Affinity transfer by CDR grafting on a nonimmunoglobulin scaffold

Neocarzinostatin (NCS) is a small “all β” protein displaying the same overall fold as immunoglobulins. This protein possesses a well‐defined hydrophobic core and two loops structurally equivalent to the CDR1 and CDR3 of immunoglobulins. NCS is the most studied member of the enediynechromoprotein family, and is clinically used as an antitumoral agent. NCS has promise as a drug delivery vehicle if new binding specificities could be conferred on its protein scaffold. Previous studies have shown that the binding specificity of the crevasse can be extended to compounds completely unrelated to the natural enediyne chromophore family. We show here that it is possible to introduce new interaction capacities to obtain a protein useful for drug targeting by modifying the immunoglobulin CDR‐like loops. We transferred the CDR3 of the VHH chain of camel antilysozyme immunoglobulin to the equivalent site in the corresponding loop of neocarzinostatin. We then evaluated the stability of the resulting structure and its affinity for lysozyme. The engineered NCS‐CDR3 presents a structure similar to that of the wild‐type NCS, and is stable and efficiently produced. ELISA, ITC, and SPR measurements demonstrated that the new NCS‐CDR3 specifically bound lysozyme.

[1]  B. Harris,et al.  Exploiting antibody-based technologies to manage environmental pollution. , 1999, Trends in biotechnology.

[2]  K. D. Hardman,et al.  Single-chain antigen-binding proteins. , 1988, Science.

[3]  A. Skerra.  Engineered protein scaffolds for molecular recognition , 2000, Journal of molecular recognition : JMR.

[4]  C. Pace,et al.  How to measure and predict the molar absorption coefficient of a protein , 1995, Protein science : a publication of the Protein Society.

[5]  P. Hudson.  Recombinant antibody constructs in cancer therapy. , 1999, Current opinion in immunology.

[6]  C. Pace.  Determination and analysis of urea and guanidine hydrochloride denaturation curves. , 1986, Methods in enzymology.

[7]  Lode Wyns,et al.  Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme , 1996, Nature Structural Biology.

[8]  M. Uhlén,et al.  Scaffolds for engineering novel binding sites in proteins. , 1997, Current opinion in structural biology.

[9]  Rapid evolution of peptide and protein binding properties in vitro. , 1992, Current opinion in biotechnology.

[10]  R. Hoess,et al.  Phage display of peptides and protein domains , 1993 .

[11]  G. Drobny,et al.  Sequential 1H NMR assignment of the complex of aponeocarzinostatin with ethidium bromide and investigation of protein-drug interactions in the chromophore binding site. , 1994, Biochemistry.

[12]  K. Constantine,et al.  Sequential 1H, 13C, and 15N NMR assignments and solution conformation of apokedarcidin. , 1994, Biochemistry.

[13]  V. Favaudon,et al.  The seven-stranded beta-barrel structure of apo-neocarzinostatin as compared to the immunoglobulin domain. , 1992, Biochimie.

[14]  M. Brigido,et al.  Humanization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen. , 2003, Molecular immunology.

[15]  Y. Zhen,et al.  C1027 chromophore, a potent new enediyne antitumor antibiotic, induces sequence-specific double-strand DNA cleavage. , 1994, Biochemistry.

[16]  S. Schaus,et al.  Gene transcription analysis of Saccharomyces cerevisiae exposed to neocarzinostatin protein– chromophore complex reveals evidence of DNA damage, a potential mechanism of resistance, and consequences of prolonged exposure , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[17]  P Van Roey,et al.  Crystal structure analysis of auromomycin apoprotein (macromomycin) shows importance of protein side chains to chromophore binding selectivity. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Andreas Plückthun,et al.  Biophysical properties of human antibody variable domains. , 2003, Journal of molecular biology.

[19]  V. Z Pletnev,et al.  Actinoxanthin Structure at the Atomic Level (Russian) , 1983 .

[20]  Lutz Riechmann,et al.  Reshaping human antibodies for therapy , 1988, Nature.

[21]  G. P. Smith,et al.  Surface presentation of protein epitopes using bacteriophage expression systems. , 1991, Current opinion in biotechnology.

[22]  H. Maeda,et al.  SMANCS and polymer-conjugated macromolecular drugs: advantages in cancer chemotherapy. , 1991, Advanced drug delivery reviews.

[23]  M. Desmadril,et al.  Reinvestigation of the Proteolytic Activity of Neocarzinostatin , 2000, Journal of bacteriology.

[24]  C. Linge,et al.  Interactions of CD80 and CD86 with CD28 and CTLA4. , 1996, Journal of immunology.

[25]  Solution structure of a novel chromoprotein derived from apo-neocarzinostatin and a synthetic chromophore. , 2002, Biochemistry.

[26]  Dominique Bourgeois,et al.  The crystal structure of a llama heavy chain variable domain , 1996, Nature Structural Biology.

[27]  K. Balamurugan,et al.  Release of the Neocarzinostatin Chromophore from the Holoprotein Does Not Require Major Conformational Change of the Tertiary and Secondary Structures Induced by Trifluoroethanol* , 2000, The Journal of Biological Chemistry.

[28]  H R Hoogenboom,et al.  Designing and optimizing library selection strategies for generating high-affinity antibodies. , 1997, Trends in biotechnology.

[29]  Andreas Plückthun,et al.  Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach. , 2003, Biochemistry.

[30]  K. Kumagai,et al.  NEOCARZINOSTATIN, AN ANTITUMOR ANTIBIOTIC OF HIGH MOLECULAR WEIGHT. ISOLATION, PHYSIOCHEMICAL PROPERTIES AND BIOLOGICAL ACTIVITIES. , 1965, The Journal of antibiotics.

[31]  L. Wyns,et al.  Camel single‐domain antibody inhibits enzyme by mimicking carbohydrate substrate , 1998, Proteins.

[32]  M. Mizugaki,et al.  Neocarzinostatin: selective tryptophan oxidation and neocarzinostatin-chromophore binding to apo-neocarzinostatin. , 1991, Chemical & pharmaceutical bulletin.

[33]  V. Favaudon,et al.  Three-dimensional solution structure of apo-neocarzinostatin from Streptomyces carzinostaticus determined by NMR spectroscopy. , 1992, European journal of biochemistry.

[34]  S. Muyldermans,et al.  Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. , 1994, Protein engineering.

[35]  P. V. von Hippel,et al.  Calculation of protein extinction coefficients from amino acid sequence data. , 1989, Analytical biochemistry.

[36]  Y. Nozaki.  The preparation of guanidine hydrochloride. , 1972, Methods in enzymology.

[37]  S. Muyldermans,et al.  Naturally occurring antibodies devoid of light chains , 1993, Nature.

[38]  Frédéric Pecorari,et al.  In vitro evolution of the binding specificity of neocarzinostatin, an enediyne-binding chromoprotein. , 2003, Biochemistry.

[39]  M. Desmadril,et al.  Key interactions in neocarzinostatin, a protein of the immunoglobulin fold family. , 2002, Protein engineering.

[40]  I. Goldberg,et al.  Roles of chromophore and apo-protein in neocarzinostatin action. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[41]  The de novo design of an antibody combining site. Crystallographic analysis of the VL domain confirms the structural model. , 1994, Journal of molecular biology.

[42]  L. Riechmann,et al.  Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability. , 1996, Protein engineering.

[43]  S E Hufton,et al.  Building novel binding ligands to B7.1 and B7.2 based on human antibody single variable light chain domains. , 2001, Journal of molecular biology.

[44]  A. Skerra,et al.  Fermenter production of an artificial fab fragment, rationally designed for the antigen cystatin, and its optimized crystallization through constant domain shuffling , 1995, Proteins.

[45]  A. Epenetos,et al.  Antibody‐guided enzyme therapy of cancer producing cyanide results in necrosis of targeted cells , 2002, International journal of cancer.