Immunotherapy is a powerful therapeutic modality that has great potential for the treatment of cancer. Considering the lack of options which exist for patients with refractory solid tumors, immunotherapy could provide patients with an alternative treatment that may be used in conjunction with existing therapies. In the last several years, it has become apparent that successful immunotherapy may require a minimum of two critical steps. First, the immune response that is generated in cancer patients needs to be amplified and involve several different cellular components including neutrophils and NK cells associated with innate immunity as wells as dendridic ceils and armed T-cells associated with antigen-directed active immunity. Secondly, negative regulatory mechanisms for suppressing the immune response against self-antigens needs to be eliminated during the time of tumor immunotherapy. In this proposal, we present evidence that combination therapy with a fusion protein consisting of a tumor targeting monoclonal antibody (TNT) and the human chemokine LEC (Liver-expressed chemokine, CCL16) and CD25+ T-regulatory cell depletion can produce complete and lasting cures of two solid tumor models of the BALB/c mouse. Evidence is presented that upon localization of LEC to necrotic areas of tumors abundant in tumor antigens, the immune system of the mouse is activated to cause significant regression of tumor accompanied by the infiltration of PMNs, dendritic cells, NK cells, and activated T-cells in both the tumor and tumor draining lymph nodes. However, if prior to the administration of the LEC fusion protein the mice were pretreated with PC61, a rat anti-CD25 monoclonal antibody to eliminate CD25+ cells, all the mice experienced complete regression of tumor which was shown to involve the induction of memory cells by rechallenge studies with 107 tumor cells. Current clinical studies with other reagents known to suppress CD25+ T-reg. activity such as CTLA-4 blockade have also shown better anti-tumor responses, but these studies have demonstrated that if CD25+ T-reg. cells are not eliminated, severe autoimmune systems are induced in mice and patients. To date, the laboratory has generated a fully human fusion protein, designated LEC/NHS76, which is currently being develop for use in patients. To complement the use of this fusion protein as well as other approaches such as tumor vaccines for tumor immunotherapy, we now propose generate a human monoclonal antibody with a biologic profile similar to PC61 which can eliminate human CD25+cells in patients. To accomplish this goal, the gene for human CD-25 will be linked to the Fc region of human lgG1 to create a fusion protein that can be produced in quantity and purified simply with Protein-A affinity chromatography. Purified human CD25-Fc will then be used in panning experiments using the MRC phage display library to identify FABs that bind specifically to the CD25 antigen (alpha chain of 11-2 receptor). Candidate FABs will then be genetically engineered into whole human lgG1/kappa antibodies and expressed using the Glutamine Synthetase Expression System in murine NS0 cells, purified by tandem protein-A affinity chromatography and ion-exchange chromatography methods. Candidate antibodies will then be tested for their ability to kill MT-1 CD25+ human tumor cells as well as PBMCs from donor individuals. Additional studies are planned to test the effectiveness of these antibodies to kill human adult T-cell leukemia cells transplanted in NOD/SCID mice with the advice and help of Dr. Thomas Waldmann who is expert in this field. If unlike PC61, the candidate human antibodies are not found to eliminate CD25+ cells, an antibody/drug conjugate will be generated using the drug auristatin which has recently been shown to produce a stable and effective drug conjugate. Along with LEC/NHS76, this new antibody/drug conjugate should be applicable to use in humans and may provide clinicians with a vital weapon in our relentless effort to treat cancer.
