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Clinical and Diagnostic Laboratory Immunology, March 1999, p. 236-242, Vol. 6, No. 2
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Specific Enzyme-Linked Immunosorbent Assays for Quantitation of Antibody-Cytokine Fusion Proteins

J. Gan,1 K. Kendra,2 M. Ricci,1 J. A. Hank,1 S. D. Gillies,3 and P. M. Sondel1,4,5,*

Lexigen Pharmaceuticals Corp., Lexington, Massachusetts 02173,3 and Department of Human Oncology,1 Medical Oncology Section, Department of Medicine,2 Department of Pediatrics,4 and Department of Medical Genetics,5 University of Wisconsin, Madison, Wisconsin 53792

Received 22 May 1998/Returned for modification 9 July 1998/Accepted 5 November 1998

Preliminary testing has shown in vitro and in vivo that antitumor activity can be obtained with fusion proteins linking tumor-reactive monoclonal antibodies to cytokines, such as granulocyte-macrophage colony-stimulating factor or interleukin 2 (IL-2). Preclinical and clinical testing of these reagents requires their in vitro and in vivo quantitation and pharmacokinetic evaluation. We have focused on the detection of a fusion protein which links one human IL-2 molecule to the carboxy terminus of each heavy chain of the tumor-reactive human-mouse chimeric anti-GD2 antibody, ch14.18. We have developed enzyme-linked immunosorbent assays (ELISAs) to evaluate intact tumor-reactive fusion proteins. By these ELISAs we can reliably measure nanogram quantities of intact ch14.18-IL-2 fusion protein and distinguish the intact protein from its components (ch14.18 and IL-2) in buffer, mouse serum, and human serum with specificity and reproducibility. The measurement of intact ch14.18-IL-2 fusion protein is not confounded by free IL-2 or free ch14.18 when 100 ng or less of total immunoglobulin per ml is used during the assay procedure. Our results indicate that these ELISAs are suitable for preclinical and clinical testing and with slight modifications are applicable to the analysis of a variety of other fusion proteins.

* Corresponding author. Mailing address: K4/448 CSC, 600 Highland Ave., Madison, WI 53792. Phone: (608) 263-9069. Fax: (608) 263-4226. E-mail: pmsondel{at}facstaff.wisc.edu.

Clinical and Diagnostic Laboratory Immunology, March 1999, p. 236-242, Vol. 6, No. 2
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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