CLONING AND MODIFICATION OF ANTI-TUMOR ANTIGEN IMMUNOGLOBULIN GENES

正在进行的研究包括重组免疫球蛋白的设计和使用

Ongoing studies involve the design and use of recombinant immunoglobulin (Ig) forms for cancer therapy and diagnosis. Murine monoclonal antibody (MAb) CC49 selectively reacts with the pancarcinoma antigen, tumor associated glycoprotein (TAG)-72. Preclinical studies and clinical trials have demonstrated the diagnostic and potential therapeutic utility of this MAb. To optimize therapeutic efficacy and minimize toxicity and host immune responses, emphasis is now being placed on the design and translational research of novel recombinant Ig forms of CC49. The genes encoding these antibodies have been cloned and mouse-human chimeric and humanized versions of these MAbs have been generated. 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. To facilitate 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. 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 and replaced with the homologous regions of human CDRs. Using these variants and sera from patients administered murine CC49 in a previous clinical trial, attempts are under way to identify those amino acid residues which contribute to the idiotopes that are the targets of patient's immune response. Collaborative studies are underway to generate specific redirected antitumor human T-cell populations. To that end, genes encoding MHC- unrestricted antigen receptors, containing single-chain antitumor Ig genes and the zeta chain of the CD3 complex of the T cell have been constructed introduced into T lymphocytes by retroviral gene transfer. CD8+ lymphocytes retrovirally transduced by the Universal receptor (UR) genes have shown specific cytolysis against TAG-72 positive tareget cells. Generation of redirected T cells against CEA positive human tumor cells is underway. Another approach for the immunotherapy of human carcinomas is based on conjugating an antigenic peptide of a tumor antigen to the anti-human FcgR1 antibody. Gene constructs encoding fusion proteins of two different peptides and the Fab of the humanized antibody specific for a site of the human FcgR1 have been made and expressed The fusion proteins are currently being characterized. These molecules will be used to target antigenic peptides to professional antigen presenting cells for internalization and presentation of the peptide in the context of MHC class 1.