Thioether-Bonded Constructs of Fab′γ and Fc γ Modules Utilizing Differential Reduction of Interchain Disulfide Bonds (1)
It is a benefit for therapeutics if we have bispecific F(ab)2 , trispecific F(ab)3 , and mouse/ human chimeras such as FabFc and FabFc2 employing a single Fab module.
Fc gamma 1 was prepared by a brief digestion of human normal IgG with papain under mild conditions. Limited digestion of mouse mAbs with pepsin, monitored by HPLC, yielded F(ab’)2 preparations that were separated from the digest by gel filtration. Reduction of all the interchain SS bonds of F(ab’)2 with DTT (2 mM, pH 8, 30 min at 30°C) yielded monomeric Fab’(-SH)5, which was purified by passage through Sephadex G-25 under conditions discouraging oxidation of SH groups: the column was equilibrated with N-saturated 50mM Na acetate, 1 mM Na2EDTA, pH 5, and run at 5°C. The protein may alternatively be separated and concentrated simultaneously by passage onto SP-Sepharose (equilibrated with the same buffer and run at 5°C), followed by elution with the equilibrating buffer made 1 M in NaCl. Fab’-SH was prepared from Fab’(-SH)5 by two SS-interchange reac- tions: first with Py-SS-Py (1 mM in 1 M DMF, 0.05 M Na acetate, pH 5, 30 min at 5°C), followed by a second reaction with DTT, this time at pH 5 (1 mM DTT for 30 min at 5°C). The product may then be separated on Sephadex G-25 or SP-Sepharose. Different reduction methods yield individual Fab’ products.
In the first interchange, the module Fab’(-SH)5 results from complete reduction of the inter- chain SS bonds. Addition of a surplus amount of disulfide reagent now recloses the gannma-light SS bonds, a result facilitated by the cysteine residues being held in close proximity by persisting pro- tein conformation. This has involved <10% of the protein when the concentration is <5 mg/ml. The reaction is at pH 5, but can be conducted over a broad range.
allowing incubation at 30°C to take place for 3 h or more, then adding two further small increments at intervals. A yield of 60 – 65% of thioether-linked F(ab’)2 is usual.
Heterodimers (bispecific F(ab,)2) were prepared. To one partner a surplus of BMP linker was added, the resulting Fab’-maleimide was separated on Sephadex G-25, and was then added to the partner Fab’-SH. After overnight incubation at pH 5 and 30°C, the products were separated on Superdex 200. All reactions were conducted at pH 5 to discourage oxidation and untoward SS interchange.In this preparation, the yield of F(ab’)2 was <45%.
All reactions to combine them took place at pH 5. First the Fab’-SH was allowed to react with a surplus of BMP linkers to form Fab’ maleimide, which was separated on Sephadex G-25. It was then incubated, in 2.5-fold molar excess, with Fab’(SH)5 overnight at 30°C. The reaction was terminated by alkylating surplus SH groups on the central module. Finally, the products were separated on Superdex 200. The yield of F(ab’)3 was 31%.
The stability of the Fab2Fc2 was compared with IgG1 in guinea pigs. They showed a similar catabolic rate of T1/2=3.8 days. Instead, Fab’ or Fab’2 has <0.5 day from previous work.
