Tumor-specific Ag Research Articles

Interruption of Thymic Activity in Adult Mice Improves Responses to Tumor Immunotherapy.

The thymus produces precursors of both conventional T cells (Tconv; also known as effector T cells) and regulatory T cells (Treg) whose interactions prevent autoimmunity while allowing efficient protective immune responses. Tumors express a composite of self-antigens and tumor-specific Ags and engage both Tconv and Treg. Along the aging process, the thymus involutes, and tumor prevalence increases, a correlation proposed previously to result from effector cell decline. In this work, we directly tested whether interruption of thymic activity in adult mice affects Foxp3-expressing Treg composition and function and alters tumor immune surveillance. Young adult mice, on two different genetic backgrounds, were surgically thymectomized (TxT) and analyzed or challenged 2 mo later. Cellular analysis revealed a 10-fold decrease in both Tconv and Treg numbers and a bias for activated cells. The persisting Treg displayed reduced stability of Foxp3 expression and, as a population, showed a compromised return to homeostasis upon induced perturbations. We next tested the growth of three tumor models from different tissue origins and/or presenting distinct degrees of spontaneous immunogenicity. In none of these conditions, adult TxT facilitated tumor growth. Rather, TxT enhanced the efficacy of antitumor immunotherapies targeting Treg and/or the immune checkpoint CTLA4, as evidenced by the increased frequency of responder mice and decreased intratumoral Treg to CD8+IFN-γ+ cell ratio. Together, our findings point to a scenario in which abrogation of thymic activities affects preferentially the regulatory over the ridding arm of the immune activities elicited by tumors and argues that higher prevalence of tumors with age cannot be solely attributed to thymic output decline.

Coexpressed Catalase Protects Chimeric Antigen Receptor-Redirected T Cells as well as Bystander Cells from Oxidative Stress-Induced Loss of Antitumor Activity.

Treatment of cancer patients by adoptive T cell therapy has yielded promising results. In solid tumors, however, T cells encounter a hostile environment, in particular with increased inflammatory activity as a hallmark of the tumor milieu that goes along with abundant reactive oxygen species (ROS) that substantially impair antitumor activity. We present a strategy to render antitumor T cells more resilient toward ROS by coexpressing catalase along with a tumor specific chimeric Ag receptor (CAR) to increase their antioxidative capacity by metabolizing H2O2. In fact, T cells engineered with a bicistronic vector that concurrently expresses catalase, along with the CAR coexpressing catalase (CAR-CAT), performed superior over CAR T cells as they showed increased levels of intracellular catalase and had a reduced oxidative state with less ROS accumulation in both the basal state and upon activation while maintaining their antitumor activity despite high H2O2 levels. Moreover, CAR-CAT T cells exerted a substantial bystander protection of nontransfected immune effector cells as measured by CD3ζ chain expression in bystander T cells even in the presence of high H2O2 concentrations. Bystander NK cells, otherwise ROS sensitive, efficiently eliminate their K562 target cells under H2O2-induced oxidative stress when admixed with CAR-CAT T cells. This approach represents a novel means for protecting tumor-infiltrating cells from tumor-associated oxidative stress-mediated repression.

Landscape of Tumor Antigens in T Cell Immunotherapy

Cancer immunotherapy is a rapidly evolving field that exploits T cell responses to tumor-associated Ags to induce tumor rejection. Molecular identification of tumor rejection Ags has helped define several classes of Ags, including tissue differentiation and tumor germline Ags. The ability to genetically engineer Ag-specific receptors into T cells provides an opportunity to translate these findings into therapies. New immunotherapy agents, notably checkpoint inhibitors, have demonstrated unprecedented efficacy in certain cancers. However, the nature of the Ags driving those beneficial immune responses remains unclear. New evidence suggests that tumors express immunogenic, tumor-specific epitopes generated from the same mutations that drive cancer development. Correlations between cancer types responding to immunotherapies and the frequency of somatic mutations may clarify what drives natural antitumor immune responses. This fusion of tumor immunology and genetics is leading to new ways to target this class of ideal tumor-specific Ags and could allow the application of immunotherapy to many cancers.

Abstract 4101: Early detection of colonic neoplasia using fluorescence microendoscopy

Abstract Purpose: The objective of this research is to integrate nanotechnology with molecular imaging for early detection of colorectal cancer (CRC). CRC undergoes a protracted asymptomatic stage before it reaches the advance stage. Therefore, detection in the early onset through regular screening will improve therapeutic outcomes and save lives. In that regard, colonoscopy is considered the golden standard for early detection. However, its effectiveness for early detection of tumor growth is mitigated by its incapacity to disclose molecular level changes. Recent studies have reported the miss rate for this tumor size-dependent technique is as high as 20% for polyps. One molecule associated with the development and progress of CRC is the Thomsen-Friedenreich (TF) antigen (Ag). High expression of TF disaccharide in CRC and its absence from normal tissue represents a unique association in CRC that exhibits the qualities of a prognostic biomarker. Method: We developed a fluorescence (FL) nanobeacon, which can bind specifically to TF-associated CRC due the presence of multiple identical copies of peanut agglutinin (PNA) molecules derivatized on the surface of the nanobeacon. The physical property of the FL nanobeacon such as size, shape, surface topography, elemental composition and polymer distribution was evaluated using dynamic light scattering, scanning electron microscopy, X-ray photoelectron spectroscopy and permeation chromatography, respectively. We assessed the sensitivity and specificity of the nanobeacon on human specimens using FL microscopy, Western blot and quantitative PCR. TF Imaging data was validated by depletion of TF expression employing a glycanase assay. In addition, we developed an orthotopic rat model of CRC using Athymic nude rats to assess the specificity of the nanobeacon using white light colonoscopy and FL microendoscopy. Furthermore, the biodistribution of the nanobeacon was assessed using an orthotopic mouse model. Results: The overall size of the nanobeacon is approximately 350 nm. It contains 0.05% coumarin 6 dye in the polystyrene center core. There are approximately 200-300 PNA molecules, which serve as molecular recognition moieties for TF Ag. The nanobeacon specifically reported CRC by recognizing the tumor-specific Ag through surface-immobilized PNA. Removal of TF from human colorectal cancer cells and tissues resulted in a significant loss of FL signal, which suggests the specificity of the probe. In vivo imaging data demonstrated that the FL colonoscopy using the nanobeacon can detect CRC much earlier than conventional colonoscopy. Finally, biodistribution study showed that the nanobeacon is not absorbed by colonic mucosa upon being sprayed topically, thus there is no register toxicity associated with this probe. Conclusion: We demonstrated the potential use of a novel nanobeacon for early detection of CRC. Data obtained in this work suggests that TF Ag can be used as a potential biomarker for imaging CRC. Citation Format: Wellington Pham, Hironori Kumagai, Ken-Ichiro Hiwatari, Seiji Koike, Etsuo Tobita, Tokio Kitamura, Kohta Mohri, Shinji Sakuma. Early detection of colonic neoplasia using fluorescence microendoscopy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4101. doi:10.1158/1538-7445.AM2014-4101

Mutated PPP1R3B Is Recognized by T Cells Used To Treat a Melanoma Patient Who Experienced a Durable Complete Tumor Regression

Adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma. However, most of the Ag targets recognized by effective melanoma-reactive TILs remain elusive. In this study, patient 2369 experienced a complete response, including regressions of bulky liver tumor masses, ongoing beyond 7 y following adoptive TIL transfer. The screening of a cDNA library generated from the autologous melanoma cell line resulted in the isolation of a mutated protein phosphatase 1, regulatory (inhibitor) subunit 3B (PPP1R3B) gene product. The mutated PPP1R3B peptide represents the immunodominant epitope recognized by tumor-reactive T cells in TIL 2369. Five years following adoptive transfer, peripheral blood T lymphocytes obtained from patient 2369 recognized the mutated PPP1R3B epitope. These results demonstrate that adoptive T cell therapy targeting a tumor-specific Ag can mediate long-term survival for a patient with metastatic melanoma. This study also provides an impetus to develop personalized immunotherapy targeting tumor-specific, mutated Ags.

Stimulation of the Glucocorticoid-Induced TNF Receptor Family-Related Receptor on CD8 T Cells Induces Protective and High-Avidity T Cell Responses to Tumor-Specific Antigens

Treatment of tumor-bearing mice with a stimulatory Ab to glucocorticoid-induced TNFR family-related receptor (GITR) has previously been shown to elicit protective T cell responses against poorly immunogenic tumors. However, the role of GITR stimulation on CD8 T cells and the nature of tumor rejection Ags have yet to be determined. In this study, we show that a stimulatory mAb to GITR (clone DTA-1) acts directly on CD8 T cells, but not on CD4(+)CD25(+) regulatory T (T(reg)) cells, in B16 tumor-bearing mice to induce concomitant immunity against secondary B16 tumors, as well as protective memory following surgical excision of the primary tumor. Melanoma growth itself induced GITR expression on tumor-specific CD8 T cells, providing a mechanism whereby these cells may respond to stimulatory anti-GITR. Unexpectedly, in contrast to T(reg) cell depletion therapy with anti-CD4, GITR stimulation induced very weak CD8 T cell responses to melanocyte differentiation Ags expressed by the tumor, and did not induce autoimmune vitiligo. Accordingly, GITR-stimulated hosts that were primed with B16 melanoma rejected B16, but not the unrelated JBRH melanoma, indicating that tumor rejection Ags are tumor-specific rather than shared. In support of this, we show that GITR stimulation induces CD8 T cell responses to a tumor-specific Ag, and that these responses are of higher functional avidity compared with those induced by T(reg) cell depletion. We conclude that stimulation of GITR on effector CD8 T cells results in high-avidity T cell responses to tumor-specific Ags, thereby inducing potent antitumor immunity in the absence of autoimmunity.

Activation of tolerogenic dendritic cells in the tumor draining lymph nodes by CD8+ T cells engineered to express CD40 ligand.

Tolerogenic dendritic cells in the tumor microenvironment can inhibit the generation and maintenance of robust antitumor T cell responses. In this study, we investigated the effects of local delivery of CD40L by tumor-reactive CD8(+) T cells on dendritic cell activation and antitumor T cell responses in the TRAMP model. To increase the immunostimulatory signal, CD40L was engineered, by deleting the majority of the cytoplasmic domain, to increase its levels of expression and duration on the surface of CD8(+) T cells. Tumor-reactive CD8(+) T cells expressing the truncated form of CD40L stimulated maturation of dendritic cells in vitro and in the prostate draining lymph nodes in vivo. Following dendritic cell maturation, a significantly higher fraction of adoptively transferred, tumor-reactive (reporter) CD8(+) T cells was stimulated to express IFN-gamma and infiltrate the prostate tissue. The antitumor CD8(+) T cell response was further enhanced if TRAMP mice were also immunized with a tumor-specific Ag. These findings demonstrate that augmented T cell responses can be achieved by engineering tumor-reactive T cells to deliver stimulatory signals to dendritic cells in the tumor microenvironment.

IFN-γ Promotes Generation of IL-10 Secreting CD4+ T Cells that Suppress Generation of CD8 Responses in an Antigen-Experienced Host

Ags characterizing tumors or chronic viral infection are generally presented to the host immune system before specific immunotherapy is initiated, and consequent generation of regulatory CD4(+) T cells can inhibit induction of desired effector CD8 T cell responses. IL-10 produced in response to ongoing Ag exposure inhibits generation of CD8 T cells in an Ag-experienced host. We now show that this IL-10 is produced by Ag experienced CD4(+) glucocorticoid-induced tumor necrosis factor receptor(+) T cells that also secrete IFN-gamma upon antigenic stimulation, that IL-10 secretion by these cells is enhanced through IFN-gamma signaling, and, unexpectedly, that IFN-gamma signaling is required for inhibition of generation of Ag-specific CD8 T cell responses in an Ag-experienced host. Systemic inhibition of both IL-10 and IFN-gamma at the time of immunization may therefore facilitate induction of effective immunotherapeutic responses against tumor specific and viral Ags.

Protective Immunosurveillance and Therapeutic Antitumor Activity of γδ T Cells Demonstrated in a Mouse Model of Prostate Cancer

In contrast to Ag-specific alphabeta T cells, gammadelta T cells can kill malignantly transformed cells in a manner that does not require the recognition of tumor-specific Ags. Although such observations have contributed to the emerging view that gammadelta T cells provide protective innate immunosurveillance against certain malignancies, particularly those of epithelial origin, they also provide a rationale for developing novel clinical approaches to exploit the innate antitumor properties of gammadelta T cells for the treatment of cancer. Using TRAMP, a transgenic mouse model of prostate cancer, proof-of-concept studies were performed to first establish that gammadelta T cells can indeed provide protective immunosurveillance against spontaneously arising mouse prostate cancer. TRAMP mice, which predictably develop prostate adenocarcinoma, were backcrossed with gammadelta T cell-deficient mice (TCRdelta(-/-) mice) yielding TRAMP x TCRdelta(-/-) mice, a proportion of which developed more extensive disease compared with control TRAMP mice. By extension, these findings were then used as a rationale for developing an adoptive immunotherapy model for treating prostate cancer. Using TRAMP-C2 cells derived from TRAMP mice (C57BL/6 genetic background), disease was first established in otherwise healthy wild-type C57BL/6 mice. In models of localized and disseminated disease, tumor-bearing mice treated i.v. with supraphysiological numbers of syngeneic gammadelta T cells (C57BL/6-derived) developed measurably less disease compared with untreated mice. Disease-bearing mice treated i.v. with gammadelta T cells also displayed superior survival compared with untreated mice. These findings provide a biological rationale for clinical trials designed to adoptively transfer ex vivo expanded autologous gammadelta T cells for the treatment of prostate cancer.

Dual T Cell Receptor Expressing CD8+ T Cells with Tumor- and Self-Specificity Can Inhibit Tumor Growth without Causing Severe Autoimmunity

The engineering of Ag-specific T cells by expression of TCR genes is a convenient method for adoptive T cell immunotherapy. A potential problem is the TCR gene transfer into self-reactive T cells that survived tolerance mechanisms. We have developed an experimental system with T cells that express two TCRs with defined Ag-specificities, one recognizing a tumor-specific Ag (LCMV-gp(33)), the other recognizing a self-Ag in the pancreas (OVA). By using tumor cells expressing high and low amounts of Ag and mice expressing high and low levels of self-Ag in the pancreas (RIP-OVA-Hi and RIP-OVA-Lo), we show that 1) tumor rejection requires high amount of tumor Ag, 2) severe autoimmunity requires high amount of self-Ag, and 3) if Ag expression on tumor cells is sufficient and low in the pancreas, successful adoptive T cell therapy can be obtained in the absence of severe autoimmunity. These results are shown with T cells from dual TCR transgenic mice or T cells that were redirected by TCR gene transfer. Our data demonstrate that the approach of adoptively transferring TCR redirected T cells can be effective without severe side effects, even when high numbers of T cells with self-reactivity were transferred.

Tumor-Associated Embryonic Antigen-Expressing Vaccines that Target CCR6 Elicit Potent CD8+ T Cell-Mediated Protective and Therapeutic Antitumor Immunity

Despite its potency, the wider use of immunotherapy for B cell malignancies is hampered by the lack of well-defined tumor-specific Ags. In this study, we demonstrate that an evolutionarily conserved 37-kDa immature laminin receptor protein (OFA-iLRP), a nonimmunogenic embryonic Ag expressed by a variety of tumors, is rendered immunogenic if targeted to the APCs using the CCR6 ligands MIP3alpha/CCL20 and mDF2beta. The CCR6 targeting facilitated efficient Ag cross-presentation and induction of tumor-neutralizing CTLs. Although the Ag targeting alone, without activation of dendritic cells (DCs), is proposed to induce tolerance, and MIP3alpha does not directly activate DCs, the MIP3alpha-based vaccine efficiently induced protective and therapeutic antitumor responses. The responses were as strong as those elicited by the OFA-iLRP fusions with moieties that activated DCs and Th1-type cytokine responses, mDF2beta, or mycobacterial Hsp70 Ag. Although the same cDNA encodes the dimerized high-affinity mature 67-kDa mLRP that is expressed in normal tissues to stabilize the binding of laminin to cell surface integrins, the vaccines expressing OFA-iLRP elicited long-term protective CD8(+) T cell-mediated memory responses against syngeneic B cell lymphoma, indicating the potential application of these simple vaccines as preventive and therapeutic formulations for human use.

A Modified Tyrosinase-Related Protein 2 Epitope Generates High-Affinity Tumor-Specific T Cells but Does Not Mediate Therapeutic Efficacy in an Intradermal Tumor Model

The generation of tumor-specific T cells is hampered by the presentation of poorly immunogenic tumor-specific epitopes by the tumor. Here, we demonstrate that, although CD8+ T cells specific for the self/tumor Ag tyrosinase-related protein 2 (TRP2) are readily detected in tumor-bearing hosts, vaccination of either tumor-bearing or naive mice with an epitope derived from TRP2 fails to generate significant numbers of tetramer-staining TRP2-specific T cells or antitumor immunity. We identified an altered peptide epitope, called deltaV, which elicits T cell responses that are cross-reactive to the wild-type TRP2 epitope. Immunization with deltaV generates T cells with increased affinity for TRP2 compared with immunization with the wild-type TRP2 epitope, although TRP2 immunization often generates a greater number of TRP2-specific T cells based on intracellular IFN-gamma analysis. Despite generating higher affinity responses, deltaV immunization alone fails to provide any greater therapeutic efficacy against tumor growth than TRP2 immunization. This lack of tumor protection is most likely a result of both the deletion of high affinity and functional tolerance induction of lower affinity TRP2-specific T cells. Our data contribute to a growing literature demonstrating the ability of variant peptide epitopes to generate higher affinity T cell responses against tumor-specific Ags. However, consistent with most clinical data, simple generation of higher affinity T cells is insufficient to mediate tumor immunity.

Predicting Tumor Outcome following Cancer Vaccination by Monitoring Quantitative and Qualitative CD8+ T Cell Parameters

Identification of reliable surrogate predictors for evaluation of cancer vaccine efficacy is a critical issue in immunotherapy. We analyzed quantitative and qualitative CD8+ T cell parameters in a large pool of BALB/c mice that were DNA-vaccinated against P1A self tumor-specific Ag. After immunization, mice were splenectomized and kept alive for a subsequent tumor challenge to correlate results of immune monitoring assays with tumor regression or progression in each individual animal, and to assess the prognostic value of the assays. The parameters tested were 1) percentage of in vivo vaccine-induced tumor-specific CD8+ T cells; 2) results of ELISPOT tests from fresh splenocytes; 3) percentage of tumor-specific CD8+ T cells in culture after in vitro restimulation; 4) in vitro increase of tumor-specific CD8+ T cell population expressed as fold of expansion; and 5) antitumor lytic activity of restimulated cultures. Except for the ELISPOT assay, each parameter tested was shown by univariate statistical analysis to correlate with tumor regression. However, multivariate analysis revealed that only in vitro percentage of Ag-specific CD8+ T cells was an independent prognostic factor that predicted tumor outcome. These findings should be considered in the design of new immune monitoring systems used in cancer immunotherapy studies.

The Role of Intercellular Adhesion Molecule-1/LFA-1 Interactions in the Generation of Tumor-Specific CD8+ T Cell Responses

The activation of naive CD4+ T cells requires both TCR engagement and a second costimulatory signal mediated by the interaction of CD28 with CD80/CD86 expressed on professional APC. However, the situation for naive CD8+ T cells is less clear. Although evidence indicates that induction of CD8+ T cell responses is also dependent on professional APC, the ability of some tumors, which do not express CD80/CD86, to induce CTL suggests that other pathways of costimulation exist for the activation of CD8+ T cells. We examined the ability of tumor cells expressing different levels of a tumor-specific Ag to directly prime CD8+ T cells. We demonstrate that CD8+ T cells are directly activated by tumor cells in a CD80/CD86-CD28 independent manner. In this system, costimulation requires ICAM-1/LFA-1 interaction. This results in the generation of CTL capable of inhibiting tumor growth in vivo, and maintaining long-term survival.

Naturally Acquired MAGE-A10- and SSX-2-Specific CD8+ T Cell Responses in Patients with Hepatocellular Carcinoma

Immunotherapy is being proposed to treat patients with hepatocellular carcinoma (HCC). However, more detailed knowledge on tumor Ag expression and specific immune cells is required for the preparation of highly targeted vaccines. HCC express a variety of tumor-specific Ags, raising the question whether CTL specific for such Ags exist in HCC patients. Indeed, a recent study revealed CTLs specific for two cancer-testis (CT) Ags (MAGE-A1 and MAGE-A3) in tumor infiltrating lymphocytes of HCC patients. Here we assessed the presence of T cells specific for additional CT Ags: MAGE-A10, SSX-2, NY-ESO-1, and LAGE-1, which are naturally immunogenic as demonstrated in HLA-A2(+) melanoma patients. In two of six HLA-A2(+) HCC patients, we found that MAGE-A10- and/or SSX-2-specific CD8(+) T cells naturally responded to the disease, because they were enriched in tumor lesions but not in nontumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, providing evidence that these CTL were selected in vivo for high avidity Ag recognition. Therefore, besides melanoma, HCC is the second solid human tumor with clear evidence for in vivo tumor recognition by T cells, providing the rational for specific immunotherapy, based on immunization with CT Ags such as MAGE-A10 and SSX-2.

Anti-idiotype x anti-CD44 bispecific antibodies inhibit invasion of lymphoid organs by B cell lymphoma.

The demonstration that Abs to adhesion molecules can block tumor metastasis suggested their use for therapy. However, such Abs affect nonmalignant cells as well. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such bifunctional Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. Therefore, we produced bispecific Abs with specificity to the adhesion molecule, CD44, and to an idiotypic determinant of the murine B cell lymphoma, 38C-13. These anti-Id x anti-CD44 bispecific Abs blocked 38C-13 cell adhesion to hyaluronic acid, while not affecting adhesion of Id-negative cells. In vivo studies demonstrated that the bispecific Abs inhibited lymphoma cell dissemination to the lymph nodes, bone marrow, and spleen, and prolonged survival of tumor-bearing mice. Migration of 38C-13 cells to the lymphoid organs was inhibited by the bispecific Abs. Thus, the bispecific Ab-mediated reduction in metastasis resulted, at least in part, from reduced homing to these organs. In contrast to anti-CD44 monospecific Abs, the anti-Id x anti-CD44 bispecific Abs did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and tumor-specific Ags may selectively block tumor metastasis in a way which may leave at least part of the immune system intact.

Definition of TCR epitopes for CTL-mediated attack of cutaneous T cell lymphoma.

Therapeutic vaccination against cutaneous T cell lymphoma (CTCL) requires the characterization of cancer cell-specific CTL epitopes. Despite reported evidence for tumor-reactive cytotoxicity in CTCL patients, the nature of the recognized determinants remains elusive. The clonotypic TCR of CTCL cells is a promising candidate tumor-specific Ag. In this study, we report that the clonotypic and framework regions of the TCRs expressed in the malignant T cell clones of six CTCL patients contain multiple peptides with anchor residues fitting the patients' MHC class I molecules. We demonstrate that TCR peptide-specific T cells from the blood of healthy donors and patients can be induced to become cytotoxic effectors after repeated stimulation with 6 of 11 selected peptides with experimentally proven affinity for HLA-A*0201. Importantly, 4 of these 6 CTL lines reproducibly recognize and lyse autologous primary CTCL cells in MHC class I/CD8-dependent fashion. These tumoricidal CTL lines are directed against epitopes from V, hypervariable, and C regions of TCRalpha. We therefore conclude that recombined as well as V framework regions of the tumor cell TCRs contain predictable epitopes for CTL-mediated attack of CTCL cells. Our data further suggest that such peptides represent valuable tools for future anti-CTCL vaccination approaches.

A MAGE-3 peptide presented by HLA-DR1 to CD4+ T cells that were isolated from a melanoma patient vaccinated with a MAGE-3 protein.

"Cancer-germline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific Ags, which have been used in therapeutic vaccination trials of cancer patients. MAGE-3 is expressed in 74% of metastatic melanoma and in 50% of carcinomas of esophagus, head and neck, bladder, and lung. We report here the identification of a new MAGE-3 peptide, which is recognized by three different CD4(+) T cell clones isolated from a melanoma patient vaccinated with a MAGE-3 protein. These clones, which express different TCRs, recognize an HLA-DR1 peptide ACYEFLWGPRALVETS, which corresponds to the MAGE-3(267-282) and the MAGE-12(267-282) protein sequences. One of the T cell clones, which expresses LFA-1 at a high level, lysed tumor cells expressing DR1 and MAGE-3. Another of these DR1-restricted CD4(+) clones recognized not only the MAGE-3/12 peptide but also homologous peptides encoded by genes MAGE-1, 2, 4, 6, 10, and 11.

Identification of a mutated receptor-like protein tyrosine phosphatase kappa as a novel, class II HLA-restricted melanoma antigen.

Recent studies increasingly point to a pivotal role of CD4(+) T cells in human anti-tumor immune response. Here we show that lymphocytes purified from a tumor-infiltrated lymph node of a melanoma patient that had remained disease free for 10 years after surgical resection of a lymph node metastasis comprised oligoclonal class II HLA-restricted CD4(+) T cells recognizing the autologous tumor cells in vitro. In fact, the CD4(+) T cell clones isolated from these lymphocytes displayed a tumor-specific, cytotoxic activity in addition to a Th1-like cytokine profile. By a genetic approach, a peptide derived from a mutated receptor-like protein tyrosine phosphatase kappa was identified as a novel HLA-DR10-restricted epitope for all the melanoma-specific CD4(+) T cell clones. The immunogenic peptide was shown to contain the mutated residue that was crucial for T cell recognition and activation. Moreover, a systemic immunity against the mutated peptide was detectable in the patient's peripheral blood T lymphocytes obtained during the disease-free period of follow-up. These findings further support the relevance of CD4(+) T cells directed against mutated epitopes in tumor immunity and provide the rationale for a possible usage of mutated, tumor-specific Ags for immunotherapy of human cancer.

Anti-idiotype x anti-LFA-1 bispecific antibodies inhibit metastasis of B cell lymphoma.

Abs to adhesion molecules can block tumor metastasis. However, they may also block the function of normal cells. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. We therefore produced a bispecific Ab with specificity to the adhesion molecule LFA-1 and to the Id of the murine B cell lymphoma 38C-13. Here we demonstrate that this Ab blocked liver metastasis in mice carrying primary s.c. tumors and partially inhibited lymph node metastasis. Migration of 38C-13 cells to liver and lymph nodes was inhibited by the bispecific Ab, while migration to spleen was not affected. Hence, the bispecific Ab-mediated reduction in liver and lymph node metastasis resulted at least in part from reduced homing to these organs. In contrast to anti-LFA-1 monospecific Abs, the anti-Id x anti-LFA-1 bispecific Ab did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and against tumor-specific Ags may selectively block tumor metastasis in a way that may leave much of the immune system intact.