Preparation and characterization of methotrexate-immunoglobulin conjugates

In the targeting of chemotherapeutic agents to specific cells via covalent conjugation of the agent to monoclonal antibodies, conjugation methods which lead to maximum binding capacity with minimum loss of activity (for both drug and antibody) are essential. A model system for evaluation of various conjugation procedures is described. Rabbit anti-bovine serum albumin immunoglobulin (IgG) was used to represent the antibody, and methotrexate (MTX) was used as the targeting agent. The procedures evaluated were coupling via water soluble 1-ethyl-3 (3'-dimethylaminopropyl) carbodiimide (EDCI), or via prior activation of MTX's carboxyl groups involving active ester (N-hydroxysuccinimide) or mixed anhydride (isobutylchloroformate) formation. The formation of covalent conjugates in all three cases was verified by (a) the lack of incorporation of MTX into IgG when the reactions were performed in the absence of EDCI, or when MTX was reacted without prior activation, and (b) the presence of a constant amount of MTX in the conjugates after exhaustive dialysis of gel-filtration through Sephadex G-50. High incorporation of MTX and high retention of antifolate activity resulted from the EDCI coupling reaction, but there was very low recovery of protein (conjugate) and antibody activity. The biological and physicochemical properties of the conjugates prepared by the two pre-activation methods did not show any significant difference, but the conjugates prepared by the active ester method had a longer shelf-life. Therefore, of the three reactions investigated, the active ester method is our method of choice for conjugating MTX to IgG.