Ongoing studies involve the design and use of novel immunological reagents for cancer therapy and diagnosis. Several murine monoclonal antibodies (MAbs) with selective reactivity to the pancarcinoma antigen, tumor associated glycoprotein (TAG)-72, have been developed in this laboratory. The genes encoding these antibodies have been cloned. Among those MAbs that react with the TAG-72 antigen, MAb CC49 has shown greatest potential for diagnostic and therapeutic utility in preclinical studies and clinical trials. To optimize therapeutic efficacy, emphasis is now being placed on the design and translational research of novel recombinant Ig forms of CC49. The mouse-human chimeric and humanized versions of this MAb have been generated. To faciliate ex vivo transfection and in vivo expression of recombinant CC49 for therapeutic use, three single-gene encoded single-chain derivatives of MAb CC49 have now been designed, generated and characterized. One of the single-gene constructs encoded an intact cCC49 MAb. The homodimeric molecule, SCacCC49, secreted from the transfectoma showed similar binding to the TAG-72 antigen as nCC49 and similar cytolytic activity to that of cCC49. We have also developed a single-gene construct encoding a single-chain immunoglobulin-IL-2 fusion protein. In vivo expression of the fusion protein in Balb/c mice was successfully demonstrated using both a transcutaneous gene gun and intramuscular injection. To minimize toxicity and host immune responses, the humanized MAb CC49 (HuCC49) was used as a prototype to develop genetically altered MAbs. A humanized CH2 domain-deleted CC49 has been constructed and demonstrates significantly faster plasma clearance and better tumor targeting than the intact CC49. In a separate study, to minimize idiotypic responses of patients to HuCC49, murine CDRs not critical for antigen binding were identified and replaced with human CDRs. Also, dispensable regions of the essential murine CDRs were identified by evaluating the relative affinity of a panel of HuCC49 variants generated by replacing one or more amino acid residues of the essential CDRs by corresponding residues of the human CDRs. Using these variants and sera from patients administered murine CC49 in a previous clinical trial, those amino acid residues which contribute to the idiotopes that are the targets of patients immune responses have been identified. Based on these studies, a final variant of the HuCC49 has been developed that maintains moderate antigen reactivity and does not react with the anti-idiotypic antibodies of the sera from patients treated with murine CC49. In a separate study to improve tumor targeting, attempts are being made to enhance the avidity of the HuCC49 MAb. A single gene has been constructed that encodes a single-chain Ig consisting of a HuCC49 diabody attached to a human gamma 1 Fc via the hinge region. The diabody, a bivalent antigen binding structure, is made up of VH/VL and VL/VH domains; the two antigen sites are linked by lateral and noncovalent association of VL of one pair with the VH of the other. Collaborative studies are under way to generate specific redirected antitumor human T-cell populations. To that end, genes encoding MHC-unrestricted antigen receptors containing single-chain Fv of antitumor antibody and the zeta chain of the CD3 complex of the T cell have been constructed and introduced into T lymphocytes by retroviral gene transfer. CD8+ lymphocytes retrovirally transduced by the HuCC49-derived Universal receptor (UR) gene have shown specific cytolysis against TAG-72-positive target cells. Redirected T cells have also been generated that show specific cytolysis against CEA-positive human tumor cells. The construct encoding this UR is derived from HuCOL-1 single-chain Ig - carcinoma, immunodiagnosis, immunotherapy, recombinant antibodies,
