395
Recombinant antibody fragments
Peter J Hudson
Recombinant antibodies and their fragments now represent
over 30% of all biological proteins undergoing clinical trials,
which recently culminated in FDA approval for the first
engineered cancer therapeutic antibody. Other successful
applications include both the effective enhancement of the
human immune response for cancer therapy and the reduction
of unwanted immune rejections following transplantation and
antibody therapy. Important advances have been made in the
methods for design, selection and production of these new
types of engineered antibodies. Innovative selection methods
have enabled the isolation of catalytic antibodies and high-
affinity viral-specific antibodies, the latter capable of
redirecting viruses for gene therapy applications. In numerous
practical applications, recombinant antibody fragments have
been fused to radioisotopes, drugs, toxins, enzymes and
biosensor surfaces.
Addresses
CRC for Diagnostic Technologies and CSIRO Molecular Science,
343 Royal Parade, Parkville,Victoria, Australia 3052;
e-mail: peter.hudson@molsci.csiro.au
URL: http://www.crc.sci.qut.edu.au and http:\\www.molsci.csiro.au
CurrentOpinionin Bioteehnology1998, 9:395-402
http://biomednet.com/elecref/0958166900900395
:c..Current Biology Publications ISSN 0958-1669
Abbreviations
CDR complementarity-determining region
Fc constantregion fragment
Fv variable region fragment
Ig immunoglobulin
scFv single chain Fv
V variable region
VH heavy chain variable region
VL light chain variable region
Introduction
A new wave of antibody fragments arc abot, t to enter the
clinic, both for diagnosis and for therapy, and the term
"cnginccrcd antibodies' will be embraced by the general
public for the first time [l°-3°]. Because it takes a decade
to dcvclop a new thcrapeutic reagent into a viable com-
mcrcial product, many of these new antibodies are con-
sidcrcd primitive designs that were conceived 10 ycars
ago. Soinc are based on fusion of murine variable region
fragments (Fvs; complcx of hcaxv chain variable region
(Vii) and light chain variable region (VI) domains) onto
human frameworks [2"]; others are cleverly designed
rcsurFacing of murinc antibodies, for example, by grafting
murine C I ) R loops onto a human antibody framcwork
[3",4"',5]. Technology for antibody design has taken
cnorn~o/is strides forward and new library display and
selection procedures have made classic hvbridoma tech-
nology obsolete [6"°,7,8]. It in now possible to select
hi~h-afTinitv antibody fraemcnts directly from a viral
culture rather than from a live mouse. One significant
advantage of thin new technology is the isolation of anti-
bodies with new binding spccificities against hitherto
rcfractorv antigens, thereby avoiding the limitations inher-
ent in the mammalian immune response. Antibodies are
still the paradigm for the design of high-affinit> protein-
based binding rcagents [4"',9",10,11]. In the past year,
antibodies have bccn rcduccd in size, dissected into min-
imal binding fragments, and rebuilt into multivalcnt
high-axiditx/ reagents [9",12,13"]. Antibody fragments
have also bccn flised with a range of molecules limited
only by the imagination, including enzymes for prodrug
therapy, toxins for cancer targeting, viruses for gene ther-
apy, lipids for improved systemic delixery and biosensors
for rcal-timc detection of target nmlecules. Clinical diag-
nostic applications of antibody fragments includc thc full
range of i# vitro immunoassavs (30% of thc 10 billion pa
diagnostic industry) through to iH vivo tt, mour and clot
imaging reagents. This revicw dcscribcs many of the
publications in the past 12 months and highlights devel-
opments in the design, production and clinical applica-
tions of rccombinant antibody fragments
Design of ' a n t i b o d y f r a g m e n t s ' and
i m p r o v e m e n t s in t h e 'antigen binding surface'
T h e Fv module comprises aligned antibody \:tt and VI.
domains and in the smallest fragment that retains the full
monovatent binding affinity of the intact parent antibody
(Figure 1). Fvs cannot be easily produced by proteolysis
of intact immunoglobulinn (Igs) and so recombinant sin-
gle-chain Fv (scFv) molecules, in which the Vll and VI.
domains arc tcthcred together with a polypeptide linker,
arc now uscd routinely to mimic Fv fragn)ents. T h e
polypeptide linker must span at least 35 A (3.5 nm)
between the carbox\ terminal of one variable region (\7)
domain to the amino terminal of the other V domain
~ithout compromising the fidclity of the V I t--\7l~ pairing
and antigcn-binding site. T h e 15 residue hydrophilic
s c q u c n c c (Glv4Ser).~ is usuall\ chosen for the linker [13"1,
but the length can xar\' between 10 and 25 amino acid
residues or be optimised b \ phagc-display [14]. T h e
amino-termini of both VII and VI. tire near to, but not part
oil the protein-binding interface, so there in little to
choose between the t~vo possible orientations, V li-Vl, or
ViTVtl. ScFxn gcncrally rctain the same affinity an the
parent Fv module and express to higher lcxcls than Fabs
in bacteria. Both ncFxn and Fabs can be expressed to over
1 g per litre using fcrmcntcrn [1°], and usually incorpo-
rate a carboxv-terminal polypeptide tail for affinity
purification [13"]. ScFv and Fab fragments provide cffec-
tixc and highly spccific iJl vivo targeting reagents [10]
and, unless conjugated to constant rcgion fragment (Fc)
domains, will not recruit the c o m p l e m e n t cascade and
related cvtotoxic functions.
396 Protein engineering

Current dogma states that the antigen-binding surface is
comprised of six C D R loops and, therefore, most of the
designed mutations for improving; affinity ha~e been tar-
gctcd to these (]DR loops or the adjacent underlying
rcsidues [10,15,16,17"]. Some of these mutants ha~e spec-
tacular affinity enhancements, cvcn cnabling the produc-
tion of an efficient catalytic antibody capable of
prcforming new aldol reactions [17°]. Perhaps the only dis-
appointmcnt has been that desig;ned interface mutations
[11] have been less effective than random mutation and
sclcction using; phage display [10,15,16,17"]. Part of thc
problem is that we do not yet know how to design com-
plementary surfaces precisel> T h e effect of solvating
buried water molecules is still in debate and it is uncertain
whether solvation of buried water molecules increases or
decreases entropy A rigorous analysis of antibody surfaces
shows that interfaces are complementary in electrostatic
potential, but not in charge complementarit% as one would
expect [18]. T h e s e reports shiny that u c arc still primitive
in our ability to design specific interface mutations that
will restllt in affinity cnhancenlent, even using relatively
high-resohition structural analysis of both the free and
antigen-bound antibody. Ftirthernlore, despite the surprise
finding that camelids produce antibodies that display high-
affinity Vlt domains rather than the F'x module, and which
therefore bind to tbeir target antigens using just three
( ; D R loops [19], most attempts to produce a similar mini-
rnal binding domain have been unsuccessfiil [12]. Perhaps
the large loop size in camelid V domains, stabilised b \ an
intra-loop distllfide bond, is a critical component in pro-
viding a sufficiently large antig;cn-binding surface [191.
Stabilised Fv molecules and improved
expression levels
T h e crystal structure of a higb-affinity somatically n~aturcd
murinc Ig (solved as a crystallised Fab fragment), whcn

Figure 1

VH L VL
I I

Fab Fv scFv

VH VL
VH L VL I

Triabody = scFv trimer

Diabody = scFv dimer

TBR T8R

LI )L3

V H Lt V L L2 VH L3 VL
VH L1 VL L2
I I I

Two scFvs joined by linker L2 Two scFvs joined by adhesive L2 domain

Current Opinion in Biotechnology

Schematic representation of an intact Ig together with Fab and Fv fragments and the recombinant forms for scFv monomers, dimers (diabodies),
trimers (triabodies) and two other types of scFv dimers. Shown are both the gene construct and a hypothetical V-domain association for each
arrangement of V H and V L domains joined by a polypeptide linker (L). The target binding region (TBR) represents the antigen or hapten binding
sites in the Fv modules. V-domains are represented by ovals (V H is shaded grey with an amino-terminal dot) and linkers are represented as a black
line. The linker L2 can be either an adhesive polypeptide or protein domain.

compared to the low-affinity parent germline Ig, showed
that the key mutations responsible for the affinity enhance-
ment were in the VIt-\:[, interface [20"]. Significant
changes in the conformation of the binding site occur upon
binding of hapten to the germline antibod>; whereas hap-
ten binds to the mature antibody by a lock-and-key fit
mechanism. T h e effect of stabilised and optimally aligned
V domains was to reduce entropy loss upon antigen binding
and significantly enhance affinity 30,000-fold [20°]. This
observation extends the concept of the original 'interf:ace
adaptor hypothesis' [21], that is, V domains are designed to
function as semi-flexible adaptors, allowing antigen to
l-aould slightly different C D R surfaces by realignment of
the two domains. Somatic mutation iH ~i~,o can then sta-
bilise the conformation with optimal hapten complemen-
tarity. T h e effect of these observations is to expand the
problem of designing targeted mutations for affinity
enhancement because the sequence search now inch]des
mutations in the V-domain interfaces. Indeed, a number of
recent studies have identified key residues in both the
interface and framework regions that improve folding and
stability and, thcrcb> enhance expression levels [22-26].
For example, Carter and co-workers [4"°,25] designed com-
plementary surfaces to enhance \'-domain association and
described these as 'knobs-into-holes' engineering. Fv frag-
ments can also be stabilised by chemical cross-linking or
engineering of disulfide bonds between chains [26,27]. "Ib
select for optimal V-domain aligmnents and Fv stabilit>
novel phage selection me-tffods were developed [8,25],
including a strategy of Fv fragment stabilisation in the pres-
ence of antigen [28]. In this latter method, VII and VI.
domains are displayed separately followed by an antigen
selection strategx/ that favours high-affinity Fv molecules
with strong \:tt-VI, pairing.
Intracellular antibodies fl)r gene or protein-targeted thera-
pies have now been designed without intradomain disul-
fides to improve the folding efficiency in the reducing
environment of the cytoplasm [29-31].
Engineered valency and flexibility in antibody
fragments
Intact antibodies are polyvalent molecules (Figure 1) with
generally enhanced functional affinity (avidity) relative to
mono~ alent Fab fragments, particularly in the case of pen-
tamcric IgM molecules. T h e r e is, therefore, a similar
increase in functional affinity when Fab or scFv fragments
arc complcxed into dimers, trimers or larger aggregates
[9"]. Flexibility between antigen-binding sites is impor-
tant; in intact lgs the 'elbow' angle within Fab arms and
'hingc' angles between arms allow significant flexibility,
and numerous cross-linking geometries [32]. Despite this
flcxibilitx; intact Igs are often not able to cross-link adja-
cent receptors on the same viral or cell surface [33,34].
Recently, cross-linked homodimers of whole lgs have been
sho~ n to cross-link adjacent receptors on the same cell sur-
face, and thereby activate intracellular signalling and apop-
tosis [34]. Manv cancer cell specific Igs, which are
RecombinantantibodyfragmentsHudson 397
currently sitting idly in lab freezers, are predicted to have
significantly improved therapeutic value when engineered
into flexible structures capable of cross-linking receptor
molecules [34]; however, cross-linked Igs are probably too
large (300 kDa) for effective turnout penetration. For these
reasons (high a~iditv and effective target cross-linking),
there have bccn many attempts to conjugate Fab or scFv
molecules into dimers or higher multimers (Figure 1) to
produce high-avidity reagents of t'~0-120 kDa in size, capa-
ble of rapid tumour penetration without fast kidney clear-
ante. T h e s e designs have included chemical cross-linking
strategies and a variety of recombinant fl, sions using adhe-
sive protein domains or peptides [9°]. Perhaps the simplest
design is a diabody [13"], in which a short 5-residue linker
b e t w e e n V domains prevents formation of aligned
V domains into a functional Fv module and instead results
in combination of the V domains of two scFv molecules
into a bivalent dimer. Recently, a surprising resuh showed
that reduction of linkers to less than three residues pre-
vents the formation of a diabodv and instead directs three
scFv moleculcs to associate into a trimer (triabody; see
Figure 1) with three flmctional antigen binding sites
[13",35,36]. T h e gain in functional affinity through multi-
valent binding (often termed avidity; [9"]) makes trimeric
scFvs particularly attractive for iH ~,i~,o tumour imaging as
an altemative reagent to diabodies [37 °] and multivalent
chemical conjugates [38].
Of course, multiple binding to surface-bound antigens is
dependent on correct alignment and orientation in the Fv
modules of diabodies and triabodies, otherwise apparent
gains in functional affinity are likely to be small. Diabodies
and triabodies have been shown to be relatively flexible
molecules from the orientation of antigen-binding sites
revealed in single-molecule EM images [39]. T h e effect of
manipulating linker length, sequence and structure on dia-
body and ttiabody stability and flexibility can now be
analysed using single-molecule imaging. T h e ability of tri-
abodies to cross-link surface receptors is unknown, and
will obviously depend on flexibility between the F~ mod-
ules and the orientation of the antigen-binding sites, as
well as the structure of the receptor.
Cancer imaging studies using multivalent
antibody fragments
I;; ~'i~,o cancer imaging data has shown that radiolabelled
diabodies (60 kDa), minibodies (dimers of s c F v - C H 3
fusions: 90 kDa) and dimerised Fabs (120 kDa) all exhibit
improved tun3our targeting, as measured by tumour-to-
blood ratios, compared to monomeric scFv (30 kDa), Fab
(60 kDa) and parcnt bivalent Ig (150 kI)a) [10,27,37°,38].
Monovalent antibody fragments (Fab and scFv) clear
rapidly from the blood due to their small size and have rcl-
ativcly high off rates due to the single binding site [10,37°].
On the other hand, whole Igs appear to be too large for
effective tumour penetration [37°]. Two recent studies
[10,37 °] concur that the higher flmctional affinity (avidity)
of multimers combined ~ith the reduced kidney clearance
398 Proteinengineering
rates when muhimers wcrc 60-90 kDa in size wcrc partic-
ularly suited to tumour targeting.
Clinical trials arc in progress to evaluate the in c'i~,o stabil-
it3. and radiotherapeutic potential of these diabodies and
minibodies. Folh)wing this trend, we would expect that
radiolabellcd triabndies (90 kI)a) would outperform dia-
bodies due to higher avidity and reduced kidney clearancc.
Improvements in tumour labelling and therapy \sere
reported for chemically conjugated Fabs (120 kI)a) com-
pared to the parent Ig and were attributed to the increase
in stability of thc maleimide cross-linkers and improved
flexibility at the hinge region, alhming better cross-linking
of adjacent receptors on the same cell surface [38]. T h e
i m p r m e d ftmctional affinit% tumour penetration and
biodistribution of these novel cross-linked antibody frag-
ments is promising fi)r the development of a n c w genera-
tion of antibody fragments for imaging and therapx.
Engineering multiple specificity in antibody
fragments
Cross-linking reagents have bccn a major fi)cus in clinical
therapy, particularly for rccrnitmcnt of cytotoxic T cells for
canccr trcatment or ancrgy of T -cclls fi)r transplant thera-
py [1",4"']. T h e r e are also numcrous diagnostic applica-
tions, as many immunoassays require cross-linking
rcagcnts [40,41]. Bispecific antibodies can bc produccd by
fusion of hvbridomas into quadroma cell lines but this
tcchniquc is complex, time consuming and often incffcc-
tixe. Far simpler strategies incorporate chemical conjuga-
tion or recombinant expression systems to couplc two
different Fab or F~ modules together (Figure 1) [9"].
Rcccntly, superb mammalian cell expression systems ha~c
been dcveh)ped for the production of bispecific 'minibod-
ics', using designed heavv chain CH3-constant domain
intcrfaccs that forcc efficient hctcrodimcrisation of two
different Fab-CH3 molcculcs [4",42].
Bispccific diabodics arc simple recombinant constructs
comprising tx~o scFv molecules that are forced to combine
together because each contains a short (five rcsiduc) link-
cr joining \:ll and VI. domains that prevents monomeric
scFx fi)nnation [43,44]. In one example [43], bispecific dia-
bodies wcrc designed to cross-link colon cancer cells to
serum lg, thereby inducing the complement cascade,
including mononuclear phagocyte respirator\ burst and
phagocytosis, and directing synergistic T-cell cytotoxicity.
Furthcrmorc, b \ virtue of binding to serum lg their half-
life (beta-phase) was incrcascd fivefold compared to a con-
trol diabody of the same molecular weight. In a second
example [441, bispccific diabodics with specificity to C lq
recruited thc complement cascade directly. In a third
example [45], a bispecific scFv dimcr was produccd that
can recruit T cells (via CI)3) to cancer cells bearing the
I tER2 antigcn. Bispccific diabodics and scFv dimers haxe
two advantages over bispecific antibodies: firstly: by lack-
ing Fc domains, bispecific diabodies will only activate
T cells ~ h e n cr()ss-linkcd to tarect (cancer) cells: secondly,
the anti-bispecific response in the host is minimised duc to
the small diabody size and, again, lack of Fc domains.
Therapeutic and diagnostic applications of
antibody fragment fusions
rl'hc original 'Magic Bullet' concept is still alive: a number
of cell-specific antibodies are being cvaluatcd as delivery
agents to dircct a cvtotoxic con]ponent to a ttlnlotlr site.
T h e original principals ha~c now been significantly
refined; exquisitely sensitive laumaniscd cancer antibod\
fragments can be fused to Fc domains to initiate cvtotoxic
effector functions, prmiding thc connecting hinge to l"c
retains thc disufide linkage and flcxibilit\ requircd for
effcctixc clustering and presentation of heax\ r chain con-
stant ((]H2) domains [46,47]. New types of immunotoxins
haxe been produccd 13\ recombinant or chemical fusion of
antibody fragments to cytotoxic drugs, pcptidcs and pro-
tcins [48,49",50-57]. I n n m a t i v e improvements to thc
imnaunotoxin concept [48,49",50] include A 1 ) E P T - - thc
concept of antibody-enzyme fusions that specifically acti-
vate a prodrug into a cytotoxic agent at the cell target site
[51,52]. Refined designs include fusions to intcrlcukin 2
for cvtokinc stimulation and T cell proliferation at the tar-
get turnout site [53], fusions to T-cell costimulator\ molc-
cults for "Fccll modulation (either actixation or ancrgy),
fusions to transmembrane cell-signalling domains as 'T-
bodies' [54] and fusions to a viral surface markcrs for viral-
targctcd genc then~py [55-57]. In gcnc thcrap.x, the
dclivered g e n t may encode a cytotoxic protcin or a surfacc
antibody (fusion) capablc of rccruiting complen~cnt or
T cells [54]. Antibody fragments ha~c been fused to lipids
[58] and alkaline phosphatase [59] for diagnostic applica-
tions. T h e antibody-fusion proteins provide vcrx high sen-
sitivity in these diagnostic assays by enabling signal
amplification at'tcr the initial antibody binding interaction.
T h c ultimate diagnostic device will be a recombinant anti-
b e d \ fused directly to a bioscnsor platform capablc of real-
time detection of target antigens. T h e current research
biosensor (BIAcoreTM: Pharmacia) dctccts molccular mass
increase on a surl'ace and, although somewhat limited in
sensitixit3, has bccn cspccially useful for rcal-timc moni-
toring of a n t i g e n - a n t i b o d y interactions [9,13°].
Recombinant designs for antibody l'usions arc rcall\ onl\
limited by thc imagination and an expression svstcna capa-
ble of producing c()rrectl\ folded protein products.
Antibody fragment libraries: construction,
display and selection
Bacteriophage libraries
Phage display technology is currentl\ the most popular in
c'/tro method for the selection of hi~h-affinity antibody
fragments (JK Scott and co-~orkcrs, see this issuc
pp 427-435: also [6°',7,8]). T h e current fd phagc and
phagcmid ~cctors cnablc coupling of affinit\ selection
(based on the displayed rcpcrtoires of antibody Fab and
scFv fragmcnts) to thc recovery of the packaged g e n t
encoding that antibody. This system is highly cffcctixc
and has been used cxtcnsixclv to isolate human Fab and

scFv fragments against a wide rangc of proteins, D N A ,
cell-surfacc markers, viruses and parasites. H u m a n anti-
body gencs can be extracted from p r e - i m m u n i s e d donors
and have alreadx' b e e n somatically m a t u r e d to high affin-
ity (Kd >10 -7 M). An alternative strategy is to sclcct anti-
b o d y fragments from very largc 'nafve" repertoires that
have b e e n e i t h e r c o n s t r u c t e d from germline V domains or
s y n t h e s i s e d with large n u m b e r s of mutations, typically in
C I ) R loops. D c s p i t c these successes, f\l vectors impose a
series of major limitations, including gene deletion, plas-
mid instability, gross phagc cross-contaminations and lim-
itcd library size.
Polysome libraries
Polvsomes arc stable p r o t e i n - r i b o s o m e - m R N A c o m p l e x -
es that can bc uscd to replace live b a c t e r i o p h a g e as the
d i s p l a y v e h i c l e for r e c o m b i n a n t a n t i b o d y f r a g m e n t s
[60"]. T h e p o l y s o m e s arc formed by p r e v e n t i n g release
of newly sx'nthesised and corrcctly fnldcd protein from
thc ribosome. As for phage display, thc selection is based
on protein affinity ~ i t h c o n c o m i t a n t r e c m c r y of the gene
(as m R N A ) that e n c o d e s the protein. Repertoire diversi-
ty is d i r e c t e d by thc m R N A population. Polysome display
has many advantages over phage-displa% including easy
synthesis of very large libraries t)3' avoiding the need for
cell transfections.
Library selection
A n t i b o d y f r a g m e n t s d i s p l a v c d on c i t h e r p h a g e or
polysomcs can be selected by binding to covalcntlv immo-
bilised antigen. The choice of affinity matrix can often
determine the kinetics of the selected antibody gone flag-
m e n t [6"',7]. Protocols have bcen established for selection
on live cells, as well as establishing competitive selection
procedures in solution, to discriminate b e t ~ e e n mutants
with as little as a txvofold affinity difference [6"'].
Affinity enhancement of antibody fragments
using libraries
(]urrcnt strategies all e m u l a t e the natural somatic hypcr-
mutation and selection process that occurs /,v F/co and are
based on the principal of introducing gcnctic mutations
follCmed by selection of the e n c o d e d antibodies that
exhibit e n h a n c e d affinity. One of the most effcctixe ran-
dora mutagenesis methods incorporates replication iJl c/co
using F:,~c~eriJzia Fo/i mutator cells [61]. Mutations can bc
targeted to tile V-domain genes by homologous g e n t rcas-
sortments [62"] or error-prone P e R [6"]. Specific window
mutagenesis targets the introduced sequence diversity to
just short regions of p o l x p c p t i d c sequence (e.g. 5-10
residues) and is the most effective m e t h o d in generating a
c o m p l e t e set of m u t a t e d residues. It is important to make
the correct choice for the short regions of sequencc and
tlsuallv thc mutations have heen directed to the (',DR
loops [ 6 - ] . Mutations in thc framework regions, however,
provide an alternative target for mutagenesis since thcv
often exhibit a pronouccd cffcct on affinity, stability and
expression levels [8,22-25].
RecombinantantibodyfragmentsHudson 399
T h e two domain structure of the Fv module enables chain
shuffling to be used both for sequential i m p r o v e m e n t s in
affinity and for hunlanisation [631. In chain shuffling, just
one V domain is altcrcd at a time, relying on promiscuous
V domain paring to crcatc ncx~ flmctional Fv modules. For
cxample, starting with an antibody of known target affini-
ty, only onc V domain is displayed as a library while the
other V domain is hcld constant to provide a defined target
affinity. R e p e a t e d cycles of chain shuffling and affinity
selection will produce high-affinity antibodies. Using this
"guidcd selection" or ' e p i t o p e imprinting' strateg 5 it has
bccn possible to sequentially replace the \; domains of a
parcnt mousc antibody to form a fully human Fv with
high-affinity to thc samc c p i t o p c [63].
New affinity maturation technologies
T h e f l m d a m c n t a l problcm in library mutagenesis strate-
gies is the librar\: size required to search the c o m p l e t e set
of residue mutations (for example, a c o m p l e t e m u t a g e n e -
sis of just six residues requires 20 ~' = 6.4 × 107 molecules).
A further limitation is that the target antigen is not itself
part of the maturation process, but is a d d e d as a static mol-
ecule in soluble or i m m o b i l i s e d form. Recently, an
improved strategy has been d e x c l o p c d , t c r m e d Selection
and Amplification of Phage (SAP: [64]) or Selective
Infection (SIP; [65,66°]). T h i s sccond-gencration format
uncouplcs the display system (currently as s c F v / F a b on
phage heads) so that a n t i g e n - a n t i b o d y recognition is an
intcgral c o m p o n c n t of the viral transfection process
[64,65,66"]. In this way, continuous evolution (affinity
maturation) of the a n t i g e n - a n t i b o d y interaction can be
theoretically achieved as affinity is now coupled to the
efficiency of viral transfection. T h c antigen is p r e s e n t e d
as a soluble fusion molecule with the amino-terminal
domain of f d - g p l I I and thc antibody fragment repertoircs
arc displaycd on phage as fusions to the carboxv-terminal
f d - g p l l l domain. Phage propagation iv ~,i~,o during con-
tinuous cell culture provides a simple selection process for
the highest affinity l i g a n d - r c c c p t o r interaction. Similar to
phage selection, the usc of c o m p e t i n g concentrations ot
antigen allows selcction of the higher affinity scFv/Fabs.
T h e ~ectors and host cclls are still under d e v e l o p m e n t
and it would be unwise to suggcst that S I P - S A P m e t h o d s
will replace the establishcd fd-phage affinit\ maturation
procedures in the short tcrm. A major hurdle is the pre-
sentation of antigen as a soluble, correctly folded fusion
molecule [66"].
Transgenic mice with human humoral immune
systems
Injection of live animals is still tile simplest route to pro-
ducc antibody fragments, dcspite the fact that phage
display' libraries can supcrsede natural i n m n m e repertoires
in size and afl'inity maturation strategies can s u p e r s e d e
sonmtic mutation in the specd of selecting high affinity
mutants. Transgenic micc have been produccd that lack
the native murine antibody repertoire and instead harbour
most of thc human antibody \:-gencs in the germline [67].
400 Protein e n g i n e e r i n g
Injcction of these animals with a foreign antigcn o r hapten
effectively evokes an immune response and a human anti-
body is produced in B -cells [67]. T h e antibody genes can
bc recovcrcd from B -cells either bv P e R and library sclec-
tion o r bv fusion into a monoclonal cell line bv classic
h vhridtmm teclmology.
Conclusions
During the past 12 months there has been a rapid increase in
both scientific and commercial interest in recombinant anti-
bodies, leading to the large number of publications covered
in this reviex~: (~linical interest is due to encouraging results
from phase 3 trials, which has led to several recent Food and
1)rug Adrninistration approvals for therapeutic antibodies.
Scientific interest has stemmed frona the ehtcidation of the
key elements required for antibody fragment design and effi-
cient expression. Many of the antibody fragments and fusion
proteins discussed in this review are now tmdergoing scale-
ttp production, some in transgenic animal or plant expression
systems, and wc cagerly a'wait the results of clinical trials. In
the next 12 months, it is probablc that wc will havc gcncric
solutions for improved Fv stabilit> leading to improved
expression yields, and generic strategies for the design of
mttltimcrs, immunotoxins and other innmative fltsion pro-
teins. I.ibrar3: display and selection strategies have been sig-
nificantly improved to the point that these i~] ~'i:,o systems
are nov, more effective than conventional hvbridonaa tech-
nology. By providing a highly stablc, protcasc-rcsistant scaf-
fold, recombinant antibody fragmcnts will continuc to be the
paradigm for selection of high-affinity protein-based
rcagents, including the deveh)pmcnt of catalytic antibodies.
Useful Internet sites for antibodies and fragments:
htrp:llx~x~.mfacn.uni-hcidclbcrg.dclSl)lSl)scF~Sirc.htnfl (1"', structure,,)
http://',~ w~.mrc-cpc.caln.ac.uk/m~t-dou (Vbasc: h u m a n anribud~, s e q u e n c e s )
http://x~ ~ ~.antibodvrcsourcc.com {t 5;A links)
Acknowledgements
I thank John :\t~cl]. {';rcA (]oia and Alex Kot'tt for their t o t n m c n r s on this
rcxicx~ and particularly, John Atwcll fi~r t:igurc 1.
References and r e c o m m e n d e d reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
° of special interest
°* of outstanding interest
1. Carter P, Merchant AM: Engineering antibodies for imaging and
• therapy. Curr Opin Biotechnol 1997, 8:449-454.
This review is an excellent summary of the clinical applications using
recombinant antibodies from January 1996 to April 1992. A comparison to
this current review (above) demonstrates the very rapid progress that has
been made in the design and engineering of recombinant antibody
fragments in 1997 and 1998.
2. Maloney DG, Grillo-Lopez A J, White CA, Bodkin D, Schilder RJ,
• Neidhart JA, Janakiraman N, Foon KA, Liles TM, Dallaire BK et al.:
IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy
in patients with relapsed low-grade non-Hodgkin's lymphoma.
Blood 1997, 90:2188-2195.
This publication describes the pharmocokinetics and Phase I, II trials for the
first FDA approved recombinant cancer therapeutic antibody. The antibody
was designed as a simple chimaera of mouse V domains fused to human
constant region C domains. It is a useful reference for initial clinical data
using recombinant antibodies and will be followed by many other
publications in 1998 describing more detailed clinical trials with a number
of therapeutic recombinant antibodies.
3. Parren PW, Burton DR: Antibodies against HIV-1 from phage
• display libraries: mapping of an immune response and progress
towards antiviral immunotherapy. Chem Immunol 1997, 65:18-56.
An excellent review describing the antiviral applications of recombinant
antibodies.
4. Datl'Acqua W, Carter P: Antibody engineering. Curr Opin Struct Biol
•. 1998, in press.
This review provides a detailed description of the most recent structural
designs and modifications that are possible with engineered antibody
fragments. It summarises the latest methods and strategies and should be
read in conjuction with this review (above).
5. Banfield M J, King DJ, Mountain A, Brady RL: V-domain rotations
in engineered antibodies-crystal structures of the Fab
fragments from two murine antitumour antibodies and their
engineered human constructs. Proteins - Struct Func Genet
1997, 29:161-171.
6. Hoogenboom HR, De Bruine AP, Hufton SE, Hoet RM, Arends J-W,
•. Roovers Re: Antibody Phage Display technology and its
applications. Immunotechnology 1998, in press.
This review provides the most recent detailed description of the strategies
for phage display and selection (see also Scott et al. for phage display of
peptides; this issue, Curr Opin Biotechnol pp 427-435). The review also
provides detailed sections for the currnet in-vogue methods and a section
on troubleshooting.
7. Rader C, Barbas CF II1: Phage display of combinatorial antibody
libraries, Curr Opin Biotechnol 1997, 8:503-508.
8. Baca M, Presta LG, O'Connor S J, Wells JA: Antibody humanization
using monovalent phage display. J Biol Chem 1997,
272:10678-10684.
9. PIL~ckthun A, Pack P: New protein engineering approaches to
• multivalent and bispecific antibody fragments. Immunotechnol
1997, 3:83-105.
A review of multivalent and multispecific antibody fragments with an
excellent explanation of the functional affinity (avidity) gains via apparently
reduced off-rates, which result from both multiple binding and rebinding to
the target antigens.
10. Adams GP, Schier R, Marshall K, Wolf EJ, McCall AM, Marks JD,
Weiner LM: Increased affinity leads to improved selective tumor
delivery of single-chain Fv antibodies. Cancer Res 1998,
58:485-490.
11. Dougan DA, Malby RL, Gruen LC, Kortt AA, Hudson PJ: Effects of
substitutions in an antibody binding surface on antigen affinity.
Protein Eng 1998, 11:65-74.
12. Cat XH, Garen A: Comparison of fusion phage libraries displaying
V H or single chain Fv antibody fragments derived from the
antibody repertoire of a vaccinated melanoma patient as a source
of melanoma-specific targeting molecules. Proc Natl Acad Sci
USA 1997, 94:9261-9266.
13. Kortt AA, Lah M, Oddie GW, Gruen LC, Burns JE, Pearce LA,
• Atwell JA, McCoy A J, Howlett G J, Metzger DW et aL: Single chain Fv
Fragments of anti-neuraminidase antibody NCl 0 containing five
and ten residue linkers form dimers and with zero residue linker a
trimer. Protein Eng 1997, 10:423-433
This publication provides the first detailed description of a triabody and
explains why scFv trimers are the preferred conformation when very short
polypeptide linkers between V domains prevent diabody formation.
14. Turner DJ, Ritter MA, George AJT: Importance of the linker in
expression of single-chain Fv antibody fragments - optirnisation
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15. De Wildt RM, Ruytenbeek R, van Venrooij WJ, Hoet RM: Heavy chain
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16. Komissarov AA, Marchbank MT, Calcutt M J, Quinn TP, Deutscher SL:
Site-specific mutagenesis of a recombinant anti-single-stranded
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17. Barbas CF III, Heine A, Zhong G, Hoffmann T, Gramatikova S
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Catalytic antibodies provide a new range of commercial opportunities for
engineered antibodies. This publication is the best demonstration to date
that describes the selection of new antibodies with hitherto unknown
enzyme (catalytic) activity and with specific catalysis of new chemical
reactions (aldol condensations).
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20. Wedemayer G J, Patten PA, Wang LH, Schultz PG, Stevens RC:
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site. Science 1997, 276:1665-1669.
This important publication demonstrates for the first time that affinity
maturation in vivo can result from mutations exclusively in the V domain
interface that stabilise V domain pairing rather than resulting from mutations
in the CDR loops that form the antigen binding surface.
21. Colman PM: Structure of antibody-antigen complexes: implications
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22. Kipriyanov SM, Moldenhauer G, Martin ACR, Kupriyanova OA,
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23. Nieba L, Honegger A, Krebber C, Pl~ckthun A: Distrupting the
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-
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24. Forsberg G, Forsgren M, Jaki M, Norin M, Sterky C, Enhorning A,
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25. Atwell S, Ridgway JB, Wells JA, Carter P: Stable heterodimers from
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26. Brinkmann U, Dicarlo A, Vasmatzis G, Kurochkina N, Beers R, Lee B,
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27. Fitzgerald K, Holliger P, Winter G: Improved turnout targeting by
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29. Uson I, Bes MT, Sheldrick GM, Schneider TR, Hartsch T, Fritz H-J:
X-ray crystallography reveals stringent conservation of protein
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immunoglobulin variable domain. Fold Des 1997, 2:357-362.
30. Rondon IJ, Marasco WA: Intracellular antibodies (intrabodies) for
gene therapy of infectious diseases. Ann Rev Microbiol 1997,
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31.. Gargano N, Cattaneo A: Inhibition of routine leukaemia virus
retrotranscription by the intracellular expression of a phage-
derived anti-reverse transcriptase antibody fragment. J Gen Virol
1997, 78:2591-2599.
32. Roux KH. Strelets L, Michaelsen TE: Flexibility of human IgG
subclasses. J Immunol 1997, 159:3372-3382.
33. Thouvenin E, Laurent S, Madelaine MF, Rasschaert D, Vautherot JF,
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34. Ghetie MA, Podar EM, Ilgen A, Gordon BE, Uhr JW, Vitetta ES:
Homodimerization of tumor-reactive monoclonal antibodies
markedly increases their ability to induce growth arrest or apoptosis
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Recombinant antibody fragments Hudson 401
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36. Pei XY, Holliger P, Murzin AG, Williams RL: The crystal structure of a
trimeric antibody fragment with non-cognate MH-VL domain pairs
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The second study on tumour imaging with bivalent diabodies, with results
that concur with the previous anti-CEA results of Anna Wu et aL
(Immunotechnol 1996, 2:21-36).
38. Casey JL, King DJ, Chaplin LC, Haines AM, Pedley RB, Mountain A,
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40. Kontermann RE, Martineau P, Cummings CE, Karpas A, Allen D,
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41. Libyh MT, Goossens D, Oudin S, Gupta N, Dervillez X, Juszczak G,
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scFv anti-Rh(D) antibody with multiple valences using a
C-terminal fragment of C4-binding protein. Blood 1997,
90:3978-3983.
42. Li E, Pedraza A, Bestagno M, Mancardi S, Sanchez R, Burrone O:
Mammalian cell expression of dimeric small immune proteins
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44. Kontermann RE, Wing MG, Winter G: Complement recruitment
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46. Brekke OH, Michaelsen TE, Sandlin R, Sandlie I: Activation of
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47. Coloma MJ, Trinh KR, Wims LA, Morrison SL: The hinge as a spacer
contributes to covalent assembly and is required for function of
IgG. J Immunol 1997, 158:733-740.
48. Ghetie MA, Ghetie V, Vitetta ES: Immunotoxins for the treatment of
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49. Zewe M, Rybak SM, Dubel S, Coy JF, Welschof M, Newton DL, Little M:
• Cloning and cytotoxicity of a human pancreatic RNase
immunofusion. Immunotechnol 1997, 3:127-136.
Clinical trials with immunotoxins have been compromised by the
immunogenicity of the conventional antibody fusion proteins to ricin or
bacterial toxins. This publication demonstates that conjugation of antibodies
to human cytotoxic proteins, RNase or onconase, is an effective and
presumably less immunogenic alternative therapeutic reagent.
50. Pastan I: Targeted therapy of cancer with recombinant
immunotoxins. Biochim Biophys Acta 1997, 1333:C1-6.
51. Martin J, Stribbling SM, Poon GK, Begent RH, Napier M, Sharma SK,
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phase I clinical trial. Cancer Chemother Pharmacol 1997,
40:189-201.
52. Siemers NO, Kerr DE, Yarnold S, Stebbins MR, Vrudhula VM,
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and activities of a single-chain antibody-beta-lactamase fusion
protein for anticancer prodrug activation. Bioconjug Chem 1997,
8:510-519.
53. Penichet ML, Harvill ET, Morrison SL: Antibody-lL-2 fusion proteins:
a novel strategy for immune protection. Hum Antibodies 1997,
8:106-118
402 Protein engineering
54. EshharZ: Tumor-specific T-bodies: towards clinical application.
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55. Wickham TJ, Lee GM, Titus JA, Sconocchia G, Bakacs T, Kovesdi I,
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56. Watkins S.I, Mesyanzhinov VV, Kurochkina LP, Hawkins RE: The
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adenoviral gene delivery. Gene Ther 1997, 4:1004-1012.
57. Chu THT, Dornburg R: Toward highly efficient cell-type specific
gene transfer with retroviral vectors displaying single-chain
antibodies. J Virol 1997, 71:720-725.
58. Kobatake E, Sasakura H, Haruyama T, Laukkanen ML, Keinanen K,
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containing genetically engineered lipid-tagged antibody. Anal
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59. Kerschbaumer RJ, Hirschl S, Kaufmann A, Ibl M, Koenig R, Himmler G:
Single chain Fv fusion proteins suitable as coating and detecting
reagents in a double antibody sandwich enzyme-linked
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60. Hanes J, Pl(Jckthun A: In vitro selection and evolution of functional
eo proteins by using ribosome display. Proc Nat/Acad Sci USA 1997,
94:4937-4942.
The first detailed description on the use of polysomes for the display of
antibody fragment repertoires.
61. Cola G, Ayres A, Lilley GG, Hudson PJ, Irving RA: Use of mutater
cells as a means for increasing production levels of a
recombinant antibody directed against Hepatitis B. Gene 1997,
201:203-209.
62. Zhao H, Giver L, Shao Z, Affholter .JA, Arnold FH: Molecular
• evolution by staggered extension process (STEP) in vitro
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An important new method that complements the DNA shuffling and
homologous recombination strategies discussed by Willem Stemmer (Curt
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63. MahlerSM, Marquis CP, Brown G, Roberts A, Hoogenboom HR:
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64. Malmborg AC, Soderlind E, Frost L, Borrebaeck CA: Selective phage
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65. PedrazziG, Schwesinger F, Honegger A, Krebber C, Pl~JckthunA:
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66. Krebber C, Spada S, Desplancq D, Krebber A, Ge L, PI0ckthun A:
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novel method to select for protein-ligand interactions. J Mol Biol
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The best description of SIP technology which may provide the first
continuous molecular evolution strategy by phage display.
6?. Mendez MJ, Green LL, Corvalan JR, Jia XC, Maynard-Currie CE,
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[Published erratum appears in Nat Genet 1997, 16:410.]