Engineered Antibodies
`
`REVIEW
`
`39
`
`Generation and Production of Engineered Antibodies
`Sergey M. Kipriyanov* and Fabrice Le Gall
`
`Abstract
`Various forms of recombinant monoclonal antibodies are being used increasingly, mainly for therapeutic
`purposes. This review specifically focuses on what is now called antibody engineering, and discusses the
`generation of chimeric, humanized, and fully human recombinant antibodies, immunoglobulin fragments, and
`artificial antigen-binding molecules. Since the production of recombinant antibodies is a limiting factor in their
`availability, and a shortage is expected in the future, different expression systems for recombinant antibodies
`and transgenic organisms as bioreactors are also discussed, along with their advantages and drawbacks.
`Index Entries: Recombinant antibody; humanization; single-chain Fv fragment; phage display; antibody
`library; expression; bioreactor.
`
`1. Introduction
`Nearly three decades ago, Georges Köhler and
`César Milstein invented a means of cloning indi-
`vidual antibodies, thus opening up the way for tre-
`mendous advances in the fields of cell biology and
`clinical diagnostics (1). However, despite their
`early promise, monoclonal antibodies (MAbs)
`were largely unsuccessful as therapeutic reagents,
`owing to their insufficient activation of human
`effector functions and to immune reactions
`against proteins of murine origin. These problems
`have recently been largely overcome through the
`use of genetic engineering techniques with the
`production of chimeric mouse/human and com-
`pletely human antibodies. Such an approach is
`particularly suitable because of the domain struc-
`ture of the antibody molecule, because it enables
`functional domains carrying antigen-binding ac-
`tivities (Fabs or Fvs) or effector functions (Fc) to
`be exchanged between antibodies (Fig. 1).
`On the basis of sequence variation, the residues
`in the variable domains (V region) of the antibody
`molecule are assigned either to the hypervariable
`complementarity-determining regions (CDRs) or
`to framework regions (FRs). It is possible to re-
`
`place much of the rodent-derived sequence of an
`antibody with sequences derived from human im-
`munoglobulins without loss of function. This new
`generation of “chimeric” and “humanized” anti-
`bodies represents an alternative to human hybri-
`doma-derived antibodies, and the new antibodies
`should be less immunogenic than their rodent
`counterparts. Furthermore, genetically truncated
`versions of the new antibodies may be produced,
`ranging in size from the smallest antigen-binding
`unit or Fv through Fab' to F(ab')2 fragments. More
`recently it has become possible to produce totally
`human recombinant antibodies derived either
`from antibody libraries (2) or from single immune
`B-cells (3), or from transgenic mice bearing hu-
`man immunoglobulin loci (4,5).
`
`2. Cloning the Antibody Variable Regions
`Significant progress has been made in the in
`vitro immunization of human B cells (6) and in
`the development of transgenic mice containing
`human immunoglobulin loci (4,7). Recombinant
`DNA technology can also be used for generating
`human MABs from human lymphocyte mRNA.
`The genetic information for antibody V regions is
`
`*Author to whom all correspondence and reprint requests should be addressed: Affimed Therapeutics AG, Technologiepark, Im Neuenheimer
`Feld 582, D-69120 Heidelberg, Germany. E-mail:s.kipriyanov@affimed.com
`Molecular Biotechnology ©2004 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2004/26:1/39–60/$25.00
`
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`Kipriyanov and Le Gall
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`Fig. 1. Domain organization of an IgG molecule. The antigen-binding surface is formed by variable domains of
`the heavy (VH) and light (VL) chains. Effector functions are determined by constant CH2 and CH3 domains. The
`picture is based on the crystal structure of an intact, monoclonal IgG2 anti-canine lymphoma antibody, MAb231
`(Protein Data Bank [PDB] entry 1IGT). The drawing was generated with the RasMac Molecular Graphics, version
`2.7.1 molecular visualization program (R. Sayle, Biomolecular Structure, Glaxo Research and Development,
`Greenford, Middlesex, UK, personal communication).
`
`generally retrieved from total cDNA preparations
`using the polymerase chain reaction (PCR) with
`antibody-specific primers (8,9). As a source of
`immunoglobulin-specific mRNA, one can use hy-
`bridoma cells (10), human peripheral blood lym-
`phocytes (PBL) (2), and even a single human B
`cell (3,11). By using the last of these approaches,
`it is possible to avoid the cumbersome hybridoma
`technology and obtain human antibody fragments
`with the original VH/VL pairing. Single bacterial
`colonies expressing antigen-specific antibody
`fragments can be identified by colony screening
`with antigen-coated membranes (12). Novel high-
`throughput selection technologies allow the
`screening of thousands of different antibody
`clones at a time (13). The appropriate VH/VL com-
`bination for a particular antibody may also be se-
`lectively enriched from a phage-displayed antibody
`library, through a series of immunoaffinity steps
`referred to as “library panning” (14,15).
`
`3. Engineered Monoclonal Antibodies
`3.1. Chimeric Antibodies With Human
`Constant Regions
`The first generation of recombinant MAbs con-
`sisted of rodent-derived V regions fused to human
`constant (C ) regions (Fig. 2). It is thought that the
`most immunogenic regions of antibodies are the
`conserved C domains (16). Because the antigen-
`binding site of the antibody is localized within the
`V regions, the chimeric rodent/human antibody
`molecules retain their binding affinity for an anti-
`gen, and acquire the function of the substituted C
`regions. The human C regions allow more effi-
`cient interaction with human complement-depen-
`dent cytotoxicity (CDC) and antibody-dependent
`cell-mediated cytotoxicity (ADCC) effector
`mechanisms. Rituximab (Rituxan; IDEC Pharma-
`ceuticals, San Diego, CA, and Genentech, Inc.,
`San Francisco, CA) is a chimeric anti-CD20 MAb
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`Engineered Antibodies
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`
`Mouse
`
`Chimeric
`
`Humanized
`
`Fig. 2. Schematic representation of murine, chimeric, and humanized IgG molecules. The murine sequences are
`shown in white and the human sequences are shown in gray. In a chimeric antibody, the mouse heavy- and light-
`chain V region sequences are joined onto human heavy chain and light-chain C regions. In a humanized antibody,
`the mouse CDRs are grafted onto human V-region FRs and expressed with human C regions.
`
`containing the V regions of the CD20-binding
`murine IgG1 MAb, IDEC-2B8, as well as human
`IgG1 and kappa C regions (17,18). Rituximab was
`the first MAb to be approved for therapeutic use
`for any malignancy. Its approval was based on a
`pivotal single-agent trial in patients with indolent
`B-cell lymphoma, in which 166 patients were en-
`rolled from 31 centers in the United States and
`Canada. Administration of Rituximab induced re-
`missions in 60% of patients with relapsed follicu-
`lar lymphomas, including 5–10% complete
`remissions (19).
`As a further step to reduce the murine content
`in an antibody, procedures have been developed
`for humanizing the Fv regions.
`3.2. Antibody Humanization (Reshaping)
`3.2.1. Humanization by CDR Grafting
`CDRs build loops close to the N-terminus of an
`antibody, where they form a continuous surface
`mounted on a rigid “scaffold” provided by the
`framework regions. Crystallographic analyses of
`several antibody/antigen complexes have demon-
`strated that antigen-binding mainly involves this
`surface (although some framework residues have
`also been found to take part in the interaction with
`antigen). Thus, the antigen-binding specificity of
`an antibody is mainly defined by the topography
`and by the chemical characteristics of its CDR
`surface. These features in turn are determined by
`the conformation of the individual CDRs, by the
`
`relative disposition of the CDRs, and by the na-
`ture and disposition of the side chains of the amino
`acids comprised in the CDRs (20).
`A large decrease in the immunogenicity of an
`antibody can be achieved by grafting only the
`CDRs of xenogenic antibodies onto human frame-
`work and C regions (21,22) (Fig. 2). However,
`CDR grafting per se may not result in the com-
`plete retention of antigen-binding properties. In-
`deed, it is frequently found that some framework
`residues from the original antibody need to be pre-
`served in the humanized molecule if significant
`antigen-binding affinity is to be recovered (23,24).
`In this case, human V regions showing the great-
`est sequence homology to murine V regions are
`chosen from a data base in order to provide the
`human framework. The selection of human FRs
`can be made either from human germline consen-
`sus sequences, or are taken from individual hu-
`man antibodies. In some rare examples, simply
`transferring CDRs onto the most identical human
`V-region frameworks is sufficient for retaining the
`binding affinity of the original murine MAb (25).
`However, in most cases, the successful design of
`high-affinity, CDR-grafted antibodies requires
`that key murine residues be substituted into the
`human acceptor framework to preserve the CDR
`conformations. Computer modeling of the anti-
`body is used to identify such structurally impor-
`tant residues, which are then included in order to
`achieve a higher binding affinity. The process of
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`42
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`Kipriyanov and Le Gall
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`identifying the rodent framework residues to be
`retained is generally unique for each reshaped an-
`tibody, and can therefore be difficult to foresee.
`To minimize the immunogenicity of humanized
`antibodies, it has been proposed to graft only resi-
`dues involved in the canonical loop structures
`onto human germline scaffolds (26), or to replace
`murine CDRs not involved in antigen binding by
`human counterparts (27). For abolishing anti-
`idiotypic responses, grafting onto the human
`frameworks only the “abbreviated” CDRs—the
`stretches of CDR residues that contain the speci-
`ficity-determining residues essential for the sur-
`face complementarity of the antibody and its
`ligand—has been proposed (28).
`CDR grafting was successfully used for hu-
`manizing a MAb 4D5 against the product of
`protooncogene HER2 (29). HER2 is a ligandless
`member of the human epidermal growth factor re-
`ceptor (EGFR) or ErbB family of tyrosine kinases.
`Overexpression of HER2 is observed in a number
`of human adenocarcinomas and results in consti-
`tutive activation of HER2. Specific targeting of
`these tumors can be accomplished with antibod-
`ies directed against the extracellular domain of
`the HER2 protein. MAb 4D5 has been fully hu-
`manized and is termed trastuzumab (Herceptin;
`Genetech, San Francisco, CA). Treatment of
`HER2-overexpressing breast cancer cell lines
`with trastuzumab results in a number of pheno-
`typic changes, such as downmodulation of the
`HER2 receptor, inhibition of tumor cell growth,
`reversed cytokine resistance, restoration of E-
`cadherin expression levels, and reduced production
`of vascular endothelial growth factor. Interaction
`of trastuzumab with the human immune system,
`via the human IgG1 Fc domain of the antibody
`may potentiate its anti-tumor activities. In vitro
`studies demonstrate that trastuzumab is very ef-
`fective in mediating antibody-dependent cell-me-
`diated cytotoxicity against HER2-overexpressing
`tumor targets (30). Trastuzumab treatment of
`mouse xenograft models results in marked sup-
`pression of tumor growth. When given in combi-
`nation with standard cytotoxic chemotherapeutic
`agents, trastuzumab generally results in statisti-
`cally superior antitumor efficacy compared to that
`
`with either agent given alone (30). Clinical data
`have demonstrated the safety and efficacy profile
`of trastuzumab in patients with metastatic breast
`and ovarian cancer (31,32).
`3.2.2. Humanization by Resurfacing (Veneering)
`A statistical analysis of unique human and mu-
`rine immunoglobulin heavy- and light-chain V
`regions revealed that the precise patterns of ex-
`posed residues are different in human and murine
`antibodies, and that most individual surface posi-
`tions have a strong preference for a small number
`of different residues (33, 34). Therefore, it may be
`possible to reduce the immunogenicity of a non-
`human FV, while preserving its antigen-binding
`properties, by simply replacing exposed residues
`in its framework regions that differ from those
`usually found in human antibodies. This would
`humanize the surface of the xenogenic antibody
`while retaining the interior and contacting resi-
`dues that influence its antigen-binding character-
`istics and interdomain contacts. Since protein
`antigenicity can be correlated with surface acces-
`sibility, replacement of the surface residues may
`be sufficient to render the mouse V region “invis-
`ible” to the human immune system. This proce-
`dure of humanization is referred to as “veneering”
`since only the outer surface of the antibody is al-
`tered, with the supporting residues remaining un-
`disturbed (35).
`Resurfacing in the V domain maintains the core
`murine residues of the Fv sequences and probably
`minimizes CDR-framework incompatibilities. This
`procedure was successfully used for the humaniza-
`tion of murine MAb N901 against the CD56 sur-
`face molecule of natural killer (NK) cells, and of
`MAb anti-B4 against CD19 (25,36). A direct com-
`parison of engineered versions of N901 humanized
`either by CDR grafting or by resurfacing showed
`no difference in binding affinity of the two versions
`for the native antigen (25,34). For the anti-B4 anti-
`body, the best CDR-grafted version required three
`murine residues at surface positions to maintain
`binding, while the best resurfaced version needed
`only one surface murine residue (25). Thus, even
`though the resurfaced version of anti-B4 has 36
`murine residues in the Fv core, it may be less im-
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`munogenic than the CDR-grafted version with nine
`murine residues in the Fv core, because it has a pat-
`tern of surface residues that is more identical to a
`human surface pattern.
`3.2.3. Deimmunization
`Deimmunization technology (www.biovation.
`co.uk) involves the identification and removal of
`T helper (Th) cell epitopes from antibody and pro-
`tein biologics. Th-cell epitopes consist of short
`peptide sequences within proteins, with the capac-
`ity to bind to major hsitocompatibility (MHC)
`class II molecules. The peptide–MHC class II
`complexes can be recognized by T cells and can
`trigger the activation and differentiation of Th
`cells, which are required to initiate and sustain im-
`munogenicity through interaction with B cells,
`thereby resulting in the secretion of antibodies that
`bind specifically to the administered biologic. For
`antibody deimmunization, the Th-cell epitopes are
`identified within the antibody sequence by a com-
`puter-based method for predicting the binding of
`peptides to human MHC class II molecules (37,
`38). The found Th cell epitopes are eliminated
`from the antibody sequence by amino acid substi-
`tutions to avoid recognition by T cells. In this way,
`the modified antibody can circumvent both human
`antimouse antibody (HAMA) and antiidiotypic
`responses.
`3.3. Choice of Constant Region
`The construction of chimeric and humanized
`antibodies offers the opportunity of tailoring the
`C region to the requirements of the antibody. IgG
`is the preferred immunoglobulin class for thera-
`peutic antibodies, for several practical reasons.
`IgG antibodies are very stable and are easily puri-
`fied and stored. In vivo, they have a long biologi-
`cal half-life that is not just a function of their size,
`but is also a result of their interaction with the neo-
`natal or Brambell receptor (FcRn), preventing
`their proteolysis in endothelium (39). This recep-
`tor seems to protect IgG from catabolism within
`cells, and recycles it back to the blood plasma. In
`addition, IgG has subclasses that can interact with
`and trigger a whole range of humoral and cellular
`effector mechanisms. Each immunoglobulin sub-
`
`class differs in its ability to interact with Fc recep-
`tors and complement, and thus to trigger cytolysis
`and other immune reactions. Human IgG1, for
`example, would contain the H region of choice for
`mediating both ADCC and CDC (40,41). On the
`other hand, if the antibody were required simply
`to activate or block a receptor, then human IgG2
`or IgG4 would probably be more appropriate. For
`example, the humanized versions of the immuno-
`suppressive antihuman CD3 MAb OKT3 were
`prepared as IgG4 antibodies (42).
`However, all four human IgG subclasses medi-
`ate at least some biological functions. To avoid
`the unwanted side effects of a particular isotype,
`it is possible to remove or modify effector func-
`tions by genetic engineering. For example, amino
`acid substitutions in the CH2 portion of an anti-
`CD3 antibody led to the retention of its immuno-
`suppressive properties, but markedly reduced the
`unwanted biological side effects associated with
`Fc receptor binding (43–45). An alternative strat-
`egy has been described whereby potent blocking
`antibodies could be generated by assembly of the
`CH2 domain from sequences derived from the
`IgG1, IgG2, and IgG4 subclasses (46).
`3.4. Alternative Strategies for Generation
`of Human Antibodies
`Other strategies for the production of fully hu-
`man antibodies include the use of phage display
`antibody libraries (47,48) or transgenic animals
`(4,7,49,50), both utilizing human V-region rep-
`ertoires.
`3.4.1. Animals Making Human Antibodies
`Several strains of mice are now available that
`have had their mouse immunoglobulin loci re-
`placed with human immunoglobulin gene segments
`(5,51,52). The early mouse strains transgenic for
`human IgH and Igκ miniloci produced mixtures
`of human and murine antibodies. Inactivation of
`the murine IgH and Igκ loci through gene target-
`ing solved this problem. The megabase size of
`many human genes, including the human germline
`IgH, Igκ ,and Igλ loci, drove the development of
`techniques for the introduction of transgenes on
`yeast artificial chromosomes (YACs) into the
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`Kipriyanov and Le Gall
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`mouse germline (51). Transgenic mice can pro-
`duce functionally important humanlike antibod-
`ies, with very high affinities, after immunization.
`Cloning and production of these antibodies can be
`achieved with the usual hybridoma technology.
`For example, high-affinity human MAbs obtained
`against the T-cell marker CD4 are potential thera-
`peutic agents for suppressing adverse immune ac-
`tivity (51). Another human MAb with an affinity
`of 5 × 10–11 M for human EGFR, was able to pre-
`vent the formation of and eradicate human epider-
`moid carcinoma xenografts in athymic mice (53).
`Further advances included the development of
`human artificial chromosome (HAC) vectors and
`the introduction of large human chromosome frag-
`ments into mice, presumably enabling the transfer
`of entire IgH and Igκ loci (54). Breeding of these
`mice with mice having inactivated IgH/Igκ loci
`generated double-transchromosomal/double-
`knockout (TC) mice. The TC mice reportedly
`have levels of circulating IgG1, IgG2, IgG3, and
`IgG4 resembling those in human serum (5). These
`mice also make human Cμ and Cα chains, but be-
`cause the IgM and IgA antibodies generated from
`the mice contain a murine J chain, they cannot be
`considered fully human. During affinity matura-
`tion, the antibodies from transgenic mice accumu-
`late somatic mutations in both their FRs and CDRs
`(52). This means that they are no longer 100% iden-
`tical to inherited human germline genes, and can
`therefore be potentially immunogenic in humans
`(55). Besides this, “human antibodies” from mice
`can be distinguished from human antibodies pro-
`duced in human cells through their state of glyco-
`sylation, particularly with respect to their
`Galα1–3Gal residue, against which human serum
`contains IgG antibody titers of up to 100 μg/mL.
`It has been argued that an antibody containing
`such residues would not survive very long in the
`human circulation (56).
`Recently, the HAC vector containing the entire
`human IgH and Igλ loci was introduced into bo-
`vine primary fetal fibroblasts through the use of a
`microcell-mediated chromosome transfer ap-
`proach. After the primary selection, the trans-
`chromosomal cells were used to produce cloned
`bovine fetuses that gave rise to four healthy trans-
`
`chromosomal calves (50). The generation of trans-
`chromosomal calves is an important step in the
`development of a system for producing of thera-
`peutic human polyclonal antibodies.
`3.4.2. Human Antibodies From Phage
`Libraries
`Rapid growth in the field of antibody engineer-
`ing occurred after it was shown that functional
`antibody fragments could be secreted into the
`periplasmic space and even into the medium of
`Escherichia coli by fusing a bacterial signal pep-
`tide to the N-terminus of an antibody (57,58).
`These findings opened the way for transferring
`into a bacterial system, principles by which the
`immune system produces specific antibodies to a
`given antigen. It was now possible to establish
`antibody libraries in E. coli that could be directly
`screened for binding to antigen.
`In order to screen large antibody libraries con-
`taining at least 108 individual members, it was
`necessary to develop a selection system as effi-
`cient as that of the immune system, in which the
`receptor of an antibody is bound to the surface of
`a B lymphocyte. After the receptor binds its anti-
`gen, the B lymphocyte is stimulated to proliferate
`and mature into an IgG-producing plasma cell. A
`similar selection system could be imitated in mi-
`croorganisms by expressing antibodies on their
`surface. Millions of microorganisms could then be
`simultaneously screened for binding to an immo-
`bilized antigen, followed by propagation and
`amplification of the selected microorganism. Al-
`though methods have been developed for protein
`display on the surface of different microorgan-
`isms, (e.g., retroviruses [59], baculoviruses [60],
`yeasts [61,62], bacteria [63], and even cell-free
`ribosome display and mRNA display technologies
`[64]), the most successful surface expression sys-
`tem was created by using filamentous bacterioph-
`ages of the M13 family (65). The phage display
`was originally reported for scFv fragments (66),
`and later for Fab fragments (67) and other anti-
`body derivatives such as diabodies (68). It became
`possible to generate antibody libraries by using
`the polymerase chain reaction (PCR) to clone the
`large collections of V-region genes, expressing
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`
`Fab
`
`Fv
`
`scFv
`
`VL VH
`
`VL VH
`
`VL
`
`VH
`
`~ ~ ~
`~ ~ ,
`
`CL CH1
`
`dsFv
`
`VH VL
`
`knob-into-hole
`Fv
`VH VL
`
`VH
`
`each of the binding sites on the surface of a differ-
`ent phage particle, and selecting the antigen-spe-
`cific binding sites by in vitro screening of the
`phage mixture with a chosen antigen. The phage
`display technology could be used to select anti-
`gen-specific antibodies from libraries made from
`human B cells taken from individuals immunized
`with the antigen (69), exposed to infectious agents
`(70), or having autoimmune diseases (2), or can-
`cer (71). Moreover, it was shown that antibodies
`against many different antigens could be selected
`from a “naïve” binding-site library, prepared from
`the VL and VH IgM-V-gene pools of B cells of
`nonimmunized healthy individuals (15,72). It was
`also shown that libraries of synthetic antibody
`genes based on human germline segments with
`randomized CDRs behave in a manner similar to
`“naïve” antibody libraries (73,74). It therefore
`became possible to use primary (naïve or syn-
`thetic) antibody libraries with huge collections of
`binding sites of different specificity for the in vitro
`selection of human antibody fragments against
`most antigens, including nonimmunogenic mol-
`ecules, toxic substances, and targets conserved
`between species (48,75).
`However, for some therapeutic applications
`whole IgGs are the preferred substances because
`of their extended serum half-life and ability to
`trigger humoral and cellular effector mechanisms.
`This necessitates recloning of the phage-display-
`derived scFvs or Fabs into mammalian expression
`vectors containing the appropriate C domains, and
`establishing cell lines for expressing the encoded
`antibodies. The specificity and affinity of the an-
`tibody fragments are generally well retained by
`the whole IgG, and in some cases the affinity may
`significantly improve because of the bivalent na-
`ture of the IgG (76). In the past few years, about a
`dozen phage-derived antibodies have begun clini-
`cal trials (77).
`4. Recombinant Antibody Fragments
`The Fv fragment, consisting only of the VH and
`VL domains, is the smallest available immunoglo-
`bulin fragment that carries the whole antigen-bind-
`ing site (see Fig. 1). However, Fvs appear to have
`lower energies of interaction of their two chains
`
`Fig. 3. Monovalent immunoglobulin fragments.
`Fab, Fv, disulfide-stabilized Fv (dsFv), and Fv frag-
`ments with a remodeled VH/VL interface (knob-into-
`hole Fv) consist of two separate chains, while the
`single VH domain and single-chain Fv (scFv) frag-
`ments are made from a single gene.
`
`than do Fab fragments, which are held together by
`the C domains CH1 and CL. To stabilize the asso-
`ciation of the VH and VL domains, they have been
`linked with peptides (78,79), disulfide bridges (80),
`and “knob-into-hole” mutations (81) (Fig. 3).
`4.1. Monovalent Antibody Fragments
`4.1.1. Single Chain Fv Fragments (scFv)
`Peptide linkers of about 3.5 nm are required to
`span the distance between the carboxy terminus
`of one domain of an antibody molecule and the
`amino terminus of the other (79). Both orienta-
`tions, VH-linker-VL or VL-linker-VH, can be used.
`The small scFvs are particularly interesting for
`clinical applications. They are only half the size
`of Fabs and thus have lower retention times in
`nontarget tissues, more rapid blood clearance, and
`better tumor penetration. They are also potentially
`less immunogenic and are amenable to fusions
`with proteins and peptides.
`Single-chain Fv antibody fragments produced
`in bacteria provide new possibilities for protein
`purification by immunoaffinity chromatography.
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`Kipriyanov and Le Gall
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`Their advantages include lower production costs,
`a higher capacity for antigen on a weight basis,
`and better penetration in a small-pore separation
`matrix. Such recombinant immunosorbents have
`proved useful for the one-step purification of a
`desired antigen from complex protein mixtures
`(82,83). Another interesting possible application
`of the scFv antibody fragments is for the purifica-
`tion or separation of toxic compounds, which can-
`not be used for the immunization of animals, with
`antibodies selected from phage-displayed anti-
`body libraries.
`4.1.2. Disulfide-Stabilized Fv Fragments (dsFv)
`Another strategy for linking VH and VL do-
`mains has been to design an intermolecular disul-
`fide bond (Fig. 4). Such a disulfide-stabilized (ds)
`Fv fragment appeared to be much more resistant
`to irreversible denaturation caused by storage at
`37°C than was the unlinked Fv. It was more stable
`than both the scFv fragment and a chemically
`crosslinked Fv (80). The two most promising sites
`for introducing disulfide bridges appeared to be
`VH44–VL100, connecting FR2 of the heavy chain
`with FR4 of the light chain, and VH105–VL43,
`which links FR4 of the heavy chain with FR2 of
`the light chain (84). Recently, RFB4(dsFv)–PE38
`(BL22), a recombinant immunotoxin containing
`an anti-CD22 dsFv fused to truncated Pseudomo-
`nas exotoxin (PE), showed high efficacy in the
`treatment of chemotherapy-resistant hairy-cell
`leukemia (85).
`4.1.3. Single Antibodylike Domains
`In some cases, the single antibody V domains
`(VH or VL) alone are able to bind antigen. For ex-
`ample, antigen-binding VH domains were isolated
`from the lymphocytes of immunized mice (86).
`Unfortunately, the poor solubility of these murine
`domains, their reduced affinity for antigen, and
`the irreproducible outcome showed that additional
`protein engineering would be required to success-
`fully generate single-domain antibody fragments.
`Fortunately, such engineering is continuously per-
`formed in nature. Part of the humoral immune re-
`sponse of camels and llamas is based largely on
`heavy-chain antibodies in which the light chain is
`totally absent. These unique antibody isotypes
`
`(scFv' '2
`
`diabody
`
`triabody
`
`miniantibody
`
`minibody
`
`scFv-streptavidin
`
`Fig. 4. Schematic representation of multivalent re-
`combinant antibody constructs. (scFv')2 is formed by
`covalent linking of two unpaired cysteine residues. Ap-
`pearance of the non-covalent scFv dimer (diabody), tri-
`mer (triabody), and tetramer (tetrabody), respectively,
`depends on the length of the linker between VH and VL
`domains and on the stability of VH/VL associations. The
`miniantibody, minibody, and scFv–streptavidin oligo-
`mers are formed through the adhesive self-associating
`peptide or protein domains (leucine zipper-derived
`amphipathic helix, CH3, streptavidin). The antibody V
`domains (VH, VL), peptide linkers (L), intermolecular
`disulfide bond (S–S) and antigen-binding sites (Ag) of
`Fv modules are indicated.
`
`interact with antigen through only one single V do-
`main, referred to as VHH (87). Despite the absence
`of the VH–VL combinatorial diversity, these heavy-
`chain antibodies exhibit a broad antigen-binding
`
`MOLECULAR BIOTECHNOLOGY
`
`Volume 26, 2004
`
`

`

`Engineered Antibodies
`
`47
`
`repertoire by enlarging their hypervariable re-
`gions. Analogously, the new antigen receptor
`(NAR) of sharks consists of a single immunoglo-
`bulin V domain attached to five C domains, and is
`hypothesized to function as an antibody (88). Both
`camelid VHH and NAR variable domains are suit-
`able scaffolds for constructing protein libraries
`with randomized CDR loops that can be used for
`the design and selection of single-domain binding
`and targeting reagents (89). The antigen-binding,
`solubility, and stability of human VH domains can
`also be improved by mimicking camelid VHH se-
`quences (90).
`In addition, other nonantibody proteins with a
`single fold have been engineered for new specific-
`ity, including a three-helix bundle domain derived
`from the IgG-binding staphylococcal protein A
`(affibody [91]), the α-amylase inhibitor tendam-
`istat (92), the tenth fibronectin type III domain
`(93), lipocalins (94), the extracellular domain of
`CTLA-4 (95), and even green fluorescent protein
`(Dr. A. Bradbury, Los Alamos National Labora-
`tory, personal communication). Potential advan-
`tages of such single-domain binding molecules
`might be their easy production, enhanced stabil-
`ity, targeting of certain antigen types (e.g., ligand-
`binding pockets of receptors), and rapid
`engineering into multimeric or multivalent re-
`agents. However, it appears that not all kinds of
`protein scaffold that may appear attractive for the
`engineering of loop regions will indeed permit the
`construction of independent ligand-binding sites
`with high affinity and specificity. Nevertheless,
`such single immunoglobulin folds and other arti-
`ficial binding sites might eventually become ma-
`jor competitors for antibodies in many of todays
`antibody applications (96).
`4.2. Bivalent and Multivalent Fv Antibody
`Constructs
`One disadvantage of scFv antibody fragments
`is the monovalency of the products made from
`them which precludes an increased avidity from
`polyvalent binding. Several therapeutically im-
`portant antigens have repetitive epitopes, result-
`ing in a higher avidity for antibodies and antibody
`fragments with two or more antigen-binding sites.
`
`Another drawback of scFv fragments is their small
`size, resulting in their rapid clearance from the
`blood through the kidneys. Recently, attention has
`focused on the generation of scFv-based mol-
`ecules with molecular weights in the range of the
`renal threshold for first-pass clearance. In one ap-
`proach, bivalent (scFv')2 fragments have been pro-
`duced from scFv containing an additional
`C-terminal cysteine, through the use of chemical
`coupling (97) or directly in the periplasm of E.
`coli, by the spontaneous site-specific dimerization
`of scFv containing an unpaired C-terminal cys-
`teine (98) (Fig. 4). Affinity measurements dem-
`onstrated that covalently linked (scFv')2 fragments
`have binding constants quite close to those of the
`parental monoclonal antibodies, and four fold
`higher than those of scFv monomers (98). In vivo,
`bivalent (scFv')2 fragments demonstrated longer
`blood retention and greater intratumoral accumu-
`lation than did scFv monomers (97).
`Alternatively, scFv fragments