Antibody engineering Part 3 - unige.it · Antibody engineering - Part 3 Alternative antibody formats. ... An overview of various recombinant antibody formats. The antibody formats

Antibody engineering - Part 3Alternative antibody formats

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Single-chain FV fragments (scFv)

Fab Fv dsFv

Little M, Kipriyanov SM, Le Gall F, Moldenhauer G. Of mice and men: hybridoma and recombinant antibodies. Immunol Today. 2000; 21(8):364–370.

disulfide stabilized

2 Current Opinion in Molecular Therapeutics 2010 Vol 12 No 2

Figure 1. An overview of various recombinant antibody formats.

The antibody formats are grouped according to their specificity (ie, monospecific or bispecific) and valency (ie, monovalent, bivalent or tetravalent). Db Diabody, dAb single-domain antibody, DVD-Ig dual variable domain Ig, scDb single-chain Db, scFv single-chain Fv, taFv tandem scFv

Bispecific

Monospecific

Tetravalent

dAb2

taFv

Db scDb

DVD-lg

Bs(scFv)4-IgG taFv-Fc scFv-Fc-scFv

Db-Fc scDb-Fc scDb-CH3

dAb-Fc-dAb

’

Monovalent Bivalent

Fab

dAb

scFv

IgG F(ab')2

dAb-Fc

scFv-Fc scFv-CH3 Db

CH3

VL VH

VH VL

(GGGGS)4

Single chain Fragment variable (scFv)

26-28 kDa

A major limitation of scFv molecules is that they monovalently bind their target antigen

Linker >12 AA (12-24) Linker >3 but <12 AA

Linker < 3 AA

scFv Diabodies

Triabodies Tetrabodies


(scFV)2


scDiabody Tandem diabody


Because scFvs are not capable of mediating effector function, the application of scFv molecules alone is restricted to indications for which an antagonistic or agonostic activity is sufficient for therapeutic intervention, such as in inflammatory disorders

Kontermann RE. Alternative antibody formats. Curr Opin Mol Ther. 2010 Apr. 1;12(2):176–183.

scFv-Fc fusion proteins (Minibodies)

In order to improve pharmacokinetics properties and increase antigen-binding activity, scFvs have been fused to the Fc region of IgG molecules, the natural effector part of an antibody

Kontermann RE. Alternative antibody formats. Curr Opin Mol Ther. 2010 Apr. 1;12(2):176–183.

Minibody


AD: self associating protein domain

ganglioside expressed at high density on the surface ofmalignant cells of neuroectodermal origin, in particular NB,melanoma and small cell lung carcinoma (SCLC) (13). Sinceits expression in normal tissues is very restricted, GD2represents an excellent target for neuroectodermal tumortargeting. Several anti-GD2 mAbs have been developed (13-15), and some of them have already been tested in clinicaltrials (16-18). In addition, anti-GD2 scFvs have also beenproduced (19,20).

In this study the genes encoding the variable regions ofboth heavy- and light-chain from a hybridoma expressing ananti-GD2 mAb were cloned and assembled to generate twodifferent forms of anti-GD2 SIPs. The first SIP is a fully murinemolecule containing the CH3 domain of mouse IgG1, whereasthe second construct is a hybrid mouse-human moleculecontaining the CH4 domain of the secretory isoform S2 ofhuman IgE (21). Both mini-antibodies were successfullyproduced and shown to retain binding specificity as well asan affinity similar to that of the original mAb.

Materials and methods

Cell lines and media. The human NB cell line ACN (22) wasdonated some years ago by Dr S. Carrel, Lausanne, Switzer-land. The murine cell lines, myeloma Sp2/0, anti-GD2hybridoma mAb 126 (HB8568, IgM) and NB Neuro-2a, aswell as the human cell lines HeLa, IMR-32 (NB), NCI-H82and NCI-H446 (SCLC) were all from the American TypeCulture Collection (Rockville, MD, USA). The murine GD2-positive NB hybrid cell line NXS2 (23) was kindly donatedby Dr R.A. Reisfeld (Scripps Clinic, La Jolla, CA, USA).The human NB cell line GI-LI-N was established at GasliniInstitute, Genoa, Italy (24). The human melanoma cell linesColo-853, MZ2-MEL and RPMI-7932 were kindly providedby Dr M. Ponzoni (Gaslini Institute, Genoa, Italy) (25).

Human NB, SCLC, melanoma and HeLa cells werecultured in RPMI 1640 medium (Sigma-Aldrich Italia, Milan,Italy) supplemented with 10% fetal calf serum (Sigma), 2 mMglutamine, penicillin-streptomycin and non-essential amino-acids (NEAA) (Bio Whittaker Italia, Caravaggio, Italy). Thehybridoma and mouse cell lines were cultured in DMEMmedium (Sigma) containing 10% fetal calf serum, 2 mMglutamine, penicillin-streptomycin and NEAA.

SIP constructs and vectors. Mouse VH (moVH) and VL (moVL)cDNAs were obtained by reverse transcription and PCR-amplification of the RNA extracted from the anti-GD2 IgM-producing hybridoma mAb 126 (26), using partially de-generated oligonucleotides as amplification primers. moVH

was amplified with primers moVHB (5'-AGGTSMARCTGCAGSAGTCWGG-3') and moCHm (5'-CATTTGGGAAGGACTGACTC-3'). moVL was obtained with primers moVkB2(5'-GATATTGTGATGACCCAGTCTCCA-3') and moCk2(5'-TGGATACAGTTGGTGCAGC-3'). Both moVH and moVL

were re-amplified using primers containing the appropriaterestriction enzyme sites for their assembly in the final construct(all the restriction enzymes used were from New EnglandBiolabs, Beverly, MA, USA). moVH was re-amplified withprimers VH/XhoI (5'-TCTCTCGAGCAAAGGTGAGGTGCAGCTGCAGGAGTCT-3') and VH/BspEI (5'-TCTATCCGG

AAGAGACAGTGACCAGAGT-3'), moVL with primersGD2-L5V/ApaLI (5'-TGTGTGCACTCTGATATTGTGATGACCCA-3') and 3LL-2/SpeI (5'-CAGGACTAGTGCTGCCTTTGATTTCCAGCTTGG-3'). The mouse IgG1 CH3dimerizing domain (mo

Á1CH3) was derived with the samemethod from murine lymphocyte RNA using primers:moCH3/BspEI (5'-CTCTTCCGGAGGCTCTGGTGGCAGACCGAAG-3') and moCH3/SacI (5'-ATCGAGCTCTAGATTATTTCCCGGGAGAGTGGGAG-3'). Primers moCH3/BspEIand VH/BspEI were designed to create a 4-amino acid linkerbetween moVH and moÁ1CH3.

The moVL, moVH and moÁ1CH3 fragments were sequencedbefore cloning in the pUT-SEC plasmid vector (11), whichcontains the gene for tetracycline resistance, the codingsequence for the 18-amino acid linker between the VL and VH

(to allow a correct folding of the antigen binding site) and asequence encoding the leader peptide required for immuno-globulin secretion in the extracellular medium.

The chimeric

ÂSIP (huÂSIP) was obtained from the murineÁSIP (moÁSIP) after substituting the moÁ1CH3 domain withthe CH4 domain of the human IgE secretory isoform S2 (21).This domain contains a cysteine residue at the carboxy-terminal end that allows the covalent stabilization of thedimeric ÂSIP through an interchain disulphide bond (9).

The complete constructs were then excised from theplasmid pUT by a cut HindIII/XbaI for the moÁSIP construct,or HindIII/EcoRI for the huÂSIP construct, and cloned in theexpression vector pcDNA3 (Invitrogen, Carlsbad, CA, USA),under the control of the CMV promoter, as shown in Fig. 1.

Cell transfection. About 1x106 Sp2/0 cells were resuspendedin 0.5 ml cold PBS (10.1 mM Na2HPO4, 1.8 mM KH2PO4,

OCCHINO et al: CHARACTERIZATION OF ANTI-GD2 DIMERIC SINGLE CHAIN ANTIBODIES3 8 4

Figure 1. Schemes of recombinant SIPs constructs. The cDNAs of mAb 126VL and VH, mouse Á1CH3 and human ÂS2CH4 were inserted at the indicatedsites in the expression vector pcDNA3, under the control of the CMVpromoter, to generate plasmids pcDNA3-moÁSIP (a), and pcDNA3-huÂSIP (b).Sequences encoding the leader peptide and the 18-amino acid linker betweenVL and VH were derived from the pUT-SEC vector.

The cDNAs of mAb 126 VL and VH, mouse γ1CH3 and human εS2CH4 were inserted at the indicated sites in the expression vector pcDNA3, under the control of the CMV promoter, to generate plasmids pcDNA3-moγSIP (a), and pcDNA3-huεSIP (b). !!Sequences encoding the leader peptide and the 18-amino acid linker between VL and VH were derived from the pUT-SEC vector

Schemes of recombinant SIPs (minibodies) constructs

Occhino M, Raffaghello L, Burrone O, Gambini C, Pistoia V, Corrias MV, et al. Generation and characterization of dimeric small immunoproteins specific for neuroblastoma associated antigen GD2. Int. J. Mol. Med. 2004 Sep.;14(3):383–388.

Weisser NE, Hall JC. Applications of single-chain variable fragment antibodies in therapeutics and diagnostics. Biotechnol Adv. 2009;27(4):502-520

Minibodies or SIPs

applications such as tumor penetration (Ghetie et al., 1997). With thisin mind, small rAb fragments (e.g. scFvs) have been engineered to notonly have high-avidity but also be of optimal size for tumorpenetration without the fast clearance. Furthermore, many scFvmultivalent formats can be easily converted into multispecific Abfragments that can associate with two different targets and thus beable to not only recruit effector functions but also deliver cytotoxiccells to the target site. Thus, scFv fragments have been engineered intoseveral types of multimeric complexes, including dimeric, trimericand tetrameric fragments, which are discussed below.

2.3.1. Diabodies, triabodies and tetrabodiesThe easiest constructs to engineer are noncovalent diabody,

triabody and tetrabody molecules that assemble according to changesin the linker length. scFvs are predominantly monomeric when the VH

and VL domains are joined by a linker of 12 or more amino acids.However, scFvs with a linker length of three to 12 residues cannot foldinto a functional Fv domains and instead associate with a second scFvmolecule to form a dimer (diabody, ~60 kDa) (Holliger et al., 1993)due to pairing of the VH of one chain to the VL of another (Fig. 2).Furthermore, reducing the linker length to 3 amino acids or less can

Fig. 2. scFv and scFv-based rAb fragments. scFvmolecules can be engineered in the VH-linker-VL or the VL-linker-VH orientation, and thesemolecules form the basis for several scFv-basedrAb fragments. Examples of bi- and multivalent and bi-specific fragments are illustrated. M, A and B refer different linkers in the scDb and taFvs.

505N.E. Weisser, J.C. Hall / Biotechnology Advances 27 (2009) 502–520

Bispecific recombinant antibodiesHannah Byrne, Paul J. Conroy, James C. Whisstock, and Richard J. O’Kennedy. A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications. Trends in Biotechnology 2013, Vol. 31, No. 11.

• Chemical conjugation, which involves chemical cross-linking

• Fusion of two different hybridoma cell lines

• Genetic approaches involving recombinant DNA technology

bsAbs are produced by three main methods

Hannah Byrne, Paul J. Conroy, James C. Whisstock, and Richard J. O’Kennedy. A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications. Trends in Biotechnology 2013, Vol. 31, No. 11.

Recombinant DNA technology has yielded the greatest range of bsAbs, through artificial manipulation of genes and represents the most diverse approach for bsAb generation (45 formats in the past two decades)


Neutralization of receptor/ligand

Kontermann R. Dual targeting strategies with bispecific antibodies. MAbs. 2012 1;4(2)

Activation of receptor/ligand


Blockage of two epitopes on receptor/ligand


ADCC/CDC, retargeting of effector cells


Targeting of loads


Despite the ongoing development of various increasingly complex bsAb designs, only two formats, BiTES and Triomabs, have made a substantial impact


Bispecific T cell engagers (BiTEs)

• BiTEs combine the minimal binding domains (Fv fragments) of two different mAbs fused together by a short flexible linker that allows free rotation of the two arms, and thus facilitates optimal antibody:antigen interaction !

• They function by forming a link between T cells (CD3 or CD19) and TAAs, inducing T cell dependent cytotoxic activity, independently of the presence of MHC I or co-stimulatory molecules


T-cell

Tumor cell

Bispecific T cell engagers (BiTEs)

Bispecific antibodies can function as NK and T-cell engagers for cancer therapy

T-cell!NK-cell

Tumor cell

IL12!Chemokine

• 100-10 000-fold higher efficacy in tumor cell lysis relative to other CD3-bispecific formats and monoclonal IgG1 antibodies.

• Induces target cell elimination by unstimulated peripheral T cells without the need for T cell co-stimuli or T cell preactivation regimens.

• Strictly targets cell dependent, polyclonal activation of most CD4+ and CD8+ T cells.

• High protein stability and homogeneity.

Advantages of the BiTE


• Trifunctional antibodies (Triomabs) are intact IgG molecules characterized by their unique ability to engage three different cells types, typically, tumor cells, T cells, and accessory cells, such as, macrophages, dendritic cells, NK cells, and other Fc-receptor-expressing cells.

• Trifunctional antibodies have two different antigen-binding specificities, most commonly CD3 and a tumor antigen. The presence of the intact Fc region facilitates interaction with receptors triggering several immune defense reactions

Trifunctional antibodies/Triomabs


Antibody-dependent cellular cytotoxicity (ADCC) in therapeutic antibody treatment

Seidel UJ, Schlegel P, Lang P. Natural killer cell mediated antibody-dependent cellular cytotoxicity in tumor immunotherapy with therapeutic antibodies. Front Immunol. 2013 Mar 27;4:76.

Trifunctional antibodies/Triomabs function

T-cell

Tumor cell

NK-cell

NAT URE BI O TEC H N O L O GY VOLUME 23 NUMBER 9 SEPTEMBER 2005 1107

heterologous expression, secretion and fold-ing, with proteolysis and antigen-antibody accessibility. Therefore, many of these display and screening systems, although elegant in nature31,32, are not widely used today for anti-bodies. However, a recently described approach bypasses most of these problems: it is based on anchoring the antibody fragment on the periplasmic face of the inner membrane of E. coli followed by disruption of the outer mem-brane, incubation with fluorescently labeled antigen and sorting of the protoplasts. This very promising and versatile display method is directly compatible with (filamentous) phage display, combines the ease of E. coli-based library constructions with the power of cell sorting, and therefore, is likely to become widely used.

Other selection platforms. Directed evolution platforms recently devel-oped for antibody fragments include retroviral display34, display based on protein-DNA linkage35,36, microbead display by in vitro compart-mentalization37, in vivo-based growth selection based on the protein fragment complementation assay (PCA)38 or other systems39 and even single-molecule sorting40. Although each of these methods will have specific theoretical advantages, to date, their validation with antibody fragment libraries has been limited, and their advantages over more established systems (e.g. regarding the truly monovalent nature of the method, eukaryotic expression advantages, increase in library size or selection efficiency) remain to be demonstrated. (For a more in-depth discussion of library-display technologies, including PCA and two-hybrid systems, that are available but have not yet been used in combi-nation with antibody fragments, see ref. 41.)

To establish a platform to select recombinant antibody libraries in the IgG format, the preferred format for many applications, researchers recently displayed small libraries of IgGs on the surface of mammalian cells. After homologous integration of a single-gene copy in each cell, the population was sorted by flow cytometry to obtain a clone with sevenfold affinity improvement (W.D. Shen, Amgen, personal commu-nication). In the future, bigger combinatorial IgG format–based libraries may be built using vaccinia virus–based vectors42, or diversity may be introduced in vivo by using B-cell lines that hypermutate a carrier anti-body gene constitutively43 or upon induction44 or that harbor induc-ible hypermutable enzymes involved in this process in nature45. Some of these newer selection and diversification methods may open novel applications for the directed evolution of antibodies and other proteins (see also accompanying review on p. 1126–1136).

Strategies to select and screen antibody librariesIndividual clones of a recombinant single-chain Fv (scFv) or Fab library theoretically can be directly screened for antigen binding, for example, using binding assays based on ELISA or filter-based screening. Screening is limited by the number of clones that can be examined, hence in many applications the frequency of antigen-reactive clones is too low, and the libraries too large (with tens of millions to billions of clones) to do this efficiently. The connection between genotype and phenotype in phage- or ribosome-display libraries provides a means to select for clones binding to a desirable antigen, thereby increasing the frequency of antigen-reactive clones, enriching the clones with best binding affinity, or the clones with certain predefined binding characteristics. Typically many more clones can therefore be sampled compared with screen-ing procedures. Many different selection methods and experimental approaches have been developed that separate clones that bind from those that do not (Fig. 3).

Selection procedures. For phage-display libraries, selection involves exposure to antigen to allow antigen-specific phage antibodies to bind their targets during biopanning. This is followed by recovery of antigen-bound phage and subsequent infection in bacteria. Although ideally, only one round of selection would be required, nonspecific binding limits the enrichment that can be achieved per selection round and therefore, in most cases, recursive rounds of selection and amplification are needed to select the best binders from the library (Fig. 2a).Phage display–based selections are now a relatively standard procedure

in many molecular biology laboratories (a more detailed description of these procedures is provided elsewhere10,46 and references therein). For more complex selections such as those using cells or tissues, it can be instructive to use enrichment studies with control phage antibodies to optimize the efficiency of the selection method and to compare different selection approaches, and then tune the selection strategy accordingly to

Phage display

Protein-mRNAlink via:

Protein-DNAdisplay

Growthselection via:

Display on:

Microbeadvia in vitro

compartmentalization

Coupling of genoto phenotype

Selective pressureon phenotype

Screening Amplification

+

Antibody gene pool

Displayed library

Selected antibody lead

Synthetic DNA

Cloning ofgenetic diversity

B-cells

Selectioncycle

Mutagenesisand

selectioncycle

-ribosome display-mRNA display

-Yeast-Bacteria-Mammalian cells-Retroviruses-.....

-Yeast 2-hybrid-Protein fragment complementation

a bSteps in antibody selection Selection platformsFigure 2 Creating and selecting recombinant antibody libraries. (a) First, antibody diversity is generated from synthetic V genes or cloned from B cells. Next, antibody phenotype (boxes in green, blue and orange) is coupled to its genotype (wavy line) via a phenotype-genotype link (green) packaged in a host (purple) (shown here schematically for phage display). As a result, each host particle expresses (or displays) a unique antibody on its surface. The repertoire of antibodies displayed on these host particles is subjected to The process is repeated and eventually antibodies binding to antigen are confirmed by screening. (b) Different selection platforms for conventional antibodies. Color code as for a (see text for details and citations).

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Antibody display platforms

1. Generation (or cloning) of genotypic diversity

2. Coupling of genotype to phenotype

3. Application of selective pressure

4. Amplification

Hoogenboom HR. Selecting and screening recombinant antibody libraries. Nat Biotechnol. 2005 23(9):1105-1116

How antibody display platforms work

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Main types of antibody display platforms


Main methods for in vitro selection for binding


• Antibody libraries from immunized donors

• Naïve libraries

• Semi-synthetic and synthetic libraries

There are different types of antibody libraries



(a) recombinant immune libraries (b) recombinant nonimmune libraries

are cloned from naturally diverse antibody gene pools and for this reason display most diversity at the center of the binding site (depicted in green), with some level of somatic mutations throughout the V regions (green dots)

Recombinant immune and nonimmune libraries


Synthetic antibody libraries are constructed entirely in vitro using oligonucleotides that introduce areas of complete or tailored degeneracy into the CDRs of one or more V genes


Synthetic libraries

For the construction of synthetic antibody libraries, synthetic diversity (in red) is introduced into (c) the CDR3 of V-gene segments (d) all the CDRs of the V-gene segments (indicated in pink) or (e) in several chosen CDR positions (indicated by red dots). Alternatively, in semi-synthetic libraries, natural and synthetic diversity is combined; for example, in (f) heavy-chain natural diversity in the CDR3s (in green) is combined with synthetic hot-spot diversity for CDR1 and CDR2 (in red) or (g) natural CDRs (in green) are reshuffled into one or more chosen antibody frameworks.

Recombinant synthetic and semi-synthetic libraries


The targeted approach includes sequentially targeting all the CDRs of an antibody via a (h) high (red) or (i) low (pink) level of mutagenesis or (j) targeting isolated hot spots of somatic hypermutations or residues suspected of affecting affinity on experimental basis or structural reasons (red dots).

Random mutations can be introduced throughout the whole V gene using (k) E. coli mutator strains, error-prone replication with DNA polymerases or RNA replicases (shown in pink), (l) the replacement of regions that are naturally diverse via DNA shuffling or similar techniques using natural partner repertoires (shown in light green) or a combination of both.

During affinity maturation strategies, diversity is introduced into the V genes in a targeted manner or via random introduction



NAT URE BI O TEC H N O L O GY VOLUME 23 NUMBER 9 SEPTEMBER 2005 1113

tissue-specific antigens123. Combining this approach with expression cloning, immunoprecipitation and mass spectrometry has already led to the identification of novel target molecules on malignant cells124–126.

Intracellular selection. Intracellular antibodies (or intrabodies), when folded properly, are valuable tools for studying biological processes and for blocking proteins inside cells127. Individual scFv antibodies can be evolved directly for stable cytoplasmic expression by growth selection in bacteria128, although it may be faster to functionally identify pools of phage-selected antibodies that have been recloned and expressed intracellularly in mammalian cells129,130. A modified yeast two-hybrid selection strategy was previously described that can directly select and isolate several functional antigen-binding intrabodies127,131,. More recently, libraries have been engineered to contain a high percentage of functional intrabodies using scFv frameworks132 or antibody heavy-chain variable domains133 selected directly in the intracellular environ-ment, which were then employed to build single-framework intrabody libraries. Although not yet applied to the screening of large libraries or affinity optimization, combinations with fully automated two-hybrid systems may eventually yield a platform suitable for generating antibod-ies of medium affinity to panels of antigens for large-scale functional proteomics projects, as recently suggested by work done with other bind-ing proteins134.

Future developmentsLibrary technology has led to one human antibody so far approved for therapy and many more antibodies in clinical and preclinical trials (Table 3). Although a full discussion on the immunogenicity of these and other engineered antibodies135 is beyond the scope of this review, it is accepted in the field that the risk of immunogenicity may be reduced

by using antibodies that are as ‘human’ as possible. With time, library designs and affinity-maturation strategies may be even more tuned towards the ideal antibody composition: an as-close-to-human germ-line sequence with optimal affinity yet with a minimal number of T-cell epitopes and a human-like heavy chain CDR3 (ref. 136). Library designs may go even further by reducing the difficulties in downstream develop-ment by avoiding potentially problematic amino acids137 in the variable regions (e.g., methionine oxidation, asparagine deamidation or aspartate isomerization). Fortunately, as has been demonstrated recently138,139, a reduction in diversity to just four or even two well-chosen amino acids, does not necessarily limit library performance. Also, future selection procedures against instability and aggregation behavior may help to reduce potential immunogenicity and increase solubility.

There are now many different molecular selection strategies for iso-lating and engineering human antibodies. The three main selection platforms (phage, ribosome/mRNA and microbial cell display) are somewhat complementary in their use, but they all fall short of the ideal: a selection system that provides within a few days a large panel of antibodies to a large number of epitopes on the target antigen of choice, a range of selected affinities, a certain level of stability and expression and a precisely targeted sequence diversity. To build a better antibody ‘molecular evolution machine,’ we need to make further refine-ments in several areas: first, improve methods and predictive designs for introducing diversification into antibody genes to build libraries with a higher quality in functionality and biophysical properties; sec-ond, build normalized selection and amplification strategies to reduce biases towards nondesirable variables (e.g., reduce advantages due to PCR, infection, growth, multimerization); third, combine affinity and expression maturation for populations rather than individual clones to increase throughput and simultaneously maintain or improve multiple

Table 3 Examples of therapeutic antibodies derived from recombinant antibody librariesName Target Indication Company Clinical phase

Humira(adalimumab)

TNFα Autoimmune diseases Abbott/CambridgeAntibody Technology (CaT)

Approved for arthritis(in phase 2/3 for others)

Numax(MEDI-524)

Respiratory syncitial virus RSV prophylaxis MedImmune Phase 3

ABT-874 Interleukin 12 Multiple sclerosis Abbott/CaT Phase 2

CAT-192B(belimumab)

Transforming growth factor β1 Systemic sclerosis Genzyme/CaT Phase 2

LymphoStat-B B-cell activating factor Lupus/rheumatoid arthritis Human Genome Sciences/CaT Phase 2

MT201 Epithelial cell adhesion molecule Breast and prostate cancer Micromet Phase 2

HGS-ETR1 TRAIL-R1 Non-Hodgkin lymphoma Human Genome Sciences/CaT Phase 2

CAT-213 Eotaxin1 Allergic rhinitis CaT Phase 2

MYO-029 Growth differentiation factor-8 Muscular dystrophy Wyeth/CaT Phase 1

ABthrax Protective antigen Anthrax Human Genome Sciences/CaT Phase 1 finished

HGS-ETR2 TRAIL-R2 Solid tumors Human Genome Sciences/CaT Phase 1

CAT-354 Interleukin 13 Asthma CaT Phase 1

1D09C3 MHC class II Non-Hodgkin lymphoma GPC Biotech/MorphoSys Phase 1

IMC-11F8 EGFR Solid tumors ImClone Phase 1

IMC-1121b VEGFR-2 Solid tumors ImClone Phase 1

GC-1008 Transforming growth factor β Idiopathic pulmonary fibrosis Genzyme/CaT Preclinical

IMC-A12 Insulin-like growth factor receptor Solid tumors ImClone Preclinical

MOR102 Intracellular adhesion molecule 1 Autoimmune diseases MorphoSys Preclinical

DX-2240 Tie-1 Cancer Dyax Preclinical

AZD3102 Undisclosed Alzheimer disease AstraZeneca/Dyax Preclinical

αGMCSFRα Granulocyte-macrophagecolony-stimulating factorreceptor α

Autoimmune diseases CaT/AMRAD Preclinical

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Phage display platformFully human antibodiesKipriyanov, S M, Little, M, Generation of recombinant antibodies Mol. Biotechnol. 1999 12(2):173-201Hoogenboom HR. Selecting and screening recombinant antibody libraries. Nat Biotechnol. 2005 23(9):1105-1116

• The crucial advantage of this technology is the direct link that exists between the experimental phenotype (the displayed protein) and its encapsulated genotype (the DNA encoding the displayed protein)

• The discovery that functional antibody binding sites (i.e. scFvs) can be displayed on the surface of bacteriophage has allowed selection of antibodies against antigens of choice without the need for hybridoma technology


Phage display platform pros

FabscFv

scFv or Fab are usually displayed on phage surface

182 Kipriyanov and Little

MOLECULAR BIOTECHNOLOGY Volume 12, 1999

by glycine-rich tetra- and pentapeptide repeatsand followed by a C-terminal membrane anchor.The two N-terminal domains appear to be directlyinvolved in the infection process (115). Peptidescan be inserted between the second and third do-mains of pIII (116) or near the N-terminus (117)without destroying its functions in morphogenesisand infection. These findings led to the genera-tion of phage peptide libraries that can be easilyscreened for binding to ligands and antibodies(118–120).

In an analogous approach, antibodies havebeen attached to the surface of filamentous ph-ages by inserting antibody DNA in the 5' end ofgene III in the phage genome (121). A major dis-advantage, however, is that the expression of theantibody cannot be regulated. Furthermore, sincelarge inserts were shown to have adverse effectson infectivity (117), there is a high risk that anti-body libraries would tend to accumulate the morerapidly propagating deletion mutants.

5.1.1. Phagemid pIII Display SystemsTo overcome these problems, DNA coding for

an antibody fragment was incorporated into a

phagemid, which is basically a plasmid with aphage intergenic region that contains the packag-ing signal (Fig. 3) (122–124). In this case, theexpression of the fusion protein DNA is regulatedby a bacterial promoter under the control of a lacoperator, so that relatively large amounts of anti-body can be obtained for analysis after inductionwith isopropyl thiogalactose (IPTG).

To display the antibody on the phage surface,the phagemid must be packaged with the proteinssupplied by a helper phage such as M13K07,which packages its own DNA less efficiently thanthe phagemid DNA (Fig. 3). Usually 10 helperphages are added per bacterium. The superinfec-tion of bacteria with helper phage might possiblybe inhibited by the binding of the pIII fusion pro-tein to the pilin subunit of F-pili, which is whysome workers prefer to delete the pIII N-terminaldomains and fuse the antibody to the remainingC-terminal domain (125). Another minor problemmight be caused by the leakiness of the lac opera-tor, which could increase the selection pressurefor the production of deletion mutants. To tightenthe repression, Breitling et al. (123) employed adouble lac operator and used E. coli transformedwith a plasmid expressing lac repressor. In an al-ternative system, the gene coding for lac repres-sor was included in the phagemid (125).

The appropriate VH/VL combination may beselectively enriched from an scFv phage librarythrough a series of immunoaffinity steps referredto as “library panning.” Nonspecific phages areremoved during the washing procedure, follow-ing which the remaining antigen-specific phagesare eluted using either acid or alkali (12). Alter-natively, proteolytic cleavage sites for trypsin(123,125) or for Genenase I (126) have been in-troduced between the antibody domain and pIII.The competitive elution of bound phage-dis-played antibodies with a monoclonal antibody hasalso been described (127). The characterizationof the selected antibody was facilitated by includ-ing an amber stop codon between the DNA cod-ing for the antibody and pIII. Only the antibodydomain will then be produced in nonsuppressorstrains of E. coli (122). To follow the productionof recombinant antibody, marker peptides recog-

Fig. 2. Schematic representation of a filamentousphage showing the capsid proteins (A) and phage withdisplayed antibody fragment (B).

Kipriyanov, S M, Little, M, Generation of recombinant antibodies Mol. Biotechnol. 1999 12(2):173-201

Generation of Recombinant Antibodies 183

MOLECULAR BIOTECHNOLOGY Volume 12, 1999

nized by monoclonal antibodies have beenincluded either at the C-terminus (102) or withinthe linker peptide between the heavy and lightchain variable domains of a single chain antibody(123). Antibodies to the N-terminus have alsoproved to be very useful for following processingduring secretion (123,128). In a more recentreport, several of the above sequences for theimproved expression, display, and identificationof recombinant antibodies were incorporated intoa single expression phagemid (129). This vectoralso provides the antibodies with a hexahistidinesequence for purification by metal chelate chro-matography and a C-terminal cysteine to facili-tate their conjugation with other molecules.5.1.2. pIII Display System for Very LargeLibraries

The display of antibodies on filamentous ph-ages has proved to be an excellent system for se-

lecting specific clones. The library size, however,depends on the efficiency of E. coli transformation.To overcome this restriction in in vitro packaging,E. coli have been transformed with plasmids en-coding a heavy chain library and then infected withphagemid particles encoding a light chain library(130). To ensure that the heavy and light chaingenes are packaged within the same particle, thelox-Cre site-specific recombination system of bac-teriophage P1 was used to bring them together onthe same phagemid. Another proposed system forthe construction of multicombinatorial antibody li-braries by association of the heavy and light chaingenes from two different vectors within an infectedbacterium is based on the use of the phage attsite-specific recombination sites (131). Thesemethods could theoretically generate a phage dis-play library containing as many different antibod-ies as the number of E. coli cells in culture.

Fig. 3. Diagram of the proposed mechanism for monovalent display of scFv on the surface of filamentousphage M13. The expression vector fuses the scFv to the C-terminal domain of the viral coat protein pIII. Thephage f1 intergenic region (f1 IR) is included in the vector to direct packaging of the phagemid in the presence ofhelper phage. The resulting phage progeny contains wt pIII from the helper phage to allow infection and the scFv-pIII fusion protein displayed at one end of the phage.

Kipriyanov, S M, Little, M, Generation of recombinant antibodies Mol. Biotechnol. 1999 12(2):173-201

(Halin et al., 2002) or chemokines (e.g. interferon-gamma inducibleprotein 10) (Guo et al., 2004) have shown to significantly enhanceantitumor activity, for example, via infiltration of the tumor mass withlymphocytes, macrophages, natural killer cells and interferon gamma(Halin et al., 2002). Furthermore, redirecting of antigen-specific T-cellactivity, that is non-HLA restricted, has been facilitated by T-bodies, orTcells, armedwith a chimeric receptor comprised of an antigen-specificscFv fused to an intracellular lymphocyte stimulating region (seeSection8.4) (Eshhar, 2008; Westwood et al., 2008).

Conjugation of scFvs to B and T cell superantigens (such protein Lfrom Peptosteptococcus magnus and staphylococcal enterotoxin A,respectively) are other strategies for stimulating local immuneactivation and have been shown to retarget serum Ig, and to recruitcomplement, phagocyte respiratory burst and phagocytosis (Eneveret al., 2005), or to recruit lymphokine-activated killer T-cells andsuppress tumor growth (Ueno et al., 2002). In conclusion, bifunctionalscFv and scFv-based fragments are excellent protein scaffolds,representing a new group of immunotherapeutics, for the targeteddelivery of toxic compounds and/or elicitation of immune cells for thetherapeutic treatment of human disease.

3. scFv generation by molecular display

Several different molecular display formats have been described,including phage-display (McCafferty et al., 1990), ribosome display(Hanes and Pluckthun, 1997; He and Taussig, 1997) and cell-surfacedisplay (Francisco et al., 1993), from which antigen-reactive Abs canbe selected and affinity matured. Each molecular display formatshares steps with Ab generation in vivo such as the generation ofgenotypic diversity, the coupling of genotype with phenotype, clonalselection pressure and amplification. Molecular display libraries arecreated by obtaining and cloning a diverse collection of rAb genes, forexample, from the B lymphocytes of immunized animals, to generate

the library format of choice and provide the phenotypic and genotypiclink (Hoogenboom, 2005). The libraries are screened for targetantigen binding, and enriched pools of binders are amplified aftereach round of selection with the target antigen (Fig. 3). After a fewselection rounds, the clones from the enriched pools are screened forantigen reactivity. Phage-display, ribosome-display andmicrobial cell-surface display are the best established molecular display formats andare described below.

3.1. Phage display

The principle of displaying foreign polypeptides on the surface offilamentous bacteriophage (i.e. phage-display) was first introduced byG. Smith in 1985 (Smith, 1985). Phage-display, the oldest and mostcommonly used molecular display technique, is used to display,enrich, and affinity mature a vast number of proteins and peptidesfrom large libraries with up to 1010 variants. The crucial advantage ofthis technology is the direct link that exists between the experimentalphenotype (the displayed protein) and its encapsulated genotype (theDNA encoding the displayed protein) (Scott and Smith, 1990).

The discovery that functional antibody binding sites (i.e. scFvs) canbe displayed on the surface of bacteriophage has allowed selection ofantibodies against antigens of choice without the need for hybridomatechnology (McCafferty et al., 1990). Phage-displayed scFv librariesconsist of a diverse array of heavy and light chain variable regiondomains that are fused to the phage minor coat protein, pIII encodedby gene III, and displayed externally as scFv (Fig. 3.4A) (Vaughan et al.,1996). These antibody fragments can be expressed rapidly, in largequantities and at a lower cost in bacterial host, such as E. coli,compared to generating whole Abs from animal cell culture. Thediscovery that functional antibody binding sites (i.e. scFvs) can bedisplayed on the surface of bacteriophage has allowed selection ofantibodies against antigens of choice without the need for hybridoma

Fig. 3. Typicalmolecular display selection processing as demonstratedwith the phage displaymethod. Following the cloning of the Ab-display library, the link between the phenotype (i.e.scFv in the shaded blue, red purple or green area) and corresponding genotype (i.e.DNA, wavymatching coloured line being Ab gene with specificity for antigen) is created by packagingin a host (e.g.M13 phage). In phage display, the scFv is displayed on theminor pIII coat protein. Following amplification of the library, the scFv are selected for binding to the target antigen,usually by immobilizing on a solid support. The non-bound phage-displayed Abs are washed away and the bound phage-displayed Abs are eluted and infected into E. coli to amplify thepool of antigen binding Abs. This process, often called panning, is repeated a number of times to enrich for a pool of antigen binding Abs. Afterwards, the enriched pool is screened forantigen binding by e.g. ELISA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

507N.E. Weisser, J.C. Hall / Biotechnology Advances 27 (2009) 502–520


First Ab from phage display library was Humira® approved in 2002, 12 years after the description of the first phage-displayed human antibody library

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!The Human Combinatorial Antibody Library HuCal GOLD

Phage display platform

As nature uses different frameworks as a source for antibody diversity and framework residues also contribute to antigen binding and influence CDR conformation, HuCal was not restricted to one framework sequence but represents, with seven VH and seven VL consensus sequences, the sequence information of each frequently used VH and VL germ-line family

Rothe C, Urlinger S, Löhning C, Prassler J, Stark Y, Jäger U, et al. The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies. J. Mol. Biol. 2008 Feb. 29;376(4):1182–1200.

Since CDR1 and CDR2 regions contribute significantly to antigen binding, a new-generation HuCal Fab library was constructed, designated HuCal GOLD, with all six CDRs diversified to match the amino acid frequencies characteristic of natural antibodies derived from the corresponding germ-line gene


• CDR libraries were synthesized applying the TRIM (trinucleotide mutagenesis) technology, that allowed the introduction of biased amino acid compositions reflecting the situation in rearranged human antibodies

• An in- frame β-lactamase selection strategy to select for clones encoding full-length Fab fragments

• CysDisplay, antibody fragments are displayed on the tip of the phage via a disulfide bridge between the phage coat protein pIII and the heavy chain of the antibody fragment.

Key strategies


CDRs were replaced by large-sized DNA stufferfragments to create vectors for library cloning. Forheavy-chain vectors, the bla gene in CDR-H3 servedas cloning stuffer.The variability within the LCDRs increases from

CDR-L2 to CDR-L1 and CDR-L3 in nature, reflectingthe contribution of the specific CDR to antigenbinding.46 The design and thus the size of theHuCAL GOLD CDR libraries were generatedaccordingly. The order of the CDR library cloningwas from lower variability to higher variability suchthat the diversity of the previously cloned CDRcassettes could be maintained as much as possible inthe subsequent cloning steps. Cloning steps, theore-tical diversity, and actual size of the libraries aredepicted in Fig. 2. Diversification of CDR-L3 was thelast step in the generation of the light-chain librariesand was subjected to selection for in-frame antibodygenes via carbenicillin selection. Carbenicillin wasused as it is supposed to be less susceptible toβ-lactamase degradation than ampicillin.47 The sizeof the kappa libraries was between 3.2×107 and1.5×108, covering the theoretical diversity of 1.6×106 for each of the kappa CDR-L3 cassettes. The sizeof the lambda libraries was between 2.2×107 and1.1×108, not completely covering the theoreticaldiversity of 8.7×109.In parallel, CDR-H1 and CDR-H2 libraries were

inserted into each of the seven HuCAL VH mastergenes containing the β-lactamase gene instead of aCDR-H3 sequence. The natural variability of theHCDRs, increasing from CDR-H1 to CDR-H2 andCDR-H3, with CDR-H3 being the most diverse of all

CDRs, reflects the contribution of the three CDRs toantigen binding.46 As for the light chains, the leastdiverse CDR-H1 libraries were inserted first, fol-lowed by CDR-H2 library cloning and selection forin-frame clones via carbenicillin, to minimize theloss of complexity.The next step was the combination of the heavy-

chain libraries diversified in CDR-H1 and CDR-H2with the light-chain libraries diversified in all threeCDRs. The seven heavy-chain families were keptseparate, but the three Vλ and the four Vκ librarieswere mixed to two light-chain pools. Hence, 14different sublibraries were created, by combin-ing each VH library fragment still carrying theβ-lactamase gene in the CDR-H3 region with thetwo light-chain libraries. This cloning step was againsubjected to carbenicillin selection. The final cloningstep was the insertion of the CDR-H3 libraries. Foreach length ranging from 4 to 22 residues, a separatetrinucleotide cassette had been generated, combinedto three different pools and inserted as three poolsinto the libraries already carrying the diversifiedCDR-L1, -L2, -L3, -H1, and -H2. The VH1A andVH1B libraries had already been combined beforethe final CDR-H3 cloning step. Altogether, a total of3.6×1010 independent clones distributed across 12different sublibraries were obtained.Both the VH and VL regions of 213 clones were

sequenced to analyze the quality of the final library.It was found that 9% of the clones had frameshiftmutations. Only 2% of the frameshift mutationswere located in one of the five CDRs, whosediversification had been controlled by carbenicillin

Fig. 2. HuCAL GOLD cloning procedure. pMOPRH18VH3VL(d)-CLbla contains one of the seven HuCAL light-chainmaster genes (Vλ1/2/3, Vκ1/2/3/4) as VL. pMOPRH18VH(d)Vκ3-CLbla contains one of the seven HuCAL heavy-chainmaster genes (VH1A/1B/2/3/4/5/6) as VH. The restriction enzymes used for CDR library cloning are shown.Theoretical diversity of the CDR libraries is indicated, as well as the actual library size after CDR cassette cloning.Carbenicillin selection was performed at cloning steps marked by an asterisk.

1187Human Combinatorial Antibody Library HuCAL GOLDVλ1/2/3 -- Vκ1/2/3/4!master genes

VH1A/1B/2/3/4/5/6!master genes


The 12 HuCAL sublibraries diversified in all six CDRs and cloned into the conventional display vector pMORPH18 were transferred into the CysDisplay vector pMORPH23

cloned via XbaI and EcoRI into pMORPH23. Thedisplay of corresponding CysDisplay phages wasanalyzed in an anti-pIII Western blot. Furthermore,the above-described VH3-Vλ1–3 library with all sixCDRs diversified was cloned from pMORPH18into pMORPH23 to yield the VH3-Vλ sublibrary ofHuCAL GOLD. The display rate was analyzed aswell (Fig. 5). Wild-type pIII protein was clearlydetectable at around 55 kDa. Even though the truemolecular mass of wild-type pIII is 42 kDa, its largerapparent size in SDS-PAGE iswell known.48 The Fd–pIII heterodimers of around 80 kDa were visibleunder nonreducing conditions and disappearedafter DTT treatment, demonstrating that the Fdchain is linked to the gIII protein via a disulfide bond.Note that the Fab fragments used here are notconnected by an interchain CH–CL disulfide bond,as the light chain does not contain the C-terminalcysteine.27 In addition to the Fd–pIII heterodimers,homodimers of Fd (not shown) as well as pIII aregenerated in the periplasm of Escherichia coli. In theimmunoblot, the disulfide-linked pIII homodimerswere efficiently detected at around 110 kDa by theanti-pIII detection antibody and disappeared afterDTT treatment as expected. The mean number ofantibody fragments displayed per phage was deter-mined by densitometric scanning31 to be 0.1 and0.3 Fabs per phage for the initial VH2-VL and VH3-VL libraries, respectively, and 0.2 for the final VH3-Vλ HuCAL GOLD sublibrary. These data areconsistent with a monovalent display of the Fab

fragments via disulfide bonds. Introduction of acysteine at the N-terminus of pIII had no impact onphage infectivity (data not shown).Binding efficiency and functionality of Fab frag-

ments presented on the phage by conventionaldisplay or by CysDisplay were compared in phageELISA. Phage preparations of two Fab fragments,Mac1–5 and Mac1–A8, expressed either in thetricistronic CysDisplay vector pMORPH23 or inthe conventional dicistronic pMORPH18 vector,were tested for binding to Mac1 i domain in ELISA(Fig. 6). Signals were similar for the pMORPH18 andthe pMORPH23 phage preparations, demonstratingthat functional Fab fragments are efficiently dis-played on phage via disulfide linkage with a phagecoat protein (Fig. 6a). ELISA signals of the CysDis-play phages were significantly reduced by additionof DTT while signals of conventional phages werenot affected (Fig. 6b).

Generation of the HuCAL GOLD library

The 12 HuCAL sublibraries diversified in all sixCDRs and cloned into the conventional display vec-tor pMORPH18 were transferred into the CysDis-play vector pMORPH23. Each sublibrary generatedin pMORPH18 and containing one heavy-chainfamily combined with either the three Vλ or thefour Vκ libraries was excised separately with XbaIand EcoRI and cloned into pMORPH23. Thus,again, 12 different sublibraries were generated,

Fig. 5. Analysis of the displayefficiency in phage Western blot.Phage preparations of the VH3-VλHuCAL GOLD sublibrary in theCysDisplay vector pMORPH23were subjected to an anti-pIII Wes-tern blot analysis. Different amountsof phages (1–7: 1×1011, 5×1010,2.5 × 1010 , 1.3 × 1010 , 6.3 ×109 ,3.1×109, and 1.6×109, respectively)were applied under nonreducing

conditions, as well as the highest amount with the addition of 20 mM DTT (DTT). Signals corresponding to pIII, pIIIhomodimers (pIII–SS–pIII), and Fd–pIII heterodimers (Fd–SS–pIII) aremarked, and bands used for the calculation of displayrate are boxed; molecular mass marker (M) is shown.

Fig. 4. Arrangement of gene III and the antibody light and heavy chain on pMORPH23. Terminal cysteines at gene IIIand the Fd chain are indicated, and the respective signal sequences, gIII-SP, OmpA, and PhoA, are marked. Some of theunique restriction sites are shown.

1189Human Combinatorial Antibody Library HuCAL GOLD


!

Empirical approaches to antibody humanizationPhage display platform

Almagro JC, Fransson J. Humanization of antibodies. Front Biosci. 2008;13:1619-1633.

• In contrast to the rational methods to humanize antibodies, empirical methods rest on the generation of large libraries of humanized variants and selection of the best clones using enrichment technologies

• Empirical methods are dependent on a reliable selection and/or screening system that should be able to search through a vast space of antibody variants

‣ FR libraries

‣ Guided selection

Almagro JC, Fransson J. Humanization of antibodies. Front Biosci. 2008;13:1619-1633.

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