Antibody Engineering

Sudhir Paul, University of Texas Health Science Center, Houston, Texas, USA
Stephanie Planque, University of Texas Health Science Center, Houston, Texas, USA

Published online: June 2011

DOI: 10.1002/9780470015902.a0001278.pub2

The antigen-binding site of antibodies is composed of the heavy and light chain variable domains (V domains). Therapeutic human antibodies specific for individual antigen can be obtained by inducing adaptive B cell development in transgenic animal or by test-tube affinity maturation of cloned antibody V domain repertoires. The V domains are recloned as full-length antibodies to improve their pharmacokinetic behaviour and incorporate effector functions residing in the constant domains. Assembly of the V domains into multivalent constructs improves the binding avidity. Linkage to enzymes, toxins or delivery proteins imparts novel functions to the constructs. Emerging engineering strategies include the development of antibodies with catalytic activity and antibodies that can target intracellular antigens.

Key Concepts:

  • Antibodies are highly adaptive structures. They are encoded by germline genes that have diversified over evolutionary time and then undergo further antigen-driven adaptive development over the life time of an individual organism.
  • The antigen combining site is composed of the antibody light and heavy chain subunit variable domains. The constant domain contributes effector functions such complement activation and Fc receptor binding.
  • Monoclonal antibodies have emerged as a major class of biological drugs for various diseases. The efficacy and safety of monoclonal antibody drugs depend on their antigen recognition affinity, specificity and in vivo pharmacokinetics.
  • Understanding the natural process underlying adaptive sequence diversification has permitted isolation of monoclonal antibodies meeting the criteria for therapeutic applications. Antibody display and cloning methods have enabled identification of individual antibodies with defined antigenic specificities from vast repertoires.
  • Improvement of antibody functions can be attained in the test tube by rational or random mutagenesis coupled with directed selection of the mutants.
  • Catalytic antibodies combining the specificity of the antigen-binding site with the turnover capability of enzymatic sites have emerged as a viable second generation technology for novel antibody applications.

Keywords: antibody expression vectors; binding affinity; catalytic antibodies; display technologies; humanised antibodies; single chain Fv; V domains

Figure 1. Schematic diagram of an IgG antibody. The bottom circle encompasses the constant domains. The variable regions and one domain each of the heavy and light chains are included in the fragment antigen binding (Fab; left circle). Comprising the fragment variable (Fv; right circle) are the VL and VH domains, within which are located the CDRs. Most of the antigen-contacting amino acids are located in the CDRs. Superantigens bind mostly at the FRs. The catalytic site is also located in part in the FRs. Mutations can be introduced into the V domains to improve antigen-binding affinity. Combinatorial VL–VH diversification is an additional means to improve antigen-recognition properties. Heavy chain constant region domains are responsible for antigen-stimulated effector functions. cDNAs from cloned antibody V domain repertoires are inserted into vectors containing the heavy and light-chain constant domains, allowing expression of full-length antibody molecules.
Figure 2. Isolation of human monoclonal antibodies. (a) Transgenic mice expressing the human antibody repertoire are immunised and monoclonal antibodies are prepared by hybridoma or repertoire cloning strategies. Antigen-specific antibodies are identified by selection and screening procedures. Patients with microbial infection or autoimmune disease can be employed as the sources of antigen-specific antibodies to microbial antigens and autoantigens, respectively. (b) Affinity maturation of antibody V domains in vitro is conducted by sequential rounds of mutagenesis and fractionation of single-chain Fv or Fab fragments displayed as fusion proteins on phage surface. Expression of phagemid DNA in a permissive bacterial host allows production of soluble antibody fragments.
Figure 3. Novel antibody variants. (a) High avidity bundle of four Fv fragments tethered by a self-associating peptide derived from the leucine zipper motif. (b) An Fv linked to a toxin via a linker peptide (see Brinkmann et al., 1992). (c) Serine protease-like catalytic triad located in the VL domain of an scFv with proteolytic activity. Ser27a, green; His93, blue; Asp1, red. Constructs shown in (a) and (c) are reviewed in Paul (1998).
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 References
    Brinkmann U, Buchner J and Pastan I (1992) Independent domain of Pseudomonas exotoxin and single-chain immunotoxins: influence of interdomain connections. Proceedings of the National Academy of Sciences of the USA 89: 3075–3079.
    Coloma MJ, Hastings A, Wims LA and Morrison SL (1992) Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction. Journal of Immunological Methods 152: 89–104.
    Durandy A (2003) Activation-induced cytidine deaminase: a dual role in class-switch recombination and somatic hypermutation. European Journal of Immunology 33: 2069–2073.
    Fukuchi K, Tahara K, Kim HD et al. (2006) Anti-Abeta single-chain antibody delivery via adeno-associated virus for treatment of Alzheimer's disease. Neurobiology of Disease 23: 502–511.
    Green LL (1999) Antibody engineering via genetic engineering of the mouse: XenoMouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies. Journal of Immunological Methods 231: 11–23.
    Hifumi E, Morihara F, Hatiuchi K et al. (2008) Catalytic features and eradication ability of antibody light-chain UA15-L against Helicobacter pylori. Journal of Biological Chemistry 283: 899–907.
    Johnson PR, Schnepp BC, Zhang J et al. (2009) Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys. Nature Medicine 15: 901–906.
    Kohler G and Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495–497.
    Lipovsek D and Plückthun A (2004) In-vitro protein evolution by ribosome display and mRNA display. Journal of Immunological Methods 290: 51–67.
    Luo GX, Kohlstaedt LA and Charles CH (2003) Humanization of an anti-ICAM-1 antibody with over 50-fold affinity and functional improvement. Journal of Immunological Methods 275: 31–40.
    Marks JD, Hoogenboom HR, Bonnert TP et al. (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. Journal of Molecular Biology 222: 581–597.
    Mitsuda Y, Planque S, Hara M et al. (2007) Naturally occurring catalytic antibodies: evidence for preferred development of the catalytic function in IgA class antibodies. Molecular Biotechnology 36: 113–122.
    Nishiyama Y, Planque S, Mitsuda Y et al. (2009) Toward effective HIV vaccination: induction of binary epitope reactive antibodies with broad HIV neutralizing activity. Journal of Biological Chemistry 284: 30627–30642.
    book Paul S (1998) "Protein engineering". In: Walker J (ed.) Molecular Biotechniques, pp. 547–566. Totowa: Humana Press.
    Paul S, Karle S, Planque S et al. (2004) Naturally occurring proteolytic antibodies: selective immunoglobulin M-catalyzed hydrolysis of HIV gp120. Journal of Biological Chemistry 279: 39611–39619.
    Paul S, Planque S, Nishiyama Y, Escobar M and Hanson C (2010) Back to the future: covalent epitope-based HIV vaccine development. Expert Reviews of vaccines 9: 1027–1043.
    Paul S, Planque S, Zhou YX et al. (2003) Specific HIV gp120 cleaving antibodies induced by covalently reactive analog of gp120. Journal of Biological Chemistry 278: 20429–20435.
    Planque S, Mitsuda Y, Taguchi H et al. (2007) Characterization of gp120 hydrolysis by IgA antibodies from humans without HIV infection. AIDS Research and Human Retroviruses 23: 1541–1554.
    Planque S, Nishiyama Y, Taguchi H et al. (2008) Catalytic antibodies to HIV: physiological role and potential clinical utility. Autoimmunity Review 7: 473–479.
    Poul MA, Becerril B, Nielsen UB, Morisson P and Marks JD (2000) Selection of tumor-specific internalizing human antibodies from phage libraries. Journal of Molecular Biology 301: 1149–1161.
    Rondot S, Koch J, Breitling F and Dübel S (2001) A helper phage to improve single-chain antibody presentation in phage display. Nature Biotechnology 19: 75–78.
    Sapparapu G, Planque S, Nishiyama Y, Foung SK and Paul S (2009) Antigen-specific proteolysis by hybrid antibodies containing promiscuous proteolytic light chains paired with an antigen-binding heavy chain. Journal of Biological Chemistry 284: 24622–24633.
    Silverman GJ and Goodyear CS (2006) Confounding B-cell defences: lessons from a staphylococcal superantigen. Nature Reviews Immunology 6: 465–475.
    Taguchi H, Planque S, Sapparapu G et al. (2008) Exceptional amyloid beta peptide hydrolyzing activity of non-physiological immunoglobulin variable domain scaffolds. Journal of Biological Chemistry 283: 36724–36733.
    Wolbank S, Kunert R, Stiegler G and Katinger H (2003) Characterization of human class-switched polymeric (immunoglobulin M [IgM] and IgA) anti-human immunodeficiency virus type 1 antibodies 2F5 and 2G12. Journal of Virology 77: 4095–4103.
    Wootla B, Christophe OD, Mahendra A et al. (2011) Proteolytic antibodies activate factor IX in patients with acquired hemophilia. Blood 117: 2257–2264.
    Zalevsky J, Chamberlain AK, Horton HM et al. (2010) Enhanced antibody half-life improves in vivo activity. Nature Biotechnology 28: 157–159.
 Further Reading
    book Borrebaeck CAK (ed.) (1995) Antibody Engineering. New York: Oxford University Press.
    book Clark M (2007) Antibody Engineering of Fc Effector Functions. London: Henry Stewart Talks.
    Krangel MS (2003) Gene segment selection in V(D)J recombination: accessibility and beyond. Nature Immunology 4: 624–630.
    book Kontermann R and Dübel S (eds) (2010) Antibody Engineering. Heidelburg; New York: Springer.
    book McCafferty J, Hoogenboom HR and Chiswell DJ (eds) (1996) Antibody Engineering: A Practical Approach. New York: Oxford University Press.

Related Sub-Topics:

Diagnostics, Therapeutics and Methods | Antibodies
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Article Title: Antibody Engineering
 
How to Cite close
Paul, Sudhir, and Planque, Stephanie(Jun 2011) Antibody Engineering. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001278.pub2]