Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme

Krauss, Juergen; Arndt, Michaela A.E.; Zhu, Zhongyu; Newton, Dianne L.; Vu, Bang K.; Choudhry, Vidita; Darbha, Ramalakshmi; Ji, Xinhua; Courtenay-Luck, Nigel; Deonarain, Mahendra P.; Richards, J. Brent; Rybak, Susanna M.

doi:10.1038/sj.bjc.6601759

Cited by 29 publications

(25 citation statements)

References 41 publications

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“…For example, R71A mutagenesis in the heavy chain of an unstable scFv dramatically increased its stability when incubated in human serum while having only a minor effect on the binding affinity of the molecule. 111 A more stable heavy chain, made through mutation, can increase the stability of the light chain, and thereby confer more stability to the whole scFv. 24 …”

Section: Stabilized Antibodies Via Mutagenesismentioning

confidence: 99%

Antibody Structure, Instability, and Formulation

Wang

Singh

Zeng

et al. 2007

Journal of Pharmaceutical Sciences

820

667

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Section: Stabilized Antibodies Via Mutagenesismentioning

confidence: 99%

Antibody Structure, Instability, and Formulation

Wang

Singh

Zeng

et al. 2007

Journal of Pharmaceutical Sciences

820

667

View full text Add to dashboard Cite

“…Addressing these issues we constructed the single-chain Fv (scFv) and Fab recombinant antibody fragments of HuHMFG-1 and assessed stability, affinity and internalisation characteristics alongside the parent antibody. The scFv was found to be insoluble and unstable and required a stabilising mutation ( Krauss et al , 2004 ). This is the first report delineating the internalisation pathway of the clinically tested HuHMFG-1 antibody with respect to antigen binding and intracellular trafficking.…”

mentioning

confidence: 99%

Characterisation and internalisation of recombinant humanised HMFG-1 antibodies against MUC1

et al. 2005

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The humanised HMFG-1 immunoglobulin has been extensively developed as a clinical immunotherapeutic agent for MUC1 expressing tumours. We have constructed a single-chain Fv (scFv) and Fab fragment from this antibody and shown that both these species retain their specificity for MUC1. The scFv was less stable and less soluble than the Fab. Detailed analyses of the binding kinetics of the whole IgG and Fab fragment show that the affinity for MUC1 synthetic peptides is low (approximately 100 n for the IgG and 10 μ for the Fab), with particularly low but similar dissociation rate constants (0.031–0.095 s−1). Binding to native antigen on the cell surface is over two orders of magnitude better. Confocal immunofluorescence microscopy shows that both the IgG and Fab are internalised rapidly (the IgG is internalised within 15 min) and colocalise to early endosomes. This work provides an appreciation of the binding, internalising and trafficking kinetics, important for the development of future therapeutics based on this antibody.

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“…Many other residues beyond CDRs have also been identified to carry diversity in a germline gene-defined manner. For instance, residue 80, a member of the V domain’s upper core ( 48 ), considered to be important for the position and conformation of H chain CDR2 ( 49 ) and stability of the V domain ( 50 ), is extensively targeted by substitution in products of some but not all germline genes (Figure 2 ) ( 38 ). Intriguingly, arginine 80 encoded by IGHV1–8 (Figures 2 A,E), but not by IGHV3–7, IGHV3–11, IGHV3–21 , or IGHV3–23 ( 38 ), is, based on diversification of H chains in vivo , a suitable target for diversification.…”

Section: Antibody Evolution Even Beyond Conventional Cdr—specific Examentioning

confidence: 99%

In Vitro Evolution of Antibodies Inspired by In Vivo Evolution

et al. 2018

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In vitro generation of antibodies often requires variable domain sequence evolution to adapt the protein in terms of affinity, specificity, or developability. Such antibodies, including those that are of interest for clinical development, may have their origins in a diversity of immunoglobulin germline genes. Others and we have previously shown that antibodies of different origins tend to evolve along different, preferred trajectories. Apart from substitutions within the complementary determining regions, evolution may also, in a germline gene-origin-defined manner, be focused to residues in the framework regions, and even to residues within the protein core, in many instances at a substantial distance from the antibody’s antigen-binding site. Examples of such germline origin-defined patterns of evolution are described. We propose that germline gene-preferred substitution patterns offer attractive alternatives that should be considered in efforts to evolve antibodies intended for therapeutic use with respect to appropriate affinity, specificity, and product developability. We also hypothesize that such germline gene-origin-defined in vitro evolution hold potential to result in products with limited immunogenicity, as similarly evolved antibodies will be parts of conventional, in vivo-generated antibody responses and thus are likely to have been seen by the immune system in the past.

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Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme

Cited by 29 publications

References 41 publications

Antibody Structure, Instability, and Formulation

Antibody Structure, Instability, and Formulation

Characterisation and internalisation of recombinant humanised HMFG-1 antibodies against MUC1

In Vitro Evolution of Antibodies Inspired by In Vivo Evolution

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