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Rational design of CXCR4 specific antibodies with elongated CDRs.

Liu T, Liu Y, Wang Y, Hull M, Schultz PG, Wang F - J. Am. Chem. Soc. (2014)

Bottom Line: These engineered antibodies selectively bind to CXCR4 expressing cells with binding affinities in the low nanomolar range.Finally, we also demonstrate that a similar strategy can be applied to other CDRs and show that a CDRH2-peptide fusion binds CXCR4 with a K(d) of 0.9 nM.This work illustrates the versatility of scaffold-based antibody engineering and could greatly expand the antibody functional repertoire in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute , La Jolla, California 92037, United States.

ABSTRACT
The bovine antibody (BLV1H12) which has an ultralong heavy chain complementarity determining region 3 (CDRH3) provides a novel scaffold for antibody engineering. By substituting the extended CDRH3 of BLV1H12 with modified CXCR4 binding peptides that adopt a β-hairpin conformation, we generated antibodies specifically targeting the ligand binding pocket of CXCR4 receptor. These engineered antibodies selectively bind to CXCR4 expressing cells with binding affinities in the low nanomolar range. In addition, they inhibit SDF-1-dependent signal transduction and cell migration in a transwell assay. Finally, we also demonstrate that a similar strategy can be applied to other CDRs and show that a CDRH2-peptide fusion binds CXCR4 with a K(d) of 0.9 nM. This work illustrates the versatility of scaffold-based antibody engineering and could greatly expand the antibody functional repertoire in the future.

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(A) Specific bindingbetween bAb-AC1–3 and CXCR4 was determinedby a Tag-lite HTRF binding assay. The binding affinities were calculatedbased on the Cheng–Prusoff equation to give Ki values of 2.1, 5.4, and 19.8 nM for bAb-AC1, bAb-AC2,and bAb-AC3, respectively. (B) Flow cytometry histogram demonstratingnearly complete inhibition of 12G5 binding to CXCR4 by a 3-fold excessof bAb-AC1.
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fig3: (A) Specific bindingbetween bAb-AC1–3 and CXCR4 was determinedby a Tag-lite HTRF binding assay. The binding affinities were calculatedbased on the Cheng–Prusoff equation to give Ki values of 2.1, 5.4, and 19.8 nM for bAb-AC1, bAb-AC2,and bAb-AC3, respectively. (B) Flow cytometry histogram demonstratingnearly complete inhibition of 12G5 binding to CXCR4 by a 3-fold excessof bAb-AC1.

Mentions: To accurately determine the binding affinity between theengineeredantibodies and CXCR4, we applied Tag-lite homogeneous time-resolvedfluorescence (HTRF) (Cisbio Bioassays).24 Specific binding of fluorescently labeled SDF-1 to labeled SNAP-tag-CXCR4results in a HTRF signal. The binding constant (Kd) between fluorescently labeled SDF-1 and the Tag-liteCXCR4 receptor was determined to be 14.2 ± 1.2 nM (Figure S3). A dose-dependent competition wasobserved between the engineered antibodies and 50 nM of labeled SDF-1(Figure 3A). Assuming a competitive bindingmode, the Kds of bAb-AC1, bAb-AC2, andbAb-AC3 to CXCR4 were calculated to be 2.1, 5.4, and 19.8 nM, respectively.25 These results indicate that bAb-AC1 with a moreflexible glycine at i + 1 position of the hairpinturn binds the best to CXCR4, which is consistent with the flow cytometryanalysis results. On the other hand, bAb-AC3, which has a β-turnpromoting sequence (Asn-Gly) added at the end of the β-hairpin,has a decreased affinity compared to bAb-AC1 and bAb-AC2 that is probablydue to spatial constraints within the CXCR4 ligand binding pocket.


Rational design of CXCR4 specific antibodies with elongated CDRs.

Liu T, Liu Y, Wang Y, Hull M, Schultz PG, Wang F - J. Am. Chem. Soc. (2014)

(A) Specific bindingbetween bAb-AC1–3 and CXCR4 was determinedby a Tag-lite HTRF binding assay. The binding affinities were calculatedbased on the Cheng–Prusoff equation to give Ki values of 2.1, 5.4, and 19.8 nM for bAb-AC1, bAb-AC2,and bAb-AC3, respectively. (B) Flow cytometry histogram demonstratingnearly complete inhibition of 12G5 binding to CXCR4 by a 3-fold excessof bAb-AC1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4120998&req=5

fig3: (A) Specific bindingbetween bAb-AC1–3 and CXCR4 was determinedby a Tag-lite HTRF binding assay. The binding affinities were calculatedbased on the Cheng–Prusoff equation to give Ki values of 2.1, 5.4, and 19.8 nM for bAb-AC1, bAb-AC2,and bAb-AC3, respectively. (B) Flow cytometry histogram demonstratingnearly complete inhibition of 12G5 binding to CXCR4 by a 3-fold excessof bAb-AC1.
Mentions: To accurately determine the binding affinity between theengineeredantibodies and CXCR4, we applied Tag-lite homogeneous time-resolvedfluorescence (HTRF) (Cisbio Bioassays).24 Specific binding of fluorescently labeled SDF-1 to labeled SNAP-tag-CXCR4results in a HTRF signal. The binding constant (Kd) between fluorescently labeled SDF-1 and the Tag-liteCXCR4 receptor was determined to be 14.2 ± 1.2 nM (Figure S3). A dose-dependent competition wasobserved between the engineered antibodies and 50 nM of labeled SDF-1(Figure 3A). Assuming a competitive bindingmode, the Kds of bAb-AC1, bAb-AC2, andbAb-AC3 to CXCR4 were calculated to be 2.1, 5.4, and 19.8 nM, respectively.25 These results indicate that bAb-AC1 with a moreflexible glycine at i + 1 position of the hairpinturn binds the best to CXCR4, which is consistent with the flow cytometryanalysis results. On the other hand, bAb-AC3, which has a β-turnpromoting sequence (Asn-Gly) added at the end of the β-hairpin,has a decreased affinity compared to bAb-AC1 and bAb-AC2 that is probablydue to spatial constraints within the CXCR4 ligand binding pocket.

Bottom Line: These engineered antibodies selectively bind to CXCR4 expressing cells with binding affinities in the low nanomolar range.Finally, we also demonstrate that a similar strategy can be applied to other CDRs and show that a CDRH2-peptide fusion binds CXCR4 with a K(d) of 0.9 nM.This work illustrates the versatility of scaffold-based antibody engineering and could greatly expand the antibody functional repertoire in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute , La Jolla, California 92037, United States.

ABSTRACT
The bovine antibody (BLV1H12) which has an ultralong heavy chain complementarity determining region 3 (CDRH3) provides a novel scaffold for antibody engineering. By substituting the extended CDRH3 of BLV1H12 with modified CXCR4 binding peptides that adopt a β-hairpin conformation, we generated antibodies specifically targeting the ligand binding pocket of CXCR4 receptor. These engineered antibodies selectively bind to CXCR4 expressing cells with binding affinities in the low nanomolar range. In addition, they inhibit SDF-1-dependent signal transduction and cell migration in a transwell assay. Finally, we also demonstrate that a similar strategy can be applied to other CDRs and show that a CDRH2-peptide fusion binds CXCR4 with a K(d) of 0.9 nM. This work illustrates the versatility of scaffold-based antibody engineering and could greatly expand the antibody functional repertoire in the future.

Show MeSH