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.

Recombinant adenovirus expressing the colorectal carcinoma-associated antigen GA733-2 (left upper corner) is injected into a host. The host cells are infected with the virus and express viral antigen (green) and tumor antigen (yellow) in context with major histocompatibility antigens (MHC) class I. The host cells can directly induce cytolytic T lymphocytes (CTL) which are directed against tumor antigen and lyse the antigen-positive tumor cells. Alternatively, the host cells can induce CTL directed to viral antigen. The latter CTL may lyse viral antigen-expressing host cells which leads into the MHC class II pathway of antigen presentation. Antigen-presenting cells (APC) express tumor or viral antigen in context with MHC class II and induce helper T cells (Th) specific for the viral or the tumor antigen. The Th cells may be of Th1 type (induction of antigen-specific CTL) or Th2 type (induction of antigen-specific B cells).

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.                                                                                     

Cloning of HLA class II restricted antigens using phage cDNA library approach.
Tumor cDNA library is expressed in T7 phage and phages are incubated with antigen presenting cells (APC) from the same donor from whom Th cell was derived. APC presents the tumor antigen to Th cell. Th cell is stimulated by the presented antigen and proliferates which is measured in proliferation and cytokine release assays.

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.

Cytolytic T cells (CTL793-A) migrate toward melanoma cells (WM793) in organotypic culture (reconstruct), leading to tumor cell destruction. Reconstruct was established by seeding melanoma cells into collagen mixed with fibroblasts; this layer was superimposed by a layer of collagen and fibroblasts, followed by a top layer of collagen, fibroblasts and CTL. There was minimal melanoma apoptosis in cultures with control lymphocytes (PHA blasts).

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.