Abstract
Methotrexate (MTX) was first conjugated to antibovine serum albumin IgG (antiBSA) or its F(ab)2 fragment to define conditions for retention of drug and antibody activity. With identical drug: protein molar ratios, incorporation in the F(ab)2 fragment was lower than in intact antiBSA, an observation consistent with analysis of the number of lysine residues (22 in F(ab)2 compared to 40 in antiBSA). In either case, up to approximately 10 mol MTX could be incorporated per mol protein, with recovery of 70% of the protein. At an incorporation ratio of 6 mol MTX per mol protein, MTX-antiBSA retained 100% of antibody activity and MTX-F(ab)2antiBSA retained 75%. MTX-antiBSA and MTX-F(ab)2antiBSA were equally potent in vitro inhibitors of dihydrofolate reductase. Conjugates prepared from antiEL4 IgG (AELG) and from F(ab)2AELG significantly increased survival in EL4 lymphoma-bearing mice compared with mice receiving equal amounts (5 mg MTX/kg) of free MTX, MTX linked to the F(ab)2 fragment of normal rabbit IgG, or a simple mixture of MTX and F(ab)2AELG. MTX-AELG at this dose level produced longer survival than MTX-F(ab)2AELG (0.005<P<0.01).
Keywords: Tumor Growth, Lysine, Methotrexate, Dose Level, Lysine Residue
Abbreviations
- AELG
antiEL4 IgG
- antiBSA
antibovine serum albumin IgG
- BSA
bovine serum albumin
- DCC
dicyclohexyl-carbodiimide
- DFA
dihydrofolic acid
- DHFR
dihydrofolate reductase
- DMF
dimethylformamide
- IP
intraperitoneal
- MTX
methotrexate
- MTX-AELG
MTX linked to AELG
- MTX-antiBSA
MTX linked to antiBSA
- MTX-F(ab)2AELG
MTX linked to the F(ab)2 fragment of AELG
- MTX-F(ab)2antiBSA
MTX linked to the F(ab)2 fragment of antiBSA
- MTX-F(ab)2NRG
MTX linked to the F(ab)2 fragment of NRG
- MTX-NRG
MTX linked to NRG
- NHS
N-hydroxysuccinimide
- NRG
normal rabbit IgG
- PBS
0.01 M sodium phosphate (pH 7.1) containing 0.45 M sodium chloride
- TAA
tumor-associated antigen
- t1/2
half-life
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