Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 12, Num. 2, 1995, pp. 96-97

REPORTE CORTO/SHORT REPORT

Presented in the Congress Biotecnologia Habana'94. La Habana, Cuba, Nov. 28 - Dec. 3, 1994

BACTERIAL EXPRESSION AND CHARACTERIZATION OF A MODIFIED scFv FRAGMENT FROM THE ANTI-CARCINOEMBRYONIC ANTIGEN MONOCLONAL ANTIBODY CB.CEA.1

Javier E. Vazquez1, Lilia Perez1, Martha Ayala1, Leonardo Canaan- Haden1, C. de Lalla2 , A. Sidoli2 and Jorge Gavilondo1.

1Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba 2H. San Raffaelle, Dipartimento di Ricerca Biologica e Tecnologica, Milano 20132, Italy.

Code Number: BA95037
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Single-chain antibody fragments (scFv) are defined as the smaller antigen-binding molecule derived from an antibody (1-2). We report the cloning bacterial expression and molecular characterization of an anti-carcinoembryonic antigen (CEA) scFv with potential use for the localization of tumors in CEA-positive carcinoma pattients.

EXPERIMENTAL PROCEDURES

The scFv gene (VH-Linker-VL) was assembled using PCR amplification (3) and cloned into expression vectors developed by our group. The vectors were denominated pPACIB.1, pPACIB.3 (4), for export to periplasm proteins displaying 6 histidines either in the N- or C- termini, respectively. Protein expression was induced in LB media and using 10 representative E. coli strains. Also a vector called pTrp/OmpA, that produces proteins without histidines but with a myc-tag peptide (5) at the C-terminus, was used for express the scFv. Active scFv was detected after induction by specific ELISA and Western blot. IMAC purifications (6) was assayed for proteins tagged with histidines, while a monoclonal antibody-based affinity chromatography was used for scFv.myc-tag protein. Association and dissociation constants to antigen were calculated using a biosensor (BIAcore), and recognition of CEA-positive carcinoma cell lines were done by Fluorescence Analysis Cell Surface (FACS).

RESULTS AND DISCUSSION

Active scFv (concentration ca. 100 mg/L) was found in the periplasm of 5-10 E. coli strain (W3110, LE392, BMH71.18, TG1 and coliB), either employing pPACIB.1 or pPACIB.3 vectors. The scFv.myc-tag was produced in the only tested strain (coliB).

One step chromatographic purification of the 6 histidine-tagged scFv by IMAC was unsatisfactory, with purities of around 60%, independently of the position of the His-tag. However, the monoclonal antibody 9E10 affinity purification rendered very pure preparations of the scFv.myc-tag protein (purity ca. 98%).

Affinity constant for the scFV fragment (107M) resulted in a circa 2 order of magnitude lower than the value calculated for the biochemically produced Fab fragments. Values in this range have been reported either for scFv (7) or Fab fragments (8). Finally, in a FACS analysis with human CEA-positive carcinoma cells (LoVo), as a preliminary step to in vivo distribution assays, the scFv.myc-tag shows a recognition pattern similar to the parental antibody CB.CEA.1, as shows figure 1.

Fig. 1.- FACS analysis of CB.CEA.1 and derived scFv fragment using CEA-expressing carcinoma cells (LoVo)

1. WINTER, G. and C. MILSTEIN (1991). Nature 349:293.

2. MILENIC, D. E. et al. (1991). Cancer Res. 51:6363-6371

3. AYALA, M. et al. (1992). BioTechniques 13:790-799

4. GAVILONDO, J. et al. (1993). Abstr. Book, 4th IBC Conf.

5. EVAN, G. I. et al. (1985). Mol & Cell. Biol. 5:3610-3616

6. WHITLOW, M. et al. (1993). Prot. Engineering 6:989-995

7. BORREBAECK, C. et al (1992). Bio-Technology 10:697-698

8. GRUEN, L. C. et al. (1993). Eur. J. Biochem 217:319-325

Copyright 1995 Sociedad Iberolatinoamericana de Biotecnologia Aplicada a la Salud

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