Dorothee Herlyn, D.V.M.

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, which are evaluated in animals for their ability to protect against tumors before they are given to cancer patients. The vaccines are composed of antibodies - called anti-idiotypic antibodies - that mimic tumor antigens or the antigens expressed in genetically engineered viruses. The specific make-up of these vaccines is based on their enhanced ability to induce an immune response in humans. The animal models the researchers have developed for preclinical evaluation of the vaccines closely mimic the condition in patients with, for example, colorectal cancer. Some of the mice used express antigens similar to human tumor antigens on their normal and tumor tissues, and some of the rats used express mutations of tumor-associated antigen on their tumors. In other studies that have reached clinical trials, the investigators have evaluated vaccinated patients' immune response. In a phase I clinical trial with anti-idiotypic antibodies mimicking a colorectal cancer antigen, vaccinated patients developed both antibodies and T cells directed at fighting the tumor. In another program based on discovering new vaccines, the researchers are identifying cancer antigens that are recognized by patients' B cells, T helper cells, and cytolytic T lymphocytes as candidates for active immunotherapy. The researchers are also investigating active immunotherapies in experiments with recombinant colorectal cancer antigens. They have so far found that genetically engineered antigens in vaccinia viruses and adenoviruses protect mice from colon carcinoma cells, but only the adenovirus antigen inhibited growth of established tumors. Research in the laboratory also addresses other types of cancer. The researchers have isolated five helper T lymphocyte lines and 10 helper T lymphocyte clones from the blood cells of five melanoma patients to develop new skin cancer vaccines. In another project, EGF-RvIII, a tumor-specific antigen expressed by certain cancers of the breast and brain (gliomas), is also being explored. The team suggests that breast cancer patients are candidates for receiving EGF-RvIII vaccines to boost the patients’ natural immune responses against the antigen, which may lead to tumor destruction.

Recent Scientific Advances

Colorectal cancer anti-idiotypic antibodies and active immunotherapies: Anti-idiotypic antibodies - antibodies that mimic tumor antigens - may structurally and functionally mimic the tumor antigen recognized by the anti-tumor antibody. In collaboration with Dr. Edith Mitchell at Thomas Jefferson University Hospital in Philadelphia , the laboratory of Dorothee Herlyn has completed a phase I clinical trial with anti-idiotypic antibodies mimicking a colorectal cancer antigen (CRC) (1). Vaccinated patients developed both antibodies and T cells directed at the tumor antigen. The clinical responses of the patients to the vaccine are difficult to evaluate in this phase I trial because all patients had advanced disease at the time of treatment. The clinical responses to the anti-idiotype vaccine will be evaluated in a phase II trial in patients with minimal disease.

The researchers are also investigating active immunotherapies in experiments with recombinant CRC antigen (Ag) (2, 3, 4). The CRC antigen 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 9-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 5-9 years. In both clinical trials, a single epitope of the Ag was targeted. The investigators 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. A clinical trial with the adenovirus recombinant is in preparation in collaboration with Dr. Ludger Staib ( University of Ulm , Germany ).

The researchers have recently developed a novel animal model of active immunotherapy against the human CO17-1A/GA733 antigen. Mice express an antigen called murine epithelial glycoprotein (mEGP) with 82% sequence homology to the human GA733 Ag. The mEGP protein is expressed on normal murine epithelial tissues, similar to the tissue distribution of the human GA733 Ag. Thus, mice provide a promising model for active immunotherapy against the CO17-1A/GA733 Ag in the immunologically tolerant host. In addition, this model allows evaluation of potential toxic effects of vaccinations with mEGP due to the expression of this Ag in normal organs. To develop this model, the researchers produced 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 or adenoviruses. In various adjuvants mEGP protein 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, recombinant adenovirus expressing mEGP and administered in conjunction with interleukin-2 (IL-2) significantly inhibited growth of established mEGP-positive tumors.

Experimental Active Immunotherapy of Melanoma (5): CD63 is a melanoma-associated Ag that is also expressed by normal tissues. The laboratory has developed a transgenic mouse model of active immunotherapy targeting CD63. CD63 expressed in vaccinia virus and administered to mice together with IL-2 was able to onhibit growth of established melanomas. This effect was mediated by both CD4 + and CD8 + T cells. Thus, CD63 has potential as a vaccine for melanoma patients.

Cytolytic and Helper T Lymphocytes Against Colon Carcinoma and Melanoma (6-8): Cytolytic and helper T lymphocyte (CTL, Th) lines and clones were established from the peripheral blood mononuclear cells (PBMC) of three patients with primary colon carcinoma by stimulating the PBMC with autologous tumor cells and IL-2 in the presence of Epstein-Barr virus (EBV)-transformed B cells as antigen-presenting cells. Several T cell lines/clones were obtained from three different patients: a) a CD4+, HLA class I-restricted CTL line that lysed the autologous tumor as well as allogeneic HLA A1- or A3-matched colon carcinoma cells, but not HLA non-matched colon carcinoma cells or autologous or allogeneic EBV-B cells; b) a CD4+ CTL clone that lysed the autologous, but not allogeneic, tumor and did not lyse autologous EBV-B cells, K562, or Daudi cells; c) two CTL clones (CD4+, CD8+), both HLA class I-restricted, which lysed the autologous tumor, but not EBV-B cells and two Th clones (CD4+), which were of Th-2 type and augmented pokeweed mitogen-induced proliferative lymphocyte responses of autologous PBMC.

A regulatory T cell line was isolated from the lymphocytes of a colorectal cancer patient. This T cell line inhibited the induction of CTL in the patients' lymphocytes and the proliferation of established CTL. The inhibitory effect was mediated by TGF- b .

The laboratory has also isolated 5 Th lines and 10 Th clones from the PBMC of five different melanoma patients, using the approach described above for CRC-derived PBMC. The Th had functional characteristics similar to the anti-CRC Th and were HLA class II-dependent in their proliferative activities. The Th were of Th1 type according to their cytokine secretion pattern. A CTL clone was derived from the PBMC of an additional melanoma patient. The CTL clone was of Th1 type and lysed autologous and HLA B57[17]-matched allogeneic melanoma cells. The antigens recognized by the CTL and Th lines/clones are currently being identified, since they may provide vaccines that induce CTL and Th in colon carcinoma and melanoma patients. CTL antigens are cloned using the COS cell expressed tumor cell cDNA libraries, and Th antigens using bacteriophage expressed libraries.

Recently (9), the laboratory has established an organotypic culture system for the isolation of CTL and Th from fresh tumor tissues under in vivo-like conditions. In this system, tumor tissues are embedded into collagen. The system enables the study of the factors that play a role in migration of T cells into tumor tissues, such as chemokines and adhesion molecules. Thus, chemokines and their receptors (ITAC and CXCR3; IP-10 and CXCR3; SDF-1 and CXCR4) were shown to play an important role in the migration of Th cells and CTL toward tumor cells (melanoma and CRC). The chemokines may be used as vaccine enhancers in cancer patients.

Identification of Tumor Antigens by Antibody Phage Display (10): The laboratory developed a method of phage display of cancer patients' antibodies in order to identify novel tumor antigens as potential cancer vaccines. Antibody libraries derived from the B cells of a melanoma patient who has been in remission for 7 years, following vaccination with tumor cells, were expressed on the surfaces of filamentous phages. The phages expressing Fab (fragment Ag binding) were absorbed to normal melanocytes (to remove phages binding to normal cells) and eluted from melanoma cells derived from various patients (to isolate phages binding to tumor cells). To select against phages binding to N-linked sugars, phages were eluted from tunicamycin-treated allogeneic tumor cells.

Five phages were characterized in detail. They bound to the surfaces and, in a few instances, also to the cytoplasm of cultured melanoma, breast carcinoma, and glioma cells derived from various patients. They also bound to sections of melanoma tissues and, in some instances, to nevic cells. One phage was tumor-specific; it did not bind to any of the four different cultured normal cells tested. The expressed Fab binds to a protein of 23 kDa that is covalently associated with a 10 kDa protein. The protein is expressed by metastatic and vertical growth phase primary melanoma cells in tissue sections but not by radial growth phase primary melanoma cells or nevi. The antigen was molecularly cloned and found to be identical with p23 previously described to be expressed by lung carcinomas and sarcomas. The p23 antigen is a tumor progression marker for melanocytic lesions and also may have therapeutic utility for the treatment of melanoma.

Mutated epidermal growth factor receptor (EGF-RvIII) (11): The laboratory has demonstrated that breast cancer patients mount specific immune responses to EGF-RvIII. These responses may be boosted by vaccines of EGF-RvIII, possibly leading to tumor destruction. Furthermore, the prognostic value of these responses is currently being evaluated.

Selected Publications

1. Birebent, B., Mitchell, E., Akis, N., Li, W., Somasundaram, R., Purev, E., Hoey, D., Mastrangelo, M., Maguire, H., Harris, D.T., Nair, S., Cai, D., Zhang, T. and Herlyn, D.M. 2003. Monoclonal anti-idiotypic antibody mimicking the gastrointestinal carcinoma-associated epitope CO17-1A elicits antigen-specific humoral and cellular immune responses in colorectal cancer patients. Vaccine 21: 1601-1612.

2. Li, W., Berencsi, K., Basak, S., Somasundaram, R., Ricciardi, R.P., Gonczol, E., Zaloudik, J., Linnenbach, A., Maruyama, H., Miniou, P., and Herlyn, D. 1997. Human colorectal cancer (CRC) antigen CO17-1A/GA733 encoded by recombinant adenovirus inhibits growth of established CRC cells in mice. J. Immunol. 159: 763-769.

3. Basak, S., Eck, S., Gutzmer, R., Smith, A.J., Birebent, B., Purev, E., Staib, L., Somasundaram, R., Zaloudik, J., Li, W., Jacob, L., Mitchell, E., Speicher, D., and Herlyn, D. 2000. Colorectal cancer vaccines: anti-idiotypic antibody, recombinant protein, and viral vector. Ann. N.Y. Acad. Sci. 910: 237-253.

4. Staib, L., Birebent, B., Somasundaram, R., Purev, E., Braumüller, H., Leeser, C., Küttner, N., Li, W., Zhu, D., Wunner, W., Speicher, D., Beger, H-G., Song, H., Diao, J., and Herlyn, D. 2001. Immunogenicity of recombinant GA733-2E antigen (CO17-1A, EGP, KS1-4, KSA, Ep-CAM) in gastrointestinal carcinoma patients. Int. J. Cancer 92: 79-87.

5. Li, J., Li, W., Liang, S., Cai, D., Kieny, M.P., Jacob, L., Linnenbach, A., Abramczuk, J., Bender, H., Sproesser, K., Swoboda, R., Somasundaram, R., Guerry, D., and Herlyn, D. 2003. Recombinant CD63/ME491/neuroglandular/NKI/C-3 antigen inhibits growth of established tumors in transgenic mice. J. Immunol. 171: 2922-2929.

6. Guerry, D., and Herlyn, D. 2000. CD4+, HLA class I-restricted, cytolytic T-lymphocyte clone against primary malignant melanoma cells. Int. J. Cancer 85: 253-259.

7. Somasundaram, R., K. Satyamoorthy, L. Caputo, H. Yssel, and D. Herlyn. 2004. Detection of HLA class II-dependent T helper antigen using antigen phage display. Clin Exp Immunol. 135:247-252.

8. Somasudaram, R., Jacob, L., Swoboda, R., Caputo, L., Song, H., Basak, S., Monos, D., Peritt, D., Marincola, F., Cai, D., Birebent, B., Bloome, E., Mastrangelo, M., and Herlyn, D. 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- b 1. Cancer Res. 62:5267-5272.

9. Zhang, T, Somasundaram, R, Berencsi, K, Caputo, L, Rani, P, Guerry, D, Furth, E, Rollins, BJ, Putt, M, Gimotty, P, Swoboda, R, Herlyn, M, and Herlyn, D. 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.

10. Li, J., Pereira, S., VanBelle, P., Tsui, P., Elder, D., Speicher, D., Deen, K., Linnenbach, A., Somasundaram, R., Swoboda, R., and Herlyn, D. 2001. Isolation of the melanoma-associated antigen p23 using antibody phage display. J. Immunol. 166: 432-438.

11. Purev, E., D. Cai, E. Miller, R. Swoboda, T. Mayer, A. Klein-Szanto, F.M. Marincola, R. Mick, L. Otvos, W. Wunner, B. Birebent, R. Somasundaram, C.J. Wikstrand, D. Bigner, A. DeMichele, G. Acs, J.A. Berlin, and D. Herlyn. 2004. Immune responses of breast cancer patients to mutated epidermal growth factor receptor (EGF-RvIII, DeltaEGF-R, and de2-7 EGF-R). J Immunol. 173:6472-6480.