Antibody Engineering


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 ., ).


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 United States of America 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.

Durova OM , Vorobiev II , Smirnov IV , et al. (2009) Strategies for induction of catalytic antibodies toward HIV‐1 glycoprotein gp120 in autoimmune prone mice. Molecular Immunology 47: 87–95.

Eryilmaz E , Janda A , Kim J , et al. (2013) Global structures of IgG isotypes expressing identical variable regions. Molecular Immunology 56: 588–598.

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.

Gu J , Congdon EE and Sigurdsson EM (2013) Two novel Tau antibodies targeting the 396/404 region are primarily taken up by neurons and reduce Tau protein pathology. Journal of Biological Chemistry 288: 33081–33095.

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.

Hifumi E , Honjo E , Fujimoto N , et al. (2012) Highly efficient method of preparing human catalytic antibody light chains and their biological characteristics. FASEB Journal 26: 1607–1615.

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.

Kou J , Yang J , Lim JE , et al. (2015) Catalytic immunoglobulin gene delivery in a mouse model of Alzheimer's disease: prophylactic and therapeutic applications. Molecular Neurobiology 51: 43–56.

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 , Karle S , Mitsuda Y , et al. (2006) Towards irreversible HIV inactivation: stable gp120 binding by nucleophilic antibodies. Journal of Molecular Recognition 19: 423–431.

Nishiyama Y , Mitsuda Y , Taguchi H , et al. (2007) Towards covalent vaccination: improved polyclonal HIV neutralizing antibody response induced by an electrophilic gp120 V3 peptide analog. Journal of Biological Chemistry 282: 31250–31256.

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 SA , Nishiyama Y , et al. (2012) Nature and nurture of catalytic antibodies. Advances in Experimental Medicine and Biology 750: 56–75.

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 SA , Mitsuda Y , Chitsazzadeh V , et al. (2014a) Deficient synthesis of class‐switched, HIV‐neutralizing antibodies to the CD4 binding site and correction by electrophilic gp120 immunogen. AIDS 28: 2201–2211.

Planque SA , Nishiyama Y , Hara M , et al. (2014b) Physiological IgM class catalytic antibodies selective for transthyretin amyloid. Journal of Biological Chemistry 289: 13243–13258.

Planque SA , Nishiyama Y , Sonoda S , et al. (2015) Specific amyloid β clearance by a catalytic antibody construct. Journal of Biological Chemistry. DOI: 10.1074/jbc.M115.641738.

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.

Sapparapu G , Planque S , Mitsuda Y , et al. (2012) Journal of Biological Chemistry 287: 36096–36104.

Sasano M , Burton DR and Silverman GJ (1993) Molecular selection of human antibodies with an unconventional bacterial B cell antigen. Journal of Immunology 151: 5822–5839.

Silverman GJ and Goodyear CS (2006) Confounding B‐cell defences: lessons from a staphylococcal superantigen. Nature Reviews Immunology 6: 465–475.

Song YC , Sun GH , Lee TP , et al. (2008) Arginines in the CDR of anti‐dsDNA autoantibodies facilitate cell internalization via electrostatic interactions. European Journal of Immunology 38: 3178–3190.

Spiess C , Zhai Q and Carter PJ (2015) Alternative molecular formats and therapeutic applications for bispecific antibodies. Molecular Immunology. DOI: 10.1016/j.molimm.2015.01.003.

Sun M , Gao QS , Kirnarskiy L , et al. (1997) Cleavage specificity of a proteolytic antibody light chain and effects of the heavy chain variable domain. Journal of Molecular Biology 271: 374–385.

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.

Tramontano A , Janda KD and Lerner RA (1986) Catalytic antibodies. Science 234: 1566–1570.

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 2 F5 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.

Xu Z , Zan H , Pone EJ , et al. (2012) Immunoglobulin class‐switch DNA recombination: induction, targeting and beyond. Nature Reviews Immunology 12: 517–531.

Zalevsky J , Chamberlain AK , Horton HM , et al. (2010) Enhanced antibody half‐life improves in vivo activity. Nature Biotechnology 28: 157–159.

Zhong G , Lerner RA and Barbas CF III (1999) Broadening the aldolase catalytic antibody repertoire by combining reactive immunization and transition state theory: new enantio‐ and diastereoselectivities. Angewandte Chemie International Edition in English 38: 3738–3741.

Zhu C , Feng W , Weedon J , et al. (2011) The multiple shark Ig H chain genes rearrange and hypermutate autonomously. Journal of Immunology 187: 2492–2501.

Further Reading

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

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.

Kontermann R and Dübel S (eds) (2010) Antibody Engineering. Heidelberg; New York: Springer.

McCafferty J , Hoogenboom HR and Chiswell DJ (eds) (1996) Antibody Engineering: A Practical Approach. New York: Oxford University Press.

Niwa R and Satoh M (2015) The current status and prospects of antibody engineering for therapeutic use: focus on glycoengineering technology. Journal of Pharmaceutical Sciences 104: 930–941.

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. [doi: 10.1002/9780470015902.a0001278.pub3]