NEW THERAPEUTIC APPROACHES TO BREAST CANCER

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Breast cancer is among the leading causes of death among women and has steadily increased during recent decades. In this interactive grant proposal we propose two interdependent projects for the generation and application of novel anti-idiotype antibody vaccines against breast cancer. In Research Project 1 (RP 1), we will initiate a Phase lb clinical trial with a recently developed anti-idiotype antibody, designated 11D10. This antibody represents the internal image of the high MW human milk fat globule (HMFG) primarily expressed at high density by tumors of the human breast and selected other cancers. This antigen is expressed in very few non-tumor tissues and at low density. This epitope is identified by the monoclonal antibody MC-10 or BrE1. MC-10 was used as the immunizing antibody, or Ab1, against which anti-ld 11D10 (or Ab2) was generated. 11D10 was used to induce tumor specific antibodies in mice, rabbits and monkeys. Also, monoclonal anti-anti-lds, or Ab3s, which bound to the HMFG antigen, have been obtained from mice immunized with 11D10. n this clinical study, we will use the alum precipitate of anti-ld 11D10 to actively immunize MC-10 positive breast cancer patients in a Phase lb clinical trial (years 1-2). The objectives of this Phase 1 clinical trial are to: 1. Determine the effects of a single monoclonal anti-ld antibody precipitated with alum on patients' ability to generate an Ab3 (Ab1') response to cytolytic T-lymphocytes specific for autologous and/or allogeneic tumor cells; 2. Determine the optimal immunomodulatory dose of the anti-ld antibody, and 3. Monitor clinical responses. At the completion of the Phase lb trial, we will begin a Phase II trial in years 2-3 using the optimum immunomodulatory dose, and in year 4 we will initiate a Phase lb clinical trial with the selected anti-id 11D10-cytokine fusion protein developed and tested in Research Project II. Research Project II (RP II) is the laboratory arm in which we will develop methodologies to construct "second generation" monoclonal anti-idiotype reagents linking the 11D10, Ab2, anti-ld antibody used in RP 1, to the cytokines IL-2 and GM-CSF. Cytokines have been shown to increase the immunogenicity of protein antigens, including idiotypes. We will create a chimeric antibody 11D10 which will contain either of the two cytokines attached to the constant domain (CH3). The variable exon of the H chain gene of 1D10 will be cloned ad sequenced. The VH gene will be incorporated into plasmids which express the constant gene fused to the genes for either IL-2 or GM-CSF/ The heavy chain loss mutant of the 11D10 hybridoma cell line will be transfected with these plasmids. The expression of functional cytokines and immunoglobulin will be monitored in transfectoma clones and high producing clones will be selected. These chimeric antibodies will be tested in mice and non-human primates as anti-idiotype tumor vaccines. The Ab3 titer in animals immunized with the chimeric antibody will be directly compared with the Ab3 titer in animals immunized with the alum precipitated Ab2, as described in RP 1. The Ab1' will also be compared in these groups of animals. We expect to observe a significantly higher Ab3 and Ab1' titer in the chimeric Ab2 immunized animals. We will also compare the proliferative response to Ab2 in both groups. If these preclinical studies with chimeric cytokine-Ab2s induce superior anti-tumor associated antigen responses in animals, we will initiate a Phase lb clinical trial in breast cancer patients.