Dorothee Herlyn, D.V.M., D.Sc.

Professor
Immunology Program and Molecular and Cellular Oncogenesis Program
215-898-3962, Office

Introduction

Dorothee Herlyn's laboratory is developing prototype vaccines against colorectal and other cancers. The vaccines are based on proteins identified only in the cancerous cells and not in healthy cells of the same type, or on proteins that are expressed in both cell types.

Research Summary

The laboratory of Dorothee Herlyn develops cancer vaccines and testing these vaccines in pre-clinical and clinical studies. The vaccines are directed against melanoma, colon carcinoma, glioma and breast carcinoma.To develop cancer vaccines, antigens recognized by cancer patients' B cells, helper T cells and cytolytic T lymphocytes are cloned and characterized as to their expression in various tumor and normal tissues. In pre-clinical studies the vaccines are evaluated for their capacity to induce humoral, cellular and protective immune responses in animal models that mimic the conditions in cancer patients (e.g. transgenic mouse models or mouse antigen homologue models). In clinical trials, colorectal cancer patients were treated with anti-idiotypic antibody and recombinant antigen vaccines. Patients developed humoral and cellular immune responses, but the evaluation of clinical responses must await randomized phase II trials. In future clinical trials vaccine effects may be augmented by chemokines which have been shown to enhance T cell migration toward tumor cells in culture models in the laboratory.

Recent Scientific Advances

Experimental Active Immunotherapy against Colorectal Carcinoma(CRC) and Melanoma

The CRC Ag CO17-1A/GA733 has proven to be a useful target in passive
immunotherapy with monoclonal antibody (MAb) and in active immunotherapy with anti-idiotypic antibodies (Ab2) in cancer patients. Patients treated with MAb CO17-1A demonstrated significantly enhanced survival as compared to control patients in a randomized trial with an 8-year follow-up. In another trial, 75% of the patients treated with Ab2 mimicking the GA733 epitope are currently without evidence of disease after a follow-up of 4-8 years. In both clinical trials, a single epitope of the Ag was targeted. We postulated that targeting the whole Ag, which contains multiple potentially immunogenic epitopes, may be more efficacious than targeting a single epitope. Therefore, the CO17-1A/GA733 Ag was molecularly cloned and expressed in baculovirus, adenovirus, and vaccinia virus. Recombinant vaccinia and adenoviruses protected mice against a challenge with CO17-1A/GA733 Ag-positive syngeneic colon carcinoma cells, but only the adenovirus recombinant inhibited growth of established tumors. In contrast, baculovirus-derived protein in various adjuvants had no effect on tumor growth. Recombinant vaccinia and adenoviruses induced Ag-specific cytotoxic antibodies, and proliferative and delayed-type hypersensitive lymphocytes. However, only the adenovirus recombinant induced Ag-specific cytolytic T lymphocytes. Thus, the recombinant adenovirus has potential as a vaccine for cancer patients.

We have recently developed a novel animal model of active immunotherapy
against the human CO17-1A/GA733 Ag. Mice express an Ag (murine epithelial glycoprotein [mEGP]) with 82% sequence homology to the human GA733 Ag. The mEGP protein is expressed on normal murine epithelial tissues, similarto the tissue distribution of the human GA733 Ag. Thus, mice provide apromising model for active immunotherapy against the CO17-1A/GA733 Ag inthe immunologically tolerant host. In addition, this model allows evaluation ofpotential toxic effects of vaccinations with mEGP in mEGP-expressing organs. To develop this model, we produced syngeneic murine colon carcinoma cells expressing mEGP after cDNA transfer to serve as targets in active immunotherapy against the Ag. To develop mEGP-derived vaccines, the protein was produced in baculovirus or expressed in vaccinia oradenoviruses. mEGP protein in various adjuvants did not protect mice against a challenge with mEGP-positive tumors, similar to the absence of protective activity of baculovirus-derived human CO17-1A/GA733 Ag in mice.

However, in contrast to adenovirus expressing the human CO17-1A/GA733 Ag, adenovirus expressing the mouse homolog mEGP did not inhibit tumor growth in mice, not even when administered together with IL-2. This emphasizes the difficulty in breaking immunological tolerance to self Ag expressed by normal organs. We are currently testing the immunotherapeutic efficacy of mEGP expressed in Salmonella typhi murium or mEGP administered with the toll-like receptor 9 stimulating adjuvant CpG or antibodies blocking negative immunoregulators, such as tumor-expressed PD1 ligand or regulatory T cells.

We are currently establishing a transgenic mouse melanoma model for immunotherapeutic targeting mutated BRAF (BRAF(V600E)) which is a tumor-specific Ag. These studies are based on previous observations in melanoma patients. In those studies, BRAF(V600E) peptides with putative binding sites for human leukocyte antigen (HLA)-A2 were used to stimulate T lymphocytes of HLA-A2-positive melanoma patients. Four of five patients with BRAF(V600E)-positive lesions showed lymphoproliferative responses to BRAF(V600E) peptide stimulation. These responses were specific for the mutated epitope and HLA-A2 restricted in three patients. Lymphocytes from these three patients were cytotoxic against HLA-A2-matched BRAF(V600E)-positive melanoma cells. None of the four patients with BRAF(V600E)-negative lesions and none of five healthy donors had lymphoproliferative responses specific for the mutated epitope. The high prevalence (approximately 50%) of HLA-A2 among melanoma patients renders HLA-A2-restricted BRAF(V600E) peptides attractive candidate vaccines for these patients.

Identification of Tumor-Associated Antigens with Vaccine Potential

A melanoma and a colon cancer antigen recognized by cytolytic T lymphocytes (CTL) were cloned using the COS cell cDNA library expression approach and identified as tRNA isopentyltransferase 1 (TRIT-1)-related protein and nucleophosmin (NPM, B23, nutramin, or NO38), respectively. TRIT-1 has been described as a tumor suppressor in lung carcinoma.Nucleophosmin has oncogenic activity, is expressed by lymphomas and upregulated by colon carcinomas, hepatomas, bladder carcinomas, and melanomas. A melanoma antigen recognized by helper T (Th) cells was cloned using a novel phage display approach which offers several advantages over the previously used invariant chain fusion approach, such as absence of requirement for knowledge of the MHC restriction element used by the Th cells for antigen recognition. The antigen was identified as ribosomal protein (RP) L8. RPL8 is expressed by melanomas, gliomas and breast carcinomas. Mutated BRAF (V600E mutation) is a tumor-specific epitope expressed by approximately 70% of melanomas derived from different patients. Peptides of mutated BRAF induced CTL in melanoma patients' lymphocytes. This study shows for the first time that TRIT-1 related protein, nucleophosmin, RPL8 and BRAF-V600E are recognized by patients' T cells. The antigens express numerous epitopes potentially associating with different class I and II human lymphocyte antigens (HLA) and therefore they may induce CTL and Th cells in patients expressing various HLA types. These antigens also have potential as vaccines for patients with tumors of various histological types.

Lymphocyte Migration

A novel 3-dimensional melanoma culture system (reconstruct) was developed to identify the chemokines and chemokine receptors involved in the migration of cytotoxic T lymphocytes (CTL) toward tumor cells. We have shown that chemokines produced by melanoma cells attract CTL expressing the corresponding chemokine receptor. This interaction leads to CTL migration toward melanoma cells and induction of melanoma cell apoptosis. We have identified chemokines and chemokine receptors involved in the migration toward melanoma cells of 4 CTL derived from 3 different melanoma patients’ PBL. The chemokines and their receptors were identified in tumor cell apoptosis inhibition experiments with antibodies to chemokines or chemokine receptors or with chemokines. CTL migration and tumor cell apoptosis by 2 CTL derived from different patients was induced by the same chemokine/chemokine receptor pair (CXCR4 and CXCL12). We are currently investigating whether: a. CTL can localize in melanomas in immunocompromized SCID/NOD/IL-2gc-/- mice in vivo; b. CTL can inhibit tumor growth in vivo; c. migration of the CTL toward melanoma cells in vivo is dependent on chemokines; and d. CTL migration and tumor destruction can be further enhanced by antibody-chemokine fusion proteins.

The chemokines that were identified may be useful for the development of novel immunotherapies which are currently under development in our laboratory and are based on chemokine gene therapy or chemokine-antibody fusion proteins.

Selected Publications

Somasundaram, R., L. Jacob, R. Swoboda, L. Caputo, H. Song, S. Basak, D. Monos, D. Peritt, F. Marincola, D. Cai, B. Birebent, E. Bloome, J. Kim, K. Berencsi, M. Mastrangelo, and D. Herlyn. 2002. Inhibition of cytolytic T lymphocyte proliferation by autologous CD4+/CD25+ regulatory T cells in a colorectal carcinoma patient is mediated by transforming growth factor-beta. Cancer Res. 62:5267-5272.

Zaloudik, J., W. Li, L. Jacob, M.P. Kieny, R. Somasundaram, B. Acres, H. Song, T. Zhang, J. Li, and D. Herlyn. 2002. Inhibition of tumor growth by recombinant vaccinia virus expressing GA733/CO17-1A/EpCAM/KSA/KS1-4 antigen in mice. Cancer Gene Ther. 9:382-389.

Somasundaram, R., R. Swoboda, L. Caputo, A. Lee, N. Jackson, F.M. Marincola, D. Guerry, and D. Herlyn. 2003. A CD4+, HLA-DR7-restricted T-helper lymphocyte clone recognizes an antigen shared by human malignant melanoma and glioma. Int J Cancer. 104:362-368.

Zhang, T., R. Somasundaram, K. Berencsi, L. Caputo, P. Rani, D. Guerry, E. Furth, B.J. Rollins, M. Putt, P. Gimotty, R. Swoboda, M. Herlyn, and D. Herlyn. 2005. CXC Chemokine Ligand 12 (Stromal Cell-Derived Factor 1{alpha}) and CXCR4-Dependent Migration of CTLs toward Melanoma Cells in Organotypic Culture. J Immunol. 174:5856-63.

Somasundaram, R, Caputo, L, Guerry, D, and Herlyn, D. 2005. CD8+, HLA-unrestricted, cytotoxic T-lymphocyte line against malignant melanoma. J Transl Med 3:41.

Zhang, T., Somasundaram, R., Berencsi, K., Caputo, L., Gimotty, P., Rani, P., Guerry, D., Swoboda, R., and Herlyn, D. 2006. Migration of cytotoxic T lymphocytes toward melanoma cells in three-dimensional organotypic culture is dependent on CCL2 and CCR4. Eur J Immunol 36:457-467.

Somasundaram, R., Swoboda, R., Caputo, L., Otvos, L., Weber, B., Volpe, P., van Belle, P., Hotz, S., Elder, D.E., Marincola, F.M., Schuchter, L., Guerry, D., Czerniecki, B.J., and Herlyn, D. 2006. Human Leukocyte Antigen-A2-restricted CTL responses to mutated BRAF peptides in melanoma patients. Cancer Res 66:3287-93.

Zhang, T., R. Somasundaram, L. Caputo, P. Van Belle, D. Elder, B. Czerniecki, S. Hotz, L. Schuchter, F.R. Spitz, K. Berencsi, P. Rani, F. Marincola, R. Qiu, and D. Herlyn. 2006. Preferential involvement of CX chemokine receptor 4 and CX chemokine ligand 12 in T-cell migration toward melanoma cells. Cancer Biol. Ther. 5:1304-1312.

Berencsi, K., N.J. Meropol, J.P. Hoffman, E. Sigurdson, L. Giles, P. Rani, R. Somasundaram, T. Zhang, J. Kalabis, L. Caputo, E. Furth, R. Swoboda, F. Marincola, and D. Herlyn. 2007. Colon carcinoma cells induce CXCL11-dependent migration of CXCR3-expressing cytotoxic T lymphocytes in organotypic culture. Cancer Immunol Immunother. 56: 359-370.

Swoboda, R.K., Somasundaram, R., Caputo, L., Ochoa, E.M., Gimotty, P.A., Marincola, F.M., Van Belle, P., Barth, S., Elder, D., Guerry, D., Czerniecki, B., Schuchter, L., Vonderheide, R.H., and Herlyn, D. 2007. Shared MHC class II-dependent melanoma ribosomal protein L8 identified by phage display. Cancer Res. 67: 3555-3559.

Wondimu, A., Zhang, T., Kieber-Emmons, T., Gimotty, P., Sproesser, K., Somasundaram, R., Ferrone, S., Tsao, C-Y, and Herlyn, D. 2007. Peptides mimicking GD2 ganglioside elicit cellular, humoral and tumor-protective immune responses in mice. Cancer Immunol Immunother, 2007. 57: 1079-1089.