 genes to create C26/IL-12/FR alpha that has been used as a cellular vaccine to treat mice bearing lung metastases of C26/FR alpha. After vaccination mice were partially splenectomized and splenic lymphocytes frozen and used retrospectively to study in vitro CD8 T cell response related to the treatment outcome. Vaccination cured 50% of mice and the effect was CD8 T cell dependent. Mice either cured (responders) or not cured (nonresponders) by vaccination developed tumor-specific CTL. However, analysis of CTL specificity and pCTL frequencies revealed that responders had a predominant CTL activity against endogenous C26-related tumor antigens, whereas nonresponders had CTL that recognized preferentially the FR alpha antigen. CD8 from responder mice were characterized to release high levels of granulocyte-macrophage (GM)-CSF upon antigen stimulation. Tumors obtained from mice that died despite vaccination lost expression of the FR alpha transgene but maintained expression of endogenous C26 antigens. Immunoselection against FR alpha antigen was not observed in tumors from non-vaccinated controls and from CD8-depleted vaccinated mice. Down-regulation of FR alpha antigen expression was due, at least in part, to methylation of retroviral vector long terminal repeat promoter since FR alpha expression was partially restored, ex vivo, by treatment with 5-aza-2'-deoxy-cytidine (aza). These results indicate that CD8 T cell-mediated immunoselection and production of GM-CSF are determining factors for the efficacy of tumor vaccines.

Tumor cells cotransduced with B7.1 and gamma-IFN induce effective rejection of established parental tumor.

Genetic modification of tumor cells with the gene for the B7.1 or with the genes for cytokines results in increased tumor cell immunogenicity. In the work reported here, immunization of naive animals with either B7.1 or gamma-IFN gene-modified MCA106 tumor cells effectively protects the host from subsequent challenge with parental tumor. The same treatment fails to induce regression of established tumors, although tumor-specific CTL are generated in the tumor-bearing animals. In contrast, a large tumor burden of the MCA106 fibrosarcoma can be successfully eliminated by treatment with MCA106 tumor cells cotransduced with the B7.1 and gamma-IFN genes. Antitumor immunity induced by the cotransductants is primarily dependent on CD8+ T cells and partly on CD4+ T cells and NK cells, and the enhanced therapeutic effect may be attributed to the in vivo increase of CTL precursors following treatment. The gamma-IFN and B7.1 genes must be expressed on the same tumor cell for optimal therapeutic effect. Our results suggest that tumor vaccines with a potent immunoprotective effect do not necessarily have therapeutic potential and that weakly immunogenic tumors may be rendered highly immunogenic by cotransfection with the genes for B7.1 and gamma-IFN.

High Frequency of Specific CD8+ T Cells in the Tumor and Blood Is Associated with Efficient Local IL-12 Gene Therapy of Cancer

Cancer immunotherapy often aims at the reactivation and expansion of tumor-specific CTL. In an attempt to correlate in situ and/or systemic tumor-specific T cell expansion with tumor regression, we investigated the effects of adenovirus-mediated IL-12 or IFN-gamma gene transfer into established P815 murine tumors. While IFN-gamma was no more potent than the vector alone, IL-12 gene transfer promoted tumor eradication. Despite this antitumor effect, no significant cytolytic activity was detectable using classical cytotoxicity assays from in vitro restimulated splenocytes. Since intratumor gene delivery may induce a localized expansion of CTL, the presence of P815-specific CD8+ T cells in situ was assessed. Using the Immunoscope approach, we found a dramatic increase in clonotypic T cells at the tumor site following IL-12, but not IFN-gamma gene delivery. Antitumor CD8+ T cell frequencies were then re-evaluated using this molecular detection technique, which revealed a comparable expansion of specific T cells in the peripheral organs, most strikingly in the blood. These data show that local IL-12 gene transfer, in contrast to IFN-gamma, mediates a potent antitumor effect that correlates to clonal tumor-specific T cell expansions in situ and in the periphery.

Eliciting T Cell Immunity Against Poorly Immunogenic Tumors by Immunization with Dendritic Cell-Tumor Fusion Vaccines

Dendritic cells (DCs) are the most effective APCs and are being studied as natural adjuvants or Ag delivery vehicles to elicit T cell-mediated antitumor immunity. This study examined whether inoculation of DCs fused with poorly immunogenic tumor cells elicited tumor-reactive T cells for adoptive immunotherapy. DCs derived from bone marrow of C57BL/6 (B6) mice were fused with syngeneic B16 melanoma or RMA-S lymphoma cells by polyethylene glycol. The B16/DC and RMA-S/DC fusion hybrids expressed MHC class I, class II Ags, costimulatory molecules, as well as DC-specific and tumor-derived surface markers. The tumor/DC hybrids were capable of processing and presenting tumor-derived Ags, and immunization of B6 mice with irradiated B16/DC or RMA-S/DC vaccine elicited tumor-specific CTL activities. Vaccination of B6 mice with irradiated B16/DC fusion preparations induced partial host protective immunity against B16 tumor challenge. Reduced tumor incidence and prolonged survival time were observed. Adoptive transfer of T cells derived from B16/DC vaccine-primed lymph nodes into B16 tumor-bearing mice greatly reduced the number of established pulmonary metastases with or without in vivo administration of IL-2. Moreover, adoptive transfer of RMA-S/DC vaccine-primed, cultured lymph node T cells eradicated disseminated FBL-3 tumor. The results demonstrate that tumor/DC fusion products are effective cellular vaccines for eliciting T cell-mediated antitumor immunity.

A Breast and Melanoma-Shared Tumor Antigen: T Cell Responses to Antigenic Peptides Translated from Different Open Reading Frames

Infusion of TIL586 along with IL-2 into the autologous patient with metastatic melanoma resulted in the objective regression of tumor. Here, we report that screening a cDNA library from the 586mel cell line using CTL clones derived from TIL586 resulted in the isolation of a gene, CAG-3 (cancer Ag gene 3). Sequence analysis revealed that CAG-3 encodes an open reading frame identical to NY-ESO-1, which was recently reported to be recognized by autologous serum from a patient with esophageal cancer. Thus, NY-ESO-1 appears to be an immune target for both Ab- and T cell-mediated responses. Significantly, NY-ESO-1-specific CTL clones were capable of recognizing two HLA-A31-positive fresh and cultured breast tumors. To our knowledge, this represents the first direct demonstration that tumor-specific CTL clones can recognize both breast and melanoma tumor cells. A 10-mer antigenic peptide ESO10-53 (ASGPGGGAPR) was identified from the normal open reading frame of NY-ESO-1 based on its ability to sensitize HLA-A31-positive target cells for cytokine release and specific lysis. Interestingly, two additional CTL clones that were sensitized with NY-ESO-1 recognized two overlapping antigenic peptides derived from an alternative open reading frame of the same gene. These findings indicate that CTLs simultaneously responded to two different gene products translated from the normal and alternative reading frames of the same gene. Understanding of this mechanism by which the alternative reading frame is translated may have important implications in tumor immunology.

In Situ Cancer Vaccination: An IL-12 Defective Vector/Replication-Competent Herpes Simplex Virus Combination Induces Local and Systemic Antitumor Activity

Intratumoral inoculation of replication-competent, attenuated herpes simplex virus (HSV) mutants inhibits tumor growth by direct cytotoxic viral replication and induction of a tumor-specific immune response. To boost the antitumor response, we describe a defective HSV vector encoding IL-12 as an adjuvant to in situ vaccination by the replication-competent HSV helper virus. The defective HSV vector system consists of defective particles containing tandem repeats of the cytokine genes (p40 and p35) in combination with a HSV helper virus. Heterodimeric IL-12 was expressed and secreted after IL-12 defective vector infection of tumor cells. In a syngeneic, bilateral established tumor model with CT26 murine colon carcinoma, unilateral intratumoral inoculation with an IL-12 defective/replication-competent HSV vector combination significantly reduced tumor growth of the inoculated and noninoculated contralateral tumors. This antitumor effect was significantly greater than with a lacZ-defective/replication-competent HSV vector combination, which itself was significantly greater than the mock inoculation. Efficacy is associated with enhancement of tumor-specific CTL activity, including specificity against the CT26 immunodominant MHC class I restricted Ag AH1, and IFN-gamma production. There was no significant tumor growth inhibition after intratumoral inoculation of s.c. CT26 tumors in athymic mice. We conclude that this defective HSV vector system is an effective method for cytokine gene delivery to tumors in situ and IL-12 expression in tumors synergizes the antitumor activity mediated by the replication-competent HSV helper virus.

Stable expression of a retrovirally transferred adhesion molecule in a human tumor-specific CTL clone. Perspectives for the ex vivo manipulation of effector cells used in adoptive immunotherapy.

The adoptive transfer of in vivo activated effector cells for cancer treatment [1] has one major drawback: in vitro activated lymphocytes display altered features of homing upon retransplantation and frequently are trapped in the spleen and the lung [2]. One possibility to restore natural homing features could be based on the in vitro transfer of appropriate homing molecules. Since cutaneous lymphoma [Wagner S, submitted], locally growing melanoma and skin metastases of malignant melanoma [3] express high levels of a CD44 variant isoform containing exon vlO, we speculate that CD44v10 may be a particular homing receptor allowing lymphocytes to infiltrate epidermal tissue. Based on these considerations we explored the possibility of transfecting a human in vitro generated melanoma-specific CTL clone with this particular CD44 isoform. We explored the feasibility of a stable gene transfer into human CTL. Here we report on a protocol where a human melanoma-specific CTL clone stably expressed a homing receptor after retroviral transfection.

Growth of P511 mastocytoma cells in BALB/c mouse brain elicits CTL response without tumor elimination: a new tumor model for regional central nervous system immunity.

We have developed a murine model to explore the tumor-specific CTL response in the immune-privileged central nervous system using P511 mastocytoma cells. Three strains with varying degrees of histocompatibility to P511 cells (CD-1, allogeneic; BALB/c, minor histoincompatible; DBA/2, syngeneic) received tumor cells (10(4)) into the putamen 7 days after cannula implantation, when the blood-brain barrier was functionally intact. Without exception, tumor formed reproducibly by day 7 in all strains. Tumor rejection occurred in CD-1 but not in BALB/c and DBA/2 mice. Using a flank injection site, both CD-1 and BALB/c, but not DBA/2 mice, ultimately rejected flank tumors. Analysis of tumor-specific CTL in BALB/c spleens revealed that P511 administration into brain or flank elicited similar responses: no fully activated CTL were detectable but a significantly expanded population of nonkilling precursors of CTL (pCTL) were present. A P511 cell-specific pCTL population was also identified at the brain tumor site 14 days post-tumor introduction, indicating that pCTL, generated in the periphery, traffic to the tumor site in brain. These data indicate that failure to reject tumor in brain is neither due to lack of afferent stimulation nor to inability of peripheral effectors (P511 cell-specific pCTL) to reach the tumor site. We hypothesize that these effector cells are prevented from developing into fully activated CTL by conditions within the central nervous system microenvironment that down-regulate CTL development.

Eradication of tumor growth via biolistic transformation with allogeneic MHC genes.

Particle acceleration-mediated biolistics transformation has been rapidly adopted as a versatile technology for gene delivery since its original application as a physical method for delivery of foreign genes into higher plants. We have designed a versatile hand-held gene gun device for the genetic immunization of animals in vivo. The gene gun is driven by air blast and therefore does not require helium gas or vacuum for its function. In this report, we have employed this gene gun device to introduce allogeneic H-2Kb DNA directly into tumor nodules of K36 tumor-bearing AKR/J mice. Expression of the exogenous H-2Kb gene led to the regression of well-established K36 tumors. This tumor regression was correlated with the generation of potent secondary CTL responses against the K36 tumor cells following expression of the exogenous H-2Kb gene in the K36 tumor cells. Furthermore, it was observed that H-2Kb stimulator cells could stimulate the generation of both allogeneic and tumor-specific secondary CTL responses whereas mitomycin-C treated tumor cells could only stimulate tumor-specific secondary CTL responses. This approach of introducing allogeneic MHC genes directly into cutaneous tumor lesions via biolistics transformation of tumor cells in vivo can potentially be developed into an effective method for cancer gene therapy.

589 Autologous tumor-specific CTL in a patient with lung adenocarcinoma. Implication of the shared antigens expressed on [formula omitted] lung cancer cells

589 Autologous tumor-specific CTL in a patient with lung adenocarcinoma. Implication of the shared antigens expressed on [formula omitted] lung cancer cells

Stimulation of Tumor-Specific Immunity Using Tumor Cell Plasma Membrane Antigen

Plasma membranes isolated from tumor cells retain biologically active class I MHC proteins on their surfaces. CD8+T-cell activation by membrane antigen is much more effective when the small membrane vesicles (<1-μm diameter) are displayed on a surface with dimensions approaching those of a cell (5-μm diameter). Previous work had shown that tumor membrane antigen incorporated onto silica microspheres could augment tumor-specific CTL responsesin vivoand significantly reduce syngeneic tumor growth. Antigen on cell-sized solid supports has been termed large multivalent immunogen (LMI). Methods are described for preparing LMI using either silica or latex microspheres. LMI made using either are activein vivoin reducing tumor growth, suggesting that the nature of the support is not critical as long as it is of the appropriate dimensions and has a surface that allows adsorption of the membrane vesicles. Latex microspheres provide some advantages over the previously described silica microspheres with respect to handling and characterization. The effects of LMI onin vivoCTL activation and tumor growth suggest that this approach may have potential for application to clinical immunotherapy of cancers.

Two tumor specific CTL clones recognise two different tumor antigens on mouse renal carcinoma cells

Two tumor specific CTL clones recognise two different tumor antigens on mouse renal carcinoma cells

Two tumor specific CTL clones recognise two different tumor antigens on mouse renal carcinoma cells

Two tumor specific CTL clones recognise two different tumor antigens on mouse renal carcinoma cells

Diversity of the cytotoxic melanoma-specific immune response: some CTL clones recognize autologous fresh tumor cells and not tumor cell lines.

Abstract In the present work, to analyze the heterogeneity of the tumor-specific cytotoxic immune response, a large number of T cell clones were generated from the infiltrate of a tumor-proximal invaded lymph node, and two kinds of melanoma-specific CD8+ CTL clones were derived. The majority of T cell clones (about a hundred) are characterized by a specific lysis of the autologous tumor cell lines. Among 34 of the latter clones, HLA-A2 molecule and MART-1(27-35) peptide have been shown to play a predominant role in tumor recognition. However, no significant amplification at the tumor site was observed for 3 of these CTL. The other kind of tumor-specific CTL (1 oligoclonal and 2 clonal cell lines) did not lyse the autologous melanoma cell lines but lysed the "fresh" autologous tumor cells in a MHC class I-dependent manner. Functional analysis of the two different CTL clones have shown that they did not lyse NK targets, autologous peripheral monocytes, activated T cells, and transformed B cells or any of the few allogeneic cultured and uncultured melanoma cells we tested. TCR repertoire analysis has shown that one of these CTL clones was significantly detectable "in situ" among tumor-infiltrating lymphocytes, while not detectable among PBMC. Such melanoma-specific lymphocytes, which could not have been picked out through conventional screening procedures using tumor cell lines, could potentially play a role in tumor rejection. These results suggest that the immune response analyzed toward melanoma cell lines does not totally reflect the in situ immune status.

IL-2 gene delivery within an established murine tumor causes its regression without proliferation of preexisting antitumor-specific CTL.

Regression of P815 tumors established on naive syngeneic mice can be obtained by the intratumoral injection of a single dose of an adenoviral vector expressing the IL-2 gene (Ad.IL2). Injection triggers local IL-2 production for at least 10 days. We measured a number of immunologic parameters in situ following intratumoral Ad.IL2 treatment. We also analyzed the situation of regression obtained upon challenge with P815 cells of mice previously immunized against the tumor and compared both systems. While IFN-gamma messenger RNA expression was found to be elevated in both situations of tumor regression, the level of infiltration by tumor-specific CTL was different. A small amount of tumor-specific CD8+ T cells were present in growing, untreated tumors. Such cells are found in much larger numbers in tumors rejected upon challenge, consistent with a CTL-mediated rejection. In contrast, they were found not to proliferate following Ad.IL2 injection. The latter caused an increased infiltration of a polyclonal, presumably nonspecific, T cell population. These results suggest that the initial regression of established P815 tumors following Ad.IL2 treatment in vivo is mostly due to nonspecific effectors.