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. 1993 Mar;78(3):379–386.

Nucleotide sequence analysis of CDR3 elements of a panel of anti-peptide monoclonal antibodies recognizing parathyroid hormone-related protein.

R Rapley 1, P S Flora 1, D J Walsh 1, M R Walker 1
PMCID: PMC1421840  PMID: 8478021

Abstract

Nucleotide sequences of heavy (VH) and light (VL) chain variable region complementarity determining regions have been determined from in vitro amplified mRNA isolated from a panel of monoclonal antibodies (mAb) raised to a synthetic 34mer peptide representing the N-terminal portion of human parathyroid hormone-related protein (PTHrP or parathyrin) reported to contain an immunodominant epitope. These mAb vary in affinity for the synthetic peptide and native PTHrP (Ka between 5.9 x 10(8) and 1.9 x 10(11)l/M). All 10 mAb studied were found were found to utilized restricted VH2, V kappa 2, JH4 and J kappa 1 family genes. Significant differences in the length and sequence of D elements were found; however 9/10 mAb utilize members of the DSP2 family. Significantly, two broad ranges of affinity could be determined based on the presence of Asp or Ala at residue 101 in JH.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alt F. W., Yancopoulos G. D., Blackwell T. K., Wood C., Thomas E., Boss M., Coffman R., Rosenberg N., Tonegawa S., Baltimore D. Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J. 1984 Jun;3(6):1209–1219. doi: 10.1002/j.1460-2075.1984.tb01955.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barbas C. F., 3rd, Kang A. S., Lerner R. A., Benkovic S. J. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):7978–7982. doi: 10.1073/pnas.88.18.7978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barden J. A., Kemp B. E. NMR study of a 34-residue N-terminal fragment of the parathyroid-hormone-related protein secreted during humoral hypercalcemia of malignancy. Eur J Biochem. 1989 Sep 15;184(2):379–394. doi: 10.1111/j.1432-1033.1989.tb15030.x. [DOI] [PubMed] [Google Scholar]
  4. Berek C., Milstein C. The dynamic nature of the antibody repertoire. Immunol Rev. 1988 Oct;105:5–26. doi: 10.1111/j.1600-065x.1988.tb00763.x. [DOI] [PubMed] [Google Scholar]
  5. Chothia C., Lesk A. M., Tramontano A., Levitt M., Smith-Gill S. J., Air G., Sheriff S., Padlan E. A., Davies D., Tulip W. R. Conformations of immunoglobulin hypervariable regions. Nature. 1989 Dec 21;342(6252):877–883. doi: 10.1038/342877a0. [DOI] [PubMed] [Google Scholar]
  6. Clarke S. H., Claflin J. L., Potter M., Rudikoff S. Polymorphism in anti-phosphocholine antibodies reflecting evolution of immunoglobulin families. J Exp Med. 1983 Jan 1;157(1):98–113. doi: 10.1084/jem.157.1.98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feeney A. J., Thuerauf D. J. Sequence and fine specificity analysis of primary 511 anti-phosphorylcholine antibodies. J Immunol. 1989 Dec 15;143(12):4061–4068. [PubMed] [Google Scholar]
  9. French D. L., Laskov R., Scharff M. D. The role of somatic hypermutation in the generation of antibody diversity. Science. 1989 Jun 9;244(4909):1152–1157. doi: 10.1126/science.2658060. [DOI] [PubMed] [Google Scholar]
  10. Heidelberger M. Precipitating cross-reactions among pneumococcal types. Infect Immun. 1983 Sep;41(3):1234–1244. doi: 10.1128/iai.41.3.1234-1244.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Huse W. D., Sastry L., Iverson S. A., Kang A. S., Alting-Mees M., Burton D. R., Benkovic S. J., Lerner R. A. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science. 1989 Dec 8;246(4935):1275–1281. doi: 10.1126/science.2531466. [DOI] [PubMed] [Google Scholar]
  12. Kaartinen M., Rocca-Serra J., Mäkelä O. Combinatorial association of V genes: one VH gene codes for three non-cross-reactive monoclonal antibodies each specific for a different antigen (phoxazolone, NP or gat). Mol Immunol. 1988 Sep;25(9):859–865. doi: 10.1016/0161-5890(88)90122-8. [DOI] [PubMed] [Google Scholar]
  13. Kabat E. A., Nickerson K. G., Liao J., Grossbard L., Osserman E. F., Glickman E., Chess L., Robbins J. B., Schneerson R., Yang Y. H. A human monoclonal macroglobulin with specificity for alpha(2----8)-linked poly-N-acetyl neuraminic acid, the capsular polysaccharide of group B meningococci and Escherichia coli K1, which crossreacts with polynucleotides and with denatured DNA. J Exp Med. 1986 Aug 1;164(2):642–654. doi: 10.1084/jem.164.2.642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kabat E. A., Wu T. T. Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites. J Immunol. 1991 Sep 1;147(5):1709–1719. [PubMed] [Google Scholar]
  15. Kang A. S., Barbas C. F., Janda K. D., Benkovic S. J., Lerner R. A. Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4363–4366. doi: 10.1073/pnas.88.10.4363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kobrin B. J., Buhl S., Shulman M. J., Scharff M. D. A V region mutation in a phosphocholine-binding monoclonal antibody results in loss of antigen binding. J Immunol. 1991 Mar 15;146(6):2017–2020. [PubMed] [Google Scholar]
  17. Larrick J. W., Danielsson L., Brenner C. A., Abrahamson M., Fry K. E., Borrebaeck C. A. Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction. Biochem Biophys Res Commun. 1989 May 15;160(3):1250–1256. doi: 10.1016/s0006-291x(89)80138-x. [DOI] [PubMed] [Google Scholar]
  18. Lavoie T. B., Drohan W. N., Smith-Gill S. J. Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme. J Immunol. 1992 Jan 15;148(2):503–513. [PubMed] [Google Scholar]
  19. McCafferty J., Griffiths A. D., Winter G., Chiswell D. J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 1990 Dec 6;348(6301):552–554. doi: 10.1038/348552a0. [DOI] [PubMed] [Google Scholar]
  20. Meek K. D., Hasemann C. A., Capra J. D. Novel rearrangements at the immunoglobulin D locus. Inversions and fusions add to IgH somatic diversity. J Exp Med. 1989 Jul 1;170(1):39–57. doi: 10.1084/jem.170.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Morrison S. L., Canfield S., Porter S., Tan L. K., Tao M. H., Wims L. A. Production and characterization of genetically engineered antibody molecules. Clin Chem. 1988 Sep;34(9):1668–1675. [PubMed] [Google Scholar]
  22. Orlandi R., Güssow D. H., Jones P. T., Winter G. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 May;86(10):3833–3837. doi: 10.1073/pnas.86.10.3833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Parhami-Seren B., Wysocki L., Margolies M. N. The amino acid residues at the VH-D-JH junctions affect the affinity of anti-p-azophenylarsonate antibodies. J Immunol. 1989 Dec 15;143(12):4090–4097. [PubMed] [Google Scholar]
  24. Rapley R., Flora S., Walker M. R. Direct PCR sequencing of murine immunoglobulin genes using E. coli single-stranded DNA-binding protein. PCR Methods Appl. 1992 Aug;2(1):99–101. doi: 10.1101/gr.2.1.99. [DOI] [PubMed] [Google Scholar]
  25. Ratcliffe W. A., Hughes S., Gilligan M. G., Heath D. A., Ratcliffe J. G. Production and characterisation of monoclonal antibodies to parathyroid hormone-related protein. J Immunol Methods. 1990 Feb 20;127(1):109–116. doi: 10.1016/0022-1759(90)90346-w. [DOI] [PubMed] [Google Scholar]
  26. Riechmann L., Clark M., Waldmann H., Winter G. Reshaping human antibodies for therapy. Nature. 1988 Mar 24;332(6162):323–327. doi: 10.1038/332323a0. [DOI] [PubMed] [Google Scholar]
  27. Roberts S., Cheetham J. C., Rees A. R. Generation of an antibody with enhanced affinity and specificity for its antigen by protein engineering. Nature. 1987 Aug 20;328(6132):731–734. doi: 10.1038/328731a0. [DOI] [PubMed] [Google Scholar]
  28. Roberts V. A., Iverson B. L., Iverson S. A., Benkovic S. J., Lerner R. A., Getzoff E. D., Tainer J. A. Antibody remodeling: a general solution to the design of a metal-coordination site in an antibody binding pocket. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6654–6658. doi: 10.1073/pnas.87.17.6654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rocca-Serra J., Matthes H. W., Kaartinen M., Milstein C., Thèze J., Fougereau M. Analysis of antibody diversity: V-D-J mRNA nucleotide sequence of four anti-GAT monoclonal antibodies. A paucigene system using alternate D-J recombinations to generate functionally similar hypervariable regions. EMBO J. 1983;2(6):867–872. doi: 10.1002/j.1460-2075.1983.tb01515.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Siekevitz M., Gefter M. L., Brodeur P., Riblet R., Marshak-Rothstein A. The genetic basis of antibody production: the dominant anti-arsonate idiotype response of the strain A mouse. Eur J Immunol. 1982 Dec;12(12):1023–1032. doi: 10.1002/eji.1830121208. [DOI] [PubMed] [Google Scholar]
  31. Tonegawa S. Somatic generation of antibody diversity. Nature. 1983 Apr 14;302(5909):575–581. doi: 10.1038/302575a0. [DOI] [PubMed] [Google Scholar]
  32. Walker M. R., Bevan L. J., Daniels J., Rottier M. M., Rapley R., Roberts A. M. Isolation and amplification of human IgE Fd encoding mRNA from human peripheral blood lymphocytes. J Immunol Methods. 1992 Apr 27;149(1):77–85. doi: 10.1016/s0022-1759(12)80051-2. [DOI] [PubMed] [Google Scholar]
  33. Winter G., Milstein C. Man-made antibodies. Nature. 1991 Jan 24;349(6307):293–299. doi: 10.1038/349293a0. [DOI] [PubMed] [Google Scholar]
  34. Wysocki L. J., Gridley T., Huang S., Grandea A. G., 3rd, Gefter M. L. Single germline VH and V kappa genes encode predominating antibody variable regions elicited in strain A mice by immunization with p-azophenylarsonate. J Exp Med. 1987 Jul 1;166(1):1–11. doi: 10.1084/jem.166.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]

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