Conformations of immunoglobulin hypervariable regions

Chothia, Cyrus; Lesk, Arthur M.; Tramontano, Anna; Levitt, Michael; Smith‐Gill, Sandra J.; Air, Gillian; Sheriff, S.; Padlan, Eduardo A.; Davies, David R.; Tulip, W.R.; Colman, Peter M.; Spinelli, S.; Alzari, P.M.; Poljak, Roberto J.

doi:10.1038/342877a0

Cited by 1,145 publications

(730 citation statements)

References 31 publications

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“…The CDRs were determined according to Kabat, except the CDR-H1, for which the combined Kabat/Chothia definition was used (Chothia et al, 1989;Kabat and Wu, 1991). Alignment of the VH and VL amino acid sequences with the Kabat database produced maximal alignment of the murine FR with the FRs of the human B43 heavy chain and human REI light chain, respectively.…”

Section: Resultsmentioning

confidence: 99%

Humanization of anti‐human insulin receptor antibody for drug targeting across the human blood–brain barrier

Boado

Zhang

et al. 2006

Biotech & Bioengineering

185

138

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A murine monoclonal antibody (MAb) to the human insulin receptor (HIR) has been engineered for use as a brain drug delivery system for transport across the human blood-brain barrier (BBB). The HIRMAb was humanized by complementarity determining region (CDR) grafting on the framework regions (FR) of the human B43 IgG heavy chain and the human REI kappa light chain. A problem encountered in the humanization process was the poor secretion of the CDR-grafted HIRMAb by myeloma cells. This problem was solved by the production of human/mouse hybrids of the engineered heavy chain variable region (VH), which led to the replacement of five amino acids in the FR3 of the VH with original murine amino acids. No replacement of FR amino acids in the light chain variable region (VL) was required. The affinity of the humanized HIRMAb for the HIR was decreased 27% relative to the murine HIRMAb. The humanized HIRMAb avidly bound to the HIR of isolated human brain capillaries, which are used as an in vitro model system of the human BBB. The HIRMAb cross reacts with the HIR of Old World primates such as the Rhesus monkey. The humanized HIRMAb was radiolabeled with 125-iodine, and injected intravenously into an adult, anesthetized Rhesus monkey. Brain scanning showed the humanized HIRMAb was rapidly transported into all parts of the primate brain after intravenous administration. The humanized HIRMAb may be used as a brain drug and gene delivery system for the targeting of large molecule therapeutics across the BBB in humans.

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Section: Resultsmentioning

confidence: 99%

Humanization of anti‐human insulin receptor antibody for drug targeting across the human blood–brain barrier

Boado

Zhang

et al. 2006

Biotech & Bioengineering

185

138

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“…In addition, during recombination events, nucleotides can be randomly deleted from or added to the ends of the gene segments, leading to length variation and even greater diversity. As a result, CDR H3 is found with medium or large surface loops with very different sequences and patterns of interaction (30). Thus, it is not surprising to see that extra flexibility is programmed into CDR H3 of the germline antibody to expand the structural diversity of the primary repertoire beyond that generated by sequence diversity alone.…”

Section: Resultsmentioning

confidence: 99%

A Comparative Analysis of the Immunological Evolution of Antibody 28B4

et al. 2001

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In an effort to gain greater insight into the evolution of the redox active, catalytic antibody 28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, and the X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 were determined. Comparison with the previously determined structures of the unliganded and hapten-bound affinity-matured Fab [Hsieh-Wilson, L. C., Schultz, P. G., and Stevens, R. C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 5363] shows that the germline antibody binds the p-nitrophenyl ring of hapten 3 in an orientation significantly different from that seen in the affinity-matured antibody, whereas the phosphonate moiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has more electrostatic and hydrophobic interactions with hapten 3 than the germline antibody and binds the hapten in a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3 and CDR L1 upon hapten binding to the germline antibody, consistent with the notion of structural plasticity in the germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G., and Stevens, R. C. (1997) Science 276, 1665]. The structural differences are reflected in the differential binding affinities of the germline Fab (K d ) 25 µM) and 28B4 Fab (K d ) 37 nM) to hapten 3. Nine replacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to its germline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 were characterized in detail in the contexts of both the germline and the affinity-matured antibodies. One of the mutations, Asp95 H Trp, leads to a change in the orientation of the bound hapten, and its presence is a prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody for hapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwise manner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germline antibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities were found to be highly homologous to the germline antibody of 28B4, consistent with the notion that certain germline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg, F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. ( ) Science 279, 1929. The ultimate specificity of the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somatic mutation. Insights into the evolution of antibody-combining sites provided by this and other structural studies are discussed.The evolution of binding affinity in the immune system involves combinatorial and mutational processes, which in many aspects parallel those that occur in the natural evolution of enzymes. Consequently, the characterization of the affinity mat...

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“…L 1 and H 1 connect strands of differ- ent sheets within the VL and VH domains, respectively. It has been suggested by Chothia et al (1989) that antibodies have only a few main chain conformations or canonical structures for some of the hypervariable loops Figure 6 is a stereo view showing the loops oriented as they are in the antibody structure. The crystal structure of the Fab fragment of the murine AN02 complexed with its hapten has been solved at 2.9 A resolution using a novel molecular replacement method and the 2.5-A resolution crystal structure of HyHel5 as a search model (Briinger et al, 1991).…”

Section: Analysis Of the Loopsmentioning

confidence: 99%

“…Recently, analysis of known structures by Chothia et al (1989) revealed that there are a small number of main chain conformations found for five of the six hypervariable loops. Evidence indicates that a few conserved residues determine the canonical conformation.…”

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confidence: 99%

Modeling the antigen combining site of an anti‐dinitrophenyl antibody, ANO2

1992

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A model structure has been constructed for a monoclonal anti-dinitrophenyl antibody. The antibody, AN02, has been sequenced and cloned (Anglister, J., Frey ) was used to model the six hypervariable loops that contain the antigen-combining site. All the possible conformations of the loop backbones were constructed and the best loop structures were selected using a combination of the CHARMM potential energy function and evaluation of the solvent-accessible surface area of the conformers. The order in which the loops were searched was carried out based on the relative locations of the loops with reference to the framework of the 6-barrel, namely, L2-Hl-L3-H2-H3-L1. The model structures thus obtained were compared to the high resolution X-ray structure (Briinger, A.T., Leahy, D.J., Hynes, T.R., & Fox, R.O., 1991, J. Mol. Bioi. 221, 239-256).Keywords: antibody modeling; canonical structures; conformational search; homology modeling; hypervariable loops Antibodies of the IgG class consist of heavy and light polypeptide chains linked by disulfide bonds and folded into independent @-sheet domains. The light chain is approximately 220 residues and contains a variable N-terminal domain (VL) and a constant C-terminal region. The heavy chain is made up of an N-terminal variable domain (VH) and three or four constant domains. The heavy chain can contain from 450 to 575 residues. The fragment containing the noncovalent dimer of VL and the VH domains is called the Fv. The VL and VH are each antiparallel @-barrels, and the contact between them forms an eightstranded @-barrel with four strands from each domain (Richardson, 1981;Novotny et al., 1983). In each variable region, three hypervariable loops are attached to a constant &sheet framework region (FR). The specificity ~ . .~

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Conformations of immunoglobulin hypervariable regions

Cited by 1,145 publications

References 31 publications

Humanization of anti‐human insulin receptor antibody for drug targeting across the human blood–brain barrier

Humanization of anti‐human insulin receptor antibody for drug targeting across the human blood–brain barrier

A Comparative Analysis of the Immunological Evolution of Antibody 28B4

Modeling the antigen combining site of an anti‐dinitrophenyl antibody, ANO2

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