Summary
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).
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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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Kulkarni, P.N., Blair, A.H., Ghose, T. et al. Conjugation of methotrexate to IgG antibodies and their F(ab)2 fragments and the effect of conjugated methotrexate on tumor growth in vivo. Cancer Immunol Immunother 19, 211–214 (1985). https://doi.org/10.1007/BF00199228
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DOI: https://doi.org/10.1007/BF00199228