Gerd Pluschke

Application of genetically-engineered anti-CEA antibodies for potential immunotherapy of colorectal cancer.

Hitherto anti-CEA monoclonal antibodies (MAbs), normally of mouse origin, have been used primarily for clinical diagnosis of colorectal cancer, either as a tumor marker in serum to monitor tumor recurrence, or latterly as a means to localize in vivo CEA-bearing tumors and metastases in patients. In vivo diagnosis using mouse anti-CEA MAbs has so far had limited clinical utility because the antibodies elicit a strong anti-mouse immunoglobulin immune response on repeated administration in man. This problem has been addressed by the development of various strategies for “humanization” of mouse anti-CEA MAbs by genetic manipulation of immunoglobulin genes. Such humanized, engineered antibodies markedly attenuate the antigenic response directed against the MAb, such that safe, repeated administration to patients has become feasible. Such humanized anti-CEA antibodies can thus be radioactively-labelled and applied for in vivo monitoring and detection of recurrent malignant disease, or used for therapeutic strategies which similarly take advantage of the ability of the antibodies to target cytotoxic agents selectively to tumor cells. The application of these novel procedures for manipulating MAb structure presents entirely new opportunities for diagnosis and treatment of human colorectal cancer.

Sequence and diversity of rat T-cell receptor α-chain-encoding genes

The o~/13 T-cell antigen receptor is formed by the combination of the o~ and 13 polypeptide chains which both consist of a constant and a variable portion. The genetic information for the variable portion on each chain is generated by rearrangements of variable (Tcr-V), diversity (Tcr-D, only for 13 chains), and joining (TCR-J) gene segments.The variable gene segments together determine the antigen specificity of a T cell. Characterization of the structure of the mouse and human Tcr gene segments, therefore, has expanded our understanding of T-cell repertoire selection and has helped to clarify the role of T cells in immune protection and T-cell-dependent pathologic processes. Although many rat major histocompatibility complex haplotypes are well defined and the rat is used as a model for a variety of inflammatory, infectious, and autoimmune diseases (Gill et al. 1989), only partial information is available about the structure and repertoire of rat Tcr genes (Kabat et al. 1991). While the structure of the rat Tcrb-D, Tcrb-J, Tcrb-C (Williams et al. 1989, 1991; Blankenhom et al. 1992), and 24 Tcrb-V (Smith et al. 1991) genes has recently been described, only two Tcra-V, -J, -C cDNA sequences are available (Morris et al. 1988; Bums et al. 1989). In this study, we have sequenced 23 rat Tcra -V, -J, -C cDNAs from the popliteal lymph node of a Lewis rat with adjuvant arthritis to build a basis for studies of Tcra gene segment usage in rats with autoimmune diseases . Five-hundred micrograms of heat-killed Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) suspended in 0.1 ml mineral oil was injected subcutaneously into the pad of the left hind foot of a Lewis rat (Versuchstierzucht CIBA, Werk Steine, Germany), as described (Yang et al. 1992). The clinical onset of

Correlation between secreted and membrane-bound IgG in mouse myeloma cells transfected with chimeric immunoglobulin heavy and light chain genes.

Mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression vectors. Double transfectants were selected using the xanthine-guanine phosphoribosyl transferase (gpt) and the neomycin (neo) selection marker genes. A broad distribution in the level of mouse-human chimeric IgG expression was observed with series of independently isolated transfectoma clones. The relative amounts of secreted to membrane-bound antibodies correlated closely, which suggested, that fluorescence-activated cell sorting could be a valuable tool for the selection of high-yielding production cell lines. However, a single cycle of cell sorting did not steer the cloning process significantly toward cells that produce enhanced amounts of recombinant IgG. Only in cases in which the polyclonal transfectoma population contained a large percentage of nonproducing cells, these were successfully separated from the IgG-producing cell population. (c) 1996 John Wiley & Sons, Inc.

Titration calorimetry study of an anti-idiotypic antibody cascade in a human melanoma-associated antigen system.

The thermodynamic parameters of interactions between six variants of the anti-idiotypic monoclonal antibody (mAb) CGP 60686 produced by the hybridoma MK2-23 with an idiotypic mAb and five different anti-anti-idiotypic mAb were studied with high sensitivity titration calorimetry. CGP 60686 recognizes an epitope in the antigen-combining region of the human high-molecular-weight-melanoma-associated antigen (HMW-MAA)-specific mouse mAb CGP 76873 produced by the hybridoma 763.74. The five HMW-MAA-specific anti-anti-idiotypic mAbs GH 464, GH 518, GH 149, GH 386 and GH 586 were generated from mice immunization with mAb CGP 60686. All interactions between the anti-idiotypic mAb and the idiotypic mAb or the anti-anti-idiotypic mAb showed large exothermic binding enthalpies between -15 and -23 kcal/mol and binding affinities larger than 6 x 10(9) M-1. Four of the five anti-anti-idiotypic mAbs tested exhibited significantly higher binding enthalpies for the interaction with the anti-idiotypic than the idiotypic mAbs. Replacement of either the heavy or the light chain variable region of the anti-idiotypic mAbs with an unrelated variable region abolished the idiotype to anti-idiotype interaction. Thus, both the heavy and the light chain variable region of the anti-idiotypic mAbs are required for binding to the idiotype. The values of the binding enthalpy showed only small variations when binding of the idiotypic mAb CGP 76873 to four variants of the anti-idiotypic mAb CGP 60686 with different immunoglobulin constant regions, but identical variable regions were compared. Furthermore, Fab fragments of the idiotypic mAbs showed almost the same binding enthalpy per binding site as the whole IgG molecules. Immunoglobulin constant regions thus had little influence on the idiotype to anti-idiotype interactions. Taken together, the observed thermodynamic parameters suggest that the idiotype to anti-idiotype interactions studied here are enthalpy-driven processes with only minor entropic contributions. High sensitivity titration calorimetry was used to monitor protein-protein interactions within an anti-idiotypic antibody cascade. It was found that the direct measurement of the interaction enthalpy allowed a quantitative characterization of the binding processes studied.