Effects of substitutions of amino acids on the thermal stability of the Fv fragments of antibodies

Yasui, Hisashi; Ito, Wataru; Kurosawa, Yoshikazu

doi:10.1016/0014-5793(94)01027-7

Cited by 27 publications

(24 citation statements)

References 10 publications

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“…Alternatively, amino acid substitutions in the loops could make the protein thermodynamically unstable, thereby rendering it susceptible to proteolytic attack. This phenomenon has also been reported by other groups (39,40). Alfthan et al (41) have observed similar correlations of expression levels and protease sensitivity with temperature stability of Fab fragments that use different murine CHI regions.…”

Section: Resultssupporting

confidence: 86%

Expression studies of catalytic antibodies.

Ulrich

Patten

Yang

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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Section: Resultssupporting

confidence: 86%

Expression studies of catalytic antibodies.

Ulrich

Patten

Yang

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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“…Although, judging from the differences in stability between the B3V H -B5V L -PE38 chimera and the B3(Fv)-PE38 variants carrying B5V L residues at positions 4 and 7 or position 4 alone, the CDR residue may also contribute to the Fv immunotoxins' stability. Yasui et al (13) have recently reported that mutations in CDR residues can influence the stability of Fvs analysis of B3(Fv)-PE38 derivatives carrying mutations V L M4L or V L S7T separately showed that replacing V L methionine 4 with leucine stabilized the immunotoxin almost as much as the B3V H -B5V L -PE38 combination, whereas replacing V L serine 7 with threonine had no stabilizing effect and was, in fact, less active than B3(Fv)-PE38 (Fig. 3).…”

Section: Role Of Specific Residues In the Lightmentioning

confidence: 99%

Identification of Residues That Stabilize the Single-chain Fv of Monoclonal Antibodies B3

Benhar

Pastan

1995

Journal of Biological Chemistry

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B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv portion of the B3 antibody in a single-chain form, which serves as the targeting moiety, is fused to PE38, a truncated form of Pseudomonas exotoxin A, which serves as the cytotoxic moiety. B3(Fv)-PE38 is specifically cytotoxic to many human cancer cell lines and is currently evaluated in a clinical trial. Monoclonal antibodies B3 (IgG1k) and B5 (IgMk) recognize related carbohydrate epitopes on human carcinoma cells. The Fv regions of these antibodies were previously cloned and expressed as the single-chain Fv-immunotoxins B3(Fv)-PE38 and B5(Fv)-PE38, respectively. The B3(Fv)-PE38 immunotoxin binds to antigen-positive cancer cells with a higher affinity than B5(Fv)-PE38 and is a more potent cytotoxic agent than B5(Fv)-PE38. However, it is less stable and rapidly aggregates upon incubation at 37°C. The V L domains of the two Fvs are very similar, differing by only three residues, the fourth and seventh Fr1 residues and the fifth CDR1 residue. The V H domains of the two Fvs vary considerably. To investigate whether any of the different V L residues may influence the stability of the B3(Fv), we constructed a chimeric immunotoxin containing the B3V H and the B5V L . This chimera had an improved stability and a higher apparent antigen binding affinity and cytotoxic activity when compared with B3(Fv)-PE38. Site-specific mutagenesis was used to show that the V L M4L mutation has an important role in stabilizing B3(Fv), although residues V L Ser-7 and V L Ile-28 also play a role in the increased stability. When tested in an in vivo model system, the chimera containing the B3V H and the B5V L had an improved antitumor activity in a human xenograft mouse model. These studies indicate that the common use of degenerate ("family-specific") primers to clone Fv fragments may introduce destabilizing mutations.Monoclonal antibodies B3 and B5 are murine antibodies directed against Lewis Y -related carbohydrate antigens, which are abundant on the surface of many carcinomas (1). The B3 IgG or its fragments are currently used as the targeting moiety of immunotoxins that are being developed as anticancer agents. Both conventional whole IgG conjugates and singlechain recombinant immunotoxins have been prepared (1-4). The single-chain Fv 1 immunotoxin of B3 is unstable at 37°C; it undergoes inactivation mainly by aggregation, especially upon incubation in PBS or in cell culture medium. In contrast, the B5(Fv)-PE38 immunotoxin is less susceptible to inactivation under those conditions, but it has lower apparent antigen binding affinity and cytotoxicity (5). We reasoned that we might be able to combine the advantages of each Fv by chimerization of their variable domains, since the Fvs of monoclonal antibody B3 and B5 bind the same carbohydrate antigen and are homologous in sequence (particularly in the V L domain, where 109 of 112 residues are identical). Therefore, we sought to gain insight on the possible involvement of individual residues of the light chain on the st...

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“…The FTIR approach used here has the advantage of providing a good biophysical measurement of thermal stabilization, while requiring very little material. The scFv denatured at 60°C, a temperature close to the 62°C found for an antilysozyme Fv [23] using circular dichroism, but above the value for an antidansyl Fv of 52.3°C [24] obtained by differential scanning calorimetry. While the Tm of the ds-scFv was increased by 9°C, the Fab still had a higher value, 75°C.…”

mentioning

confidence: 94%

“…The 15°C difference between the Fv Tm and Fab Tm is the same as in the antidansyl antibody [24]. Yasui et al [23] also observed VL-VH dissociation at lower temperatures and found that single mutations in the CDRs could dramatically lower the thermal stability of their Fv. Since the interaction between VL and VH involves residues from the CDR loops, each Fv can be expected to have a unique Tm and VL-VH dissociation behaviour.…”

mentioning

confidence: 99%

Thermal stabilization of a single‐chain Fv antibody fragment by introduction of a disulphide bond

et al. 1995

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A disulphide bond was introduced into a single-chain Fv form of the anticarbohydrate antibody, Se155-4 by replacing Ala-L57 of the light chain and Asp-H106 of the heavy chain with cysteines, by site-directed mutagenesis. To maintain the saltbridge from the latter residue to Arg-H98, Tyr-107 was also altered to Asp. The resulting ds-scFv was shown to retain full antigen-binding activity, by enzyme immunoassay and surface plasmon resonance analysis of binding kinetics. Compared with the parent scFv, the disulphide bonded form was shown to have enhanced thermal stability, by Fourier transform IR spectroscopy. The Tm was raised from 60°C to 69°C. The ds-scFv form thus combines the stable monomeric form of the disulphide form wifh the expression advantages of the scFv.

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Effects of substitutions of amino acids on the thermal stability of the Fv fragments of antibodies

Cited by 27 publications

References 10 publications

Expression studies of catalytic antibodies.

Expression studies of catalytic antibodies.

Identification of Residues That Stabilize the Single-chain Fv of Monoclonal Antibodies B3

Thermal stabilization of a single‐chain Fv antibody fragment by introduction of a disulphide bond

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