Antibody framework residues affecting the conformation of the hypervariable loops

Foote, Jefferson; Winter, Greg

doi:10.1016/0022-2836(92)91010-m

Cited by 495 publications

(329 citation statements)

References 45 publications

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“…Thus, more of the mutational induced change in AAGD in this series is reflected in the dissociation rate constants. This appears to be the more common observation in mutational analyses of protein/protein interactions (e.g., Friguet et al, 1989;Foote & Winter, 1992;Schreiber & Fersht, 1995;England et al, 1997).…”

Section: Aspartate 101mentioning

confidence: 84%

“…Association rate constants, k,,, are 105-109 M" s-' (reviewed by Janin & Chothia, 1990; see also Schreiber & Fersht, 1995;Janin, 1997), but the range is smaller for monoclonal antibodyprotein antigen complexes: 105-106 M" s" (Friguet et al, 1989;Janin & Chothia, 1990;Foote &Winter, 1992;Raman et al, 1992;Janin, 1995;Glaser & Hausdorf, 1996;England et al, 1997). The variation within the KO values is thus primarily due to the large range in the corresponding dissociation rate constants (1-10" s-I) (Janin & Chothia, 1990).…”

Section: Fig 1 Schematic View Of the Interactions Of H E W Residuesmentioning

confidence: 99%

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Kinetic epitope mapping of the chicken lysozyme.HyHEL‐10 fab complex: Delineation of docking trajectories

1998

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Section: Aspartate 101mentioning

confidence: 84%

Section: Fig 1 Schematic View Of the Interactions Of H E W Residuesmentioning

confidence: 99%

Kinetic epitope mapping of the chicken lysozyme.HyHEL‐10 fab complex: Delineation of docking trajectories

1998

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“…Several methods of humanization of murine antibodies have been described previously, demonstrating a list of sites that have been frequently identified to be important to maintain the CDR loop conformation. 16,[68][69][70] Of the sites that differ between the humanized and murine RK35 variable domains, McCafferty et al 70 included only the H94 and L48 positions as residues being important for canonical conformation. Both the R_H94_K and I_L48_L mutation are very conservative.…”

Section: Discussionmentioning

confidence: 99%

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

Apgar

Mader

Agostinelli

et al. 2016

mAbs

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Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted antimyostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall "humanness" was increased for both the light and heavy chain variable regions.

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“…A scattered pattern of amino acid substitutions is commonly found in recombinant antibodies selected for improved affinity or physicochemical stability (Daugherty et al 2000;Jermutus et al 2001;Zahnd et al 2004). It has been reported that amino acid substitutions outside the CDRs that result in increased affinity can stabilize CDR loops in a conformation that is more suitable for antigen binding (Foote and Winter 1992;Padlan 1994;Wedemayer et al 1997). Possibly, the higher proteolytic stability of the newly selected VHHs is similarly caused by reduced flexibility of CDR loops.…”

Section: Discussionmentioning

confidence: 99%

Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy

Harmsen

Solt

Bemmel³

et al. 2006

Appl Microbiol Biotechnol

107

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We previously demonstrated that oral application of the recombinant single-domain antibody fragment (VHH) clone K609, directed against Escherichia coli F4 fimbriae, reduced E. coli-induced diarrhoea in piglets, but only at high VHH doses. We have now shown that a large portion of the orally applied K609 VHH is proteolytically degraded in the stomach. Stringent selection for proteolytic stability identified seven VHHs with 7-to 138-fold increased stability after in vitro incubation in gastric fluid. By DNA shuffling we obtained four clones with a further 1.5-to 3-fold increased in vitro stability. These VHHs differed by at most ten amino acid residues from each other and K609 that were scattered over the VHH sequence and did not overlap with predicted protease cleavage sites. The most stable clone, K922, retained 41% activity after incubation in gastric fluid and 90% in jejunal fluid. Oral application of K922 to piglets confirmed its improved proteolytic stability. In addition, K922 bound to F4 fimbriae with higher affinity and inhibited fimbrial adhesion at lower VHH concentrations. K922 is thus a promising candidate for prevention of piglet diarrhoea. Furthermore, our findings could guide selection and improvement by genetic engineering of other recombinant antibody fragments for oral use.

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Antibody framework residues affecting the conformation of the hypervariable loops

Cited by 495 publications

References 45 publications

Kinetic epitope mapping of the chicken lysozyme.HyHEL‐10 fab complex: Delineation of docking trajectories

Kinetic epitope mapping of the chicken lysozyme.HyHEL‐10 fab complex: Delineation of docking trajectories

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy

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