Antibody fragment technology and avian IgY antibodies: a powerful combination

These antibodies are generally mammalian in origin (Figure 1A) and have been selected from naïve or immune libraries for their high binding affinity and their ability to modulate the activity of their biological target. In parallel, within the research laboratory, polyclonal and monoclonal antibodies are routinely used in western blotting techniques, ELISA assays, immunoprecipitations and localisation of proteins within cells, usually by fluorescence conjugation. Classically, antibodies are raised in rabbits, horse, donkey and mice; however, despite their widespread use suffer from a series of disadvantages such as variable cost, issues of cross-reactivity, inconsistency between batches and limited supply of the isolated antibody. In this review we will highlight the powerful combination of using phage display to produce customised recombinant antibody fragments for research and the advantages of exploring the avian immune repertoire for the generation of novel research tools.

Recombinant antibody libraries: case studies

Antibody fragments were originally defined as stable proteolytic cleavage products of full-length antibodies. Some of these fragments – Fab, Fv (Figure 1B) – retain the ability to recognise an epitope with great specificity and affinity while becoming less complex at the molecular level, displaying less flexibility and simpler folding. These properties make antibody fragments the ideal molecules for use as crystallisation chaperones in structure determination of proteins, with particular success in the case of membrane proteins^2-5.

In addition, the properties of antibody fragments make them amenable for the generation of recombinant libraries that can be explored using careful selection strategies. An example of the application of this approach includes the selection of single chain variable fragment (scFv) antibodies that recognise specific conformational states of GPCR proteins⁶. Another application is the selection of antibody fragments that identify key intermediates in pathways leading to amyloid formation and disease progression.  Neurodegenerative diseases such as Parkinson’s disease, Huntington’s disease, Creutzfeldt-Jakob disease and Alzheimer’s disease are disorders typified by aberrant expression of an insoluble form of a normally soluble protein which leads to protein aggregation and deposits of amyloid fibrils in a variety of organs and tissues⁷.

Normal Huntington protein (HTT) is expressed within the cell cytoplasm whereas the mutant Huntington protein (mHTT), which has elongated poly-glutamine (polyQ) repeats, undergoes abnormal folding, proteolytic cleavage and aggregation which eventually gives rise to neuronal inclusion bodies⁸. It has been shown that the regions surrounding the polyQ tract also regulate the toxicity of the protein; the first 17 amino acids promote aggregation whereas the C terminal proline rich region inhibits protein aggregation.  A naïve human spleen scFv phage-display library was screened against the N-terminal 17 amino acids of HTT and the scFv-C4 intrabody was selected⁹. This intrabody is conformational-specific as it does not recognise the wild-type HTT protein, maybe due to epitope accessibility.  The scFv-C4 antibody was re-engineered to increase its folding stability in the cell cytoplasm and was shown to bind to the mHTT protein and reduce aggregation. Furthermore, addition of a C terminal proteosomal targeting sequence (PEST) to scFv-C4 has demonstrated that the mHTT protein can be successfully targeted for degradation, removing toxic forms of mHTT from cells¹⁰. The molecular details of how scFv-C4 interacts with the first 17 amino acids of HTT have recently been revealed in the co- crystallisation and NMR structures of these complexes¹¹. Another example is focused on α-synuclein protein, a natively unfolded cytoplasmic protein which in a mutated form can exist as small aggregates, larger oligomers or Lewy bodies linked with Parkinson’s disease. It has been demonstrated that morphologically distinct conformations of a-synuclein can be distinguished by isolated scFv fragments; D5-scFv which recognises dimeric and tetrameric forms¹² whereas syn10H-scFv antibody binds trimeric and hexameric aggregates¹³.  

An important advantage of the use of molecules selected from recombinant antibody libraries is the possibility of re-engineering the selected molecules by adding new domains that confer specific properties to the antibody-target complex. A clear example was described above, where a sequence targeting the protein to the proteasome was engineered at the C-terminus of the antibody so that its cognate partner would be directed for degradation. Other possible modifications include chemical modification by reactive moieties such as fluorescent probes and the creation of antibody fragment fusions with a SNAP-tag domain for fluorescence labelling or with mutant BirA ligase, a biotinylating-enzyme, for a proximity assay. The clear message is that antibody fragment technology is a very powerful tool to be used in the study of molecular processes.

Attributes of the Avian Model for generation of antibodies

An apparent stumbling block for the widespread use of recombinant antibody fragment libraries is the perceived difficulty involved in their creation. However, advances in molecular immunology, antibody engineering and transgenic animals have opened new opportunities to explore birds, such as chickens, geese and quails, for antibody discovery campaigns. Indeed, birds have unique mechanisms of immune diversification and a distinctive organisation of their germline antibody genes enabling fast generation of robust recombinant antibody fragments.

The use of avian antibodies as research tools is not a novel concept. Historically, the presence of protective avian antibodies (IgY) in the yolk of immunised hens was demonstrated by Klemperer in the late 1800s¹⁴. This observation was largely neglected until the 90s when the need to refine alternatives to animal testing boosted the IgY-technology¹⁵. As a consequence, specific antibodies directly extracted from egg yolk have been successfully used in research (Figure 2), diagnostics and therapies¹⁶ and holds great potential for the fields of biomedicine and biotechnology¹⁷.

Birds present three classes of immunoglobulins – IgA, IgM and IgY¹⁹ and all of them are incorporated into the egg. The first two are similar to their mammalian counterparts and are transferred into the egg white in trace amounts. However, IgY, the functional equivalent of mammalian IgG, is transferred in large amounts to the egg yolk via specific receptors at the yolk membrane²⁰. The IgY concentration in chicken serum, for example, ranges from 8-10mg/ml²¹ and can go up to 10-20mg/ml in the egg-yolk²² making it effectively the sole immunoglobulin isotype in the egg yolk. This has an impact on subsequent antibody isolation and is distinct from what happens in mammalian blood serum. A single chicken egg can deliver 100-150mg of IgY, meaning that one hen can produce five to 10 times the amount of antibody of a rabbit per year. Remarkably, in the case of an immunised hen, one can typically expect 2-10% of antigen-specific antibodies, which can be more than is obtained from mammalian sources (~5%).These properties have made extraction of specific antibodies from egg yolk a successful strategy for research, diagnostics and therapies¹⁶.

A clear advantage of using avian antibodies relates to the evolutionary distance from different mammalian species. The phylogenetic distance that separates humans from birds is about 300 million years (Myr) contrasting with the 90 Myr of other mammalian species¹⁸. It has been shown that the immune response in an antibody-producing animal tends to increase as its phylogenetic difference with the animal used as the antigen source increases. For this reason it is more likely to obtain a robust immune response against highly conserved mammalian proteins from an avian host.   

Genetic and functional studies of the IgY molecule position it as common ancestor to mammalian IgG and IgE²³. Overall, the IgY molecule keeps a common immunoglobulin architecture, with two heavy and two light chains (Figure 1A). However, IgY rather resembles mammalian IgE, having a higher molecular mass (170KDa) due to the presence of an extra constant region (CH2) in the heavy chain; it also lacks a flexible hinge-region, harbours identical CH1/CH2 interchain disulphide bond patterns and additional glycosylation sites²⁴. At the level of the antigen recognition and binding sites, avian IgY antibodies tend to present larger complementarity determining regions (CDR loops) than mammals and present a unique population of cysteine-rich CDRs, which are particularly observed in CDR3 of the variable heavy chain²⁵. These CDR differences are only now being thoroughly explored at the structural level²⁶, revealing their full potential for the generation of antibodies, unlike the ones from mammalian sources.

Figure 1: Antibody and recombinant fragment structures. A: Comparison of domain organisation of avian IgY (MW: ~170KDa) antibody with mammalian IgE (MW: ~190KDa) and IgG (MW: ~150KDa) antibodies. IgY has an equivalent functional role to mammalian IgG, being responsible for general protective immune responses, but also mediating anaphylaxis usually associated with the IgE molecule in mammalian systems. Constant (C) and Variable (V) domains of the Heavy (H) and Light (L) chains of the antibodies are presented in different colors; complementarity determining regions are shown as red lines and interchain disulphide bonds as green lines; black circles indicate glycosylation sites. Fc (fragment crystallisable), Fab (fragment antigen-binding) and Fv (variable fragment) regions are indicated. B: Examples of monovalent antibody fragments that can be obtained by recombinant antibody engineering: Fab (50KDa), Fv (25KDa) and single-chain variable fragment, scFv (25KDa).

The Fc region of avian IgY has also diverged significantly from mammalian IgG and underlies a number of important features. The IgY-Fc does not bind to naturally occurring anti-mammalian antibodies, such as rheumatoid factor, nor to mammalian Fc receptor, and it does not activate the mammalian complement system. These features are in part responsible for the delayed anti-IgY response observed in pharmacokinetic studies performed on intravascular administration of  IgY in rabbits²⁷ and support the use of yolk antibodies in systemic neutralisation of venoms or toxins^28,29. Additionally, IgY does not bind to standard immunoglobulin-binding resins, such as Protein-A (Staphylococcus aureus), Protein-G (Streptococcus sp.) or Protein–L (Peptostreptococcus magnus), and requires purification using an affinity column of the purified antigen.

A distinctive aspect of birds is their mechanism of immune diversification. Unlike mammals, they present a single functional V_H or V_L element in their heavy and light chain loci, respectively, and diversity is achieved by mechanisms of somatic hypermutation and gene conversion³⁰. The latter involves incorporation of sequences from pseudo V-gene segments, which lack the transcription regulatory and signal-recognition sequences, into the functional V-elements. The organisation of the antibody genes in single functional elements simplifies the molecular cloning of avian immune repertoires and becomes an attractive feature for preparation of antibody libraries using in vitro display technologies such as phage display³¹. Immunised chickens, for example, can present an antigen-specific response in four to six weeks; at this stage their lymphoid organs such as Bursa of Fabricius, spleen and femur bone-marrow, harbouring antibody-producing B-cells, can be collected and processed to make cDNA (Figure 2). Libraries of recombinant avian antibody fragments can easily be generated using a single pair of cloning oligonucleotides for either light- and heavy-chain genes. This contrasts with the cloning of mammalian antibody libraries, which becomes rather complex due to the presence of more than one functional gene element per chain.

Figure 2: Overall potential of Avian IgY antibodies. Immunised birds naturally deposit protective antibodies in the yolk of their eggs. These are termed IgY antibodies, they are polyclonal and can be used in a vast number of passive immunotherapies in humans and animals as well as in research and diagnostic immunoassays. Furthermore, B-cell population recovered from avian lymphoid organs can be processed as bulk, leading to naïve or immune libraries of recombinant antibody fragments. Subsequent screening of antigen-specific monoclonal antibodies can be performed by standard phage-display methods. Alternatively, sophisticated single-cell sorting methods can be applied to the initial B-cell population in order to select B-cells producing an antibody of interest.

A number of antibody fragments can be generated of varying molecular size (Figure 1) and depending on the particular task required. The Fab fragment (~50kDa) for antigen recognition has been thoroughly explored as a stable molecule that tends to be monovalent in character. However, when recombinant Fab protein is expressed in E. coli their yields generally are very low. Currently, a commonly-used recombinant antibody fragment is the single-chain variable fragment (scFv) molecule (~25kDa) and when expressed or delivered inside a cell is called an intrabody. In this molecule the variable light (V_L) and heavy (V_H) domains of an antibody are linked by a flexible linker sequence (GS)n which should be long enough to allow these domains to interact.  Both V_H-V_L or V_L-V_H configurations can be generated (Figure 1B) allowing scFv molecules to retain their ability to recognise specific antigens. The smaller size of the scFv molecule has an advantage in that higher yields can be attained from expression within the periplasm of E.coli. Most scFv molecules tend to multimerise; however this can be controlled by the length of the linker sequence between the variable domains and in some cases may be desired to enhance the avidity to the antigen target.

The availability of surface display vectors which allow the presentation of proteins on the surface of bacteria and filamentous bacteriophage by phage display³² has created a way to specifically select particular antibody properties during multiple cycles of screening and selection. Furthermore, selected positive antibody fragments can be matured for enhanced functions using random mutagenesis of the CDR regions and further phage selection rounds. The coupling of varying selection strategies during phage display screening with in vitro affinity maturation is a powerful technique to tailor specificity and affinity in the final antibody fragment.

In conclusion, immune and naïve antibody libraries derived from mammalian and avian sources are currently combined with sophisticated procedures, such as phage display screening, affinity maturation and molecular re-engineering of useful attributes, to enable the fine tuning of a robust antibody for a particular application. Importantly, fragment-based antibody technologies can easily be established in the research laboratory when used in combination with birds as an immune source. The distinctive immunological advantages of birds enables the generation of highly specific and unique antibodies, creating powerful research tools that allows the capture of distinct conformational protein states that can help to understand biological systems at the molecular level. The challenge for the future is to expand the creative use of antibodies to probe fundamental molecular mechanisms in biology, and birds are definitely going to play a part in this path.

Biographies

DR CAROL HARLEY obtained her PhD from the Biochemistry department at the University of Edinburgh, Scotland, UK (1994). She held postdoctoral positions at the University of Massachusetts Medical School and Albert Einstein College of Medicine in the USA. Dr Harley joined Pfizer Pharmaceuticals, Groton CT (2001) as a Group Leader in the High Throughput Compound Screening Facility where she headed a team that interacted with multiple therapeutic areas. In this role she developed both in vitro and cell-based assays in collaboration with multi-disciplinary teams, and screened the Pfizer compound file to identify novel compounds for lead development. Since returning to Europe in 2007 she has been a Research Scientist at the IBMC, Instituto de Biologia Molecular e Celular and at i3S, Instituto de Investigação e Inovação em Saúde in Porto, Portugal, where she is interested in the identification and characterisation of antibodies that can modulate ion channel function.

RICARDO S. VIEIRA-PIRES is an Assistant Investigator at the Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Portugal. Dr Vieira-Pires obtained his PhD from Joseph Fourier University, Grenoble, France, in the field of structural biology (2008). He held postdoctoral positions at IBMC, Instituto de Biologia Molecular e Celular, Porto, Portugal, where he played a key role in the structural and functional characterisation of ion channels and transporters by X-ray crystallography. He is currently responsible for the Structural Biotechnology Laboratory at CNC that studies proteins and molecular assemblies involved in key processes of bacterial infectious diseases. The laboratory is particularly focused on targeting microbial surface-exposed virulent factors using antibodies. Dr Vieira-Pires also coordinates the Avian Antibody platform that explores bird immune repertoires for antibody discovery.

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