Antibody Engineering

Abstract

Antibodies contain sites in the heavy and light chain variable domains (V domains) with antigen‐binding activity largely independent of their constant domains (C domains). Recombinant antibodies that bind with pathological proteins are used widely as drugs. In addition, antibodies containing V domain nucleophilic sites that bind protein targets irreversibly or catalyse target breakdown. Target‐specific therapeutic antibodies 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, restore the C domain‐dependent catalytic activity and incorporate effector functions residing in the C 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. Uptake of antibodies by internalisation of membrane‐bound antigen forms can render intracellular antigens sensitive to antibody targeting.

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 as complement activation and Fc receptor binding.
  • Monoclonal IgG antibodies that bind protein target reversibly have emerged as a major class of biological drugs for various diseases. The efficacy and safety of 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 IgG antibody functions can be attained in the test tube by rational or random mutagenesis coupled with directed selection of the mutants.
  • The new electrophilic immunisation approach has enabled on‐demand production of next‐generation monoclonal IgGs with superior protein target neutralisation capacity made possible by irreversible target binding.
  • Combined innate and adaptive immunity algorithms have been applied to produce monoclonal IgM, IgA and V domains that rapidly and specifically catalyse the degradation of disease‐associated protein targets, making feasible the development of more efficacious and safer therapeutic antibodies.

Keywords: antibody expression vectors; binding affinity; catalytic antibodies; irreversible 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. FRs also contribute catalytic residues. 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. Expression of full‐length recombinant antibody molecules can be accomplished by inserting cDNAs of the V domains into bacterial vectors containing the heavy and light chain constant domains, allowing expression of full‐length antibody molecules. (b) The monomer and pentamer form of IgM are primordial antibodies found in jawed fish. The pentamer structure is stabilised by the disulphide bonds and the J chain (J). IgNAR (immunoglobulin new antigen receptor) is a single V domain primordial antibody with a dimeric constant domain scaffold.
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 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. Properties of nucleophilic antibodies. (a) Nucleophilic catalytic triad located in the VL domain of an Fv with proteolytic activity. Ser27a, green; His93, blue and Asp1, red (Sun ., ). (b) Functional consequences of V domain nucleophilicity. The noncovalent antibody (Ab)‐antigen (Ag) complex is converted to irreversible complex 1 and complex 2 states by covalent pairing of the V domain nucleophile and weak electrophile in protein targets. Formation of irreversible complex 2 also releases ‐terminal protein target fragment. Unlike IgM/IgA, IgG molecules do not support water attack on the complex, and the irreversible IgG complexes do not proceed into catalytic cycle. H, nucleophile; Ag1‐NH‐CH(R)‐CO2H, ‐terminal antigen fragment and NH2‐Ag2, ‐terminal antigen fragment. (c) Irreversible monoclonal antibodies (designated MAb) inactivate protein targets permanently, which is predicted to result in superior therapeutic efficacy compared to reversible. The turnover capability of catalytic monoclonal antibodies should increase the therapeutic efficacy further, and catalytic monoclonal antibodies may also be safer therapeutic agents because they remove the protein target directly without activating inflammatory cells.
Figure 4. Engineered antibody variants. (a) High avidity bundle of four scFv 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 ., ).
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Further Reading

Borrebaeck CAK (ed.) (1995) Antibody Engineering. New York: Oxford University Press.

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Nuñez‐Prado N , Compte M , Harwood S , et al. (2015) The coming of age of engineered multivalent antibodies. Drug Discovery Today. DOI: 10.1016/j.drudis.2015.02.013.

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Paul, Sudhir, Planque, Stephanie, and Massey, Richard(Jul 2015) Antibody Engineering. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001278.pub3]