132 TIBTECH - APRIL 1991 [Vol.

9]

antigen co-crystals have been solved,

Single chain antibody these aligned hypervariable se-
quences are shown to make contacts
variable regions with the antigen.
Because the three-dimensional
structures of framework sequences
Robert E. Bird and Barbara Webb W~lker of an antibody variable region are so
similar, it is possible to use a specific
variable domain as representative of
The use of antibodies or antibody fragments for targeting tumors the principles. We therefore used the
{eith~:r for tumor imaging or as carriers for drugs or toxins}, has structure of MCPC6038'9 to design
encountered problems of clearance, and non-specific or inefficient our first scFv. The first step in scFv
binding in clinical trials. A novel approach, linking two antibody design is deciding where to truncate
variable fragments (Fvs), with a short peptide to generate a each chain to define the C-termini of
continuous polypeptide chain, may be able to overcome some of the VH and VL regions (Fig. ld). The
these problems. Since these single chain antibody variable regions point of truncation defines the C-
{scFvs}, are transcribed from constructed 'genes', large-scale produc- terminus - one end point of the scFv
tion in, for example, E. coli, should be straightforward. molecule - and the N-terminus of
the other chain defines the other end
point. The two truncated fragments
Single chain antibody variable (V) the N-terminus of the other, without are then linked by a peptide care-
regions (scFvs) are novel recombi- disrupting antigen binding, orig- fully chosen so as not to interfere
nant proteins composed of two anti- inated from an understanding of the with the correct folding of the vari-
body variable regions [from the light structure of antibody variable able domain and subsequent antigen
(L) and heavy (H) chains, V~ and VH, regions, and the underlying require- binding. The peptide linker (or
respectively] linked to each other by ments for accurate folding and func- tether) joins the end points of the
a peptide of approximately 15 to 25 tion. Analysis of the immense quan- truncated VH and VL regions such
amino acid residues, such that a tity of sequence data on the variable that the scFv molecule forms a con-
continuous polypeptide chain is regions of immunoglobulins tinuous polypeptide chain, starting
formed 1.2 (Fig. ld). scFvs have a gathered by Kabat and his col- with the N-terminus of one chain
number of unique properties which leagues 4'5 indicated that each vari- and ending with the C-terminus of
may be useful in overcoming able region consisted of three hyper- the other. The first scFv constructs
problems encountered with the use variable regions for the heavy and used link sequences chosen either
of antibodies or antibody fragments light chains, while the remainder from peptides in the Brookhaven
(see Box 1) for targeting tumors. was relatively less variable (Fig. la). protein structure databaseI or ones
These problems include (1) slow The hypervariable regions were which were designed de h o v e 1'2.
clearance times from the blood and proposed to form the antigen-bind- Designing scFvs as continuous poly-
tissues (this may be partially allevi- ing portion of the molecules with the peptide chains means that they can
ated by the use of specific antibody less variable region acting as the easily be produced, in large quantity,
fragments which clear more rapidly), structural framework. Subsequent by transcription and translation of
(2) non-specific binding to normal elucidation of the crystal structures synthetic 'genes'. Once the scFv
organs and tissues, and (3) the low of several antibody variable regions sequence is decided, the scFv
proportion of the total injected dose showed that the framework variable gel,~ may be constructed using
which actually binds to the tumor 3. regions fold into almost identical variable-region cDNAs.
This review summarizes the design structures (the antibody folds), and
and production of scFvs, the use of the hypervariable regions bind anti- Construction of scFv genes
scFvs in targeting tumors as exemp- genB as proposed by Kabat. Cloning of variable region cDNAs
lified by imaging human tumor Each domain of the variable region The cloning of antibody variable-
xenografts in mice, and a discussion is folded into nine strands of closely region DNA from hybridoma cells is
of the additional potential appli- packed (3-sheets. This overall folding straightforward since these cell lines
cations of scFvs in diagnostics and pattern is preserved from one anti- usually produce high levels of the H
therapeutics. body to another, even though there is and L chains and, therefore, contain
variation in sequence of the variable high concentrations of their re-
Design ofscFvs domains, particularly in the hyper- spective mRNAs. Oligonucleotide
The idea that the variable regions variable regions 6.7. The less variable primers from the constant (C) region
of an antibody could be tethered framework sequences determine the adjacent to the variable region were
from the C-terminus of one chain to folding of the variable domain and used to prime first-strand cDNA syn-
the hypervariable sequences are thesis using mRNA from the hybrid-
found in loops at one end of this oma as template 1°. A primer can be
R. E. Bird is at Molecular Oncology, Inc., domain. The hypervariable se- chosen based on published sequence
19 Firstfield Road, Gaithersburg, MD quences of each domain thus occur data, where available for a specific
20878, USA. B. W. Walker is at 5206 at the ends of G-strands at the same antibody l°'u, or deduced from a
Russett Road, Rockville, MD 20853, side of the molecule. In the cases consensus of immunoglobulin se-
USA. where the structure of antibody- quences s. Following second-strand
© 1991, ElsevierScience PublishersLtd (UK) 0167- 8~0/91/$2.00
TIBTECH - APRIL 1991 [Vol. 9] 133

- - F i g . 1 ....

synthesis 12, the cDNA is cloned, and

v " ~ v,
a
the correct clones selected by
hybridization with a second oligo-
nucleotide (which is 5' to the first
primer in the C region) 11. The hy-
bridization-positive clones are then
sequenced, and the amino acid
sequence of the variable region may
be deduced from the nucleotide
sequence. Confirmation of the N-
terminal sequence of the two chains F(ab')z
from the antibody is essential since
it is possible to clone other, incorrect
immunoglobulin sequences. d
Recently, methods to amplify
immunoglobulin variable domain
cDNAs using the polymerase chain or
reaction (PCR) have appeared 13.14. ~V L
These methods can he used to
amplify the variable region cDNAs H2N-Vt-Linker-VH-COOH H2N-VH-Linker-VL-COOH
before cloning but this is generally
not necessary since sufficient H and Fab Single chain Fvs
L chain mRNAs are produced.

Construction of the genes

Previously, we and others ~'z have The relationship between naturally occurring antibodies and antigen-binding
inserted restriction sites at the ends proteins generated artificially by cleavage of the antibody molecule. (a) The
antibody is composed of two heavy (H) and two light (L) chains. The variable
of cDNAs by oligonucleotide- regions include the antigen combining sites and the constant regions are
directed site-specific mutagenesis 15 indicated. (b) Digestion of an antibody with the protease pepsin removes the
in order to assemble the gene. The CH2 and CHa (constant) regions leaving the F(ab')~ fragment which is
linker may then be inserted using composed of the two Fab fragments (c), joined by at least one disulfide
synthetic oligonucleotides incor- bridge. Digestion of an antibody with the protease papain removes these
porating restriction sites at the ends. same constant regions and, in addition, the disulfide bridges linking the two
Now, the restriction sites can be heavy chains leaving two Fab fragments. The F(ab')2 fragment is bivalent and
inserted simultaneously with the the lab fragment is monovalent with respect to antigen binding. (d) Two
PCR amplification of the DNA frag- different configurations of scFvs are shown, with either the linker running
ment by appropriate primers ~a'14. In from the C-terminus of the VH (variable region) to the N-terminus of the VL or
the C-terminus of the VL linked to the N-terminus of VH.
fact, using primers that overlap and
code for the linker, the entire gene
can be prepared by PCR16. An
example of an scFv gene is shown in
Fig. 2a. Both methods have comparable be 'noted that the scFv using a more
yields (in the range of 1-10 mg 1-1 recently designed linker (B6.2; V~-
Production of scFvs culture). Gly-Ser-Thr-Ser-Gly-Lys-Ser-Ser-
In general, scFvs are produced in In two cases, one using Bacillus Glu-Gly-Lys-Gly-VH, Ref. 29) has an
E. coli using a variety of promoters subtilis 19 and the other using affinity slightly higher than the cor-
such as phage ~ OffPR (Ref. 1), E. cob17, the scFvs have been syn- responding Fat). This linker has also
LAC17, TAC2, or phage T7 (Ref. 18). thesized and secreted as active sol- been shown to increase the affinity
However, most of these proteins uble proteins. If the yields can be of an anti-fluorescein scFv over
made in E. coli are insoluble, necessi- increased (to >100 mg 1-1 culture) the previously published values
tating solubilization and refolding. using these secretion systems, these (Pantoliano et al., unpublished).
Their insolubility can be used to proteins could be produced econ- Thus, the sequence of any mono-
recover and partially purify the pro- omically because they eliminate clonal antibody may be used to
teins. The insoluble fraction is re- the refolding step. design and synthesize a small anti-
suspended in urea or guanidine to gen-binding protein with the same
completely unfold the protein, and scFv-antigen binding specificity (and similar affinity) 1'2 as
then either the denaturing agent is The scFvs to a number of antigens the starting monoclonal antibody. In
removed by dialysis, or the solubil- (both haptens and intact proteins) addition, it does not matter whether
ized fraction is diluted into renatur- have now been made. A comparison VH or VL is at the N-terminus since
ing buffer, thus permitting protein of the antigen affinities of scFvs and scFvs have been produced with
refolding. The active fraction of un- their corresponding Fabs from the variable regions in either con-
folded scFv protein is purified either which they are derived {Table 1) figuration 1.2, and the orientations
by affinity chromatography ~'2'~7, shows that, in all cases except one, of the variable regions seems not to
or ion-exchange chromatography 18. scFvs have a lower affinity. It should affect either affinity or specificity.
134 TIBTECH- APRIL 1991 [Vol. 9]

--Box I

Antibody structure and binding properties of antibody fragments

Antibody molecules are composed of two types of
polypeptide chains referred to as heavy (H) and light (L)
chains. Light chains are the smaller of the two types (Mr
-25000) and are common to all classes of immuno-
globulins whereas heavy chains (Mr -'50 000-77 000) are
structurally distinct for each immunoglobulin class or
subclass. The basic unit of an immunoglobulin is com-
posed of two L and two H chains linked by covalent and
non-covalent interactions (Fig. la). The heavy chain
polypeptides are joined together by one or more di-
sulfide bonds positioned at the branch point of the
structure. Each individual light chain is covalently linked
to a heavy chain by a disulfide bond. Each arm of the
immunoglobulin molecule contains two distinct domains
referred to as the constant (C) and variable (V) regions
reflecting the degree of polypeptide sequence variation
in the respective heavy and light chains. The antigen-
binding specificity of the antibody is determined by the
variable region.
Immunoglobulin molecules can be selectively cleaved
into antibody fragments using either papain or pepsin 3s.
Papain cleavage produces two Fab fragments containing
the antigen-binding site and the first constant region and
the Fc fragment (Fig. lc). Pepsin cleaves the immuno-
globulin to produce the F(ab')2 fragment which contains
both antigen-binding sites of the starting molecule and a
fragment which contains most of the Fc (Fig. lb). These
methods of producing fragments are at best imprecise
because the enzymes used have limited specificity and
additional sites may be cleaved, resulting in total degra-
dation, or molecules with significantly altered affinities.
In general Fab fragments have a lower affinity for antigen
than the whole antibody; possibly due to the loss of
avidity, loss of the advantage of having a bivalent
molecule, or due to damage to the Fab by additional
cleavage by the protease. In addition, some antibodies
seem to lack the specific cleavage sites as we are unable
to produce fragments by these methods.
Fab fragments have been produced as recombinant
proteins in animal cells 32,33, in E. COIP7, and in yeast 3e.
Recombinant light-chain genes and truncated heavy-
chain genes are introduced on expression vectors into
the respective hosts, and the recombinant Fab fragment
is secreted as an active molecule into the culture
medium. The best yield to date ( - 2 mg 1-1 culture) is
obtained with the E. coli expression system 37.
Fv fragments consist of only the variable domain of the
heavy and light chains and have been produced by
proteolytic digestion 39 and by synthesis in animal cells 4°
and in E. coli41. The Fvs produced by proteolytic diges-
tion and in E. coli reportedly bound the target antigen
(phosphorylcholine), with similar affinity (-10 s a -1) to
the intact antibody 39'41. No data on affinity was given for
animal cell Fvs.
Antigen binding with VH alone. Winter et al. have
proposed that the variable domain of the heaw chain is
sufficient for high-affinity binding to antigen 42. In fact
they have cloned amplified variable region cDNAs made
with mRNA from the spleen of a mouse immunized with
lysozyme into an expression vector and generated
clones which produce a heavy-chain variable region
which binds lysozyme. While feasible for some antigens,
it is unlikely that this approach will be generally appli-
cable since some high-affinity antibodies (e.g. the mono-
clonal 4-4-20) binds fluorescein (with an affinity of 2 x
10TMa -1) and has important light-chain-fluorescein con-
tacts in addition to heavy-chain-fluorescein contacts in
the three-dimensional crystal structure 34.
Selection of antibody repertoires in E. coli. To select
new binding specificities from cloned antibody se-
quences synthesized in E. coli, Lerner and colleagues
produced combinatorial libraries of antibody variable
regions in a bacteriophage ~. vector 43. V L and VH se-
quences are prepared by polymerase chain reaction
(PCR) from mRNA isolated from the spleen of a mouse
immunized with p-nitrophenyl phosphonamidate (NPN)
coupled to keyhole limpet haemocyanin (KLH). These
amplified sequences are inserted into a phage vector
such that each vector contains one VL and one VH
sequence linked to a constant region such that a Fab is
produced by expressing both genes. Plaques from this
combinatorial library can then be screened for new
binding specificities: Lerner et al. 43 isolated about 100
plaques that produced Fabs which bound NPN from a
library of 106 members. This system could replace the
current method of selecting new antibody specificities,
bypassing the long procedure of selecting and cloning
hybridomas.

Tumor targeting with an tumor so slowly that images of the tumor site 3. This is a particular
anti-tumor scFv tumors are not obtained until several problem in the use of antibodies for
In vivo diagnosis of cancer days after the injection of the radio- therapy where a large dose of radio-
Monoclonal antibodies against labeled imaging reagent. Clearance nuclide, toxin or drug must be de-
tumor antigens have been used in times can, however, be shortened livered to the tumor site. While some
the in vivo imaging of cancer to carry considerably by using F(ab')z or Fab results have been obtained using
the imaging agent, usually a radio- fragments of the monoclonal anti- whole antibodies or fragments gener-
nuclide, to the site of the target bodyZ°-zs; the smaller th~ antigen ated by protease digestion, a smaller
tumors. An ideal carrier for the imag- binding molecule, the shorter the antigen binding molecule such as an
ing agent should clear rapidly from clearance time. scFv might be better yet at imaging.
the blood and concentrate in the (2) The intact antibody and anti-
target tissue. However, in the cours~ body fragments clear slowly from the Tumor imaging in mice
of human tumor-imaging trials a normal tissues of some organs, thus ScFvs have been made from the
number of problems have emerged raising local background levels in variable region sequences of the
when using whole monoclonal anti- the images. monoclonal antibody B6.2, to
bodies. (3) Only a small fraction (--0.001- demonstrate that a B6.2 scFv can
(1) Intact monoclonal antibodies 0.01%) of the injected dose of radio- find and bind its antigen target in
clear from the body and enter the labeled antibody actually reaches rive 29. The monoclonal antibody
TIBTECH - APRIL 1991 [Vol. 9]
B6.2 recognizes an antigen on breast
tumor cells, and was subsequently
shown to bind to the same antigen in
some colon carcinomas 3°. A tumor-
cell line, LS174T, which expresses
this antiRen was injected subcu-
taneously into athymic mice and
tumors allowed to develop. B6.2 scFv
labeled with 12sI and B6.2 Fab'
labeled with 13~I were injected into
the tumor-bearing mice. The mice
were sacrificed at different times and
the amount of each radionuclide in
various organs and tissues was
measured. The B6.2 scFv initially
clears the blood (with a tl/Z of 2.4
rain) - about seven times faster than
the Fab' molecules, indicating that
the scFv reaches the tissues and
organs more quickly than the Fab'.
The B6.2 scFv localizes to tumor
better than the Fab' with a tumor-to-
liver ratio of 11.8 after 24 hours, com-
pared to a rath) of 7.95 for the Fab'. In
addition, the Fab' does not clear fzom
the kidney such that, after 24 hours,
the tumor-to-kidney ratio of Fab' is
0.55, compared with 5.38 for the B6.2
scFv 29. In a control experiment using
xenografts of the human melanoma
cell ~ine A375 (which does not ex-
press the B6.2 antigen), the B6.2 scFv
did not bind to tumor; however, the
control using a radiolabeled non-
specific scFv in mice bearing the
LS174T ~umors was not done. These
results imply a significant advantage
of the scFvs over their Fab counter-
parts in tumor imaging.
This first attempt to use an anti-
tumor scFv to localize tumors
heterotransplanted into mice was an
outstanding success. However, the
B6.2 scFv is valuable only for the
proof of principle: although the B6.2
scFv is specific for the antigen recog-
nized by the B6.2 monoclonal, this
antigen, unfortunately, is also ex-
pressed in normal human cells and
the B6.2 monoclonal antibody also
binds normal tissues in humans -
thus preventing use of B6.2 to treat
tumors. The success of this first
model experiment should provide
the impetus for the construction of
scFvs which bind more specifically
to tumors and would, therefore, lead
to clinical trials. Recombinant anti-
body variable regions which will
eventually prove useful in the clinic
may be expected to result from
modification or refinement of the
basic approach of scFvs discussed
here.
135
--Fig. 2
vL ILl v.
b
5' [ Vl. [ L J vH 1 Toxin J3'
Constructing scFv genes, cDNAs of the antibody variable regions are
prepared by priming first-strand synthesis using oligonucleotide primers
homologous to the nearby constant regions", the second strand is
synthesized, and the double-stranded cDNAs cloned. Confirmation that the
cDNA clones correspond to the correct variable (V) regions for a particular
monoclonal antibody is obtained using N-terminal sequence of the isolated
protein chains from the purified ,?Qnoclonal antibody. (a} Restriction sites
(marked by black arrows) are inserted using oligonucleotide-directed
mutagenesis Is at the 5" and 3" end of each variable region clone. The unique
restriction site at the 5" end is determined by the promoter which will be used
to transcribe the gene. The sites at the 3" end of VL, and the 5' end of VH are
used to insert oligonucleotides coding for linker sequences. The site at the 3"
end of the VH includes a termination codon and will be used to insert the
sequence in the expression vector. The small antigen-binding protein gene
(scFv gene) (a) may be further modified by linking to sequences encoding
toxin ~8 thus generating an scFv immunotoxin (b).
If the sequences of the variable regions are known but clones or the cell
line not available, the designed gene can be constructed from multiple
oligonucleotides, though this is laborious.
ScEv fusion proteins as TAC-PE40 protein was approxi-
!mmunoconjugates mately 100 times more efficient in a
ScFvs can be further modified by protein-synthesis inhibition assay
fusing the 'gene' encoding the scFv using HUT-102 cells than was the
to the gene for a peptide toxin (Fig. anti-TAC-antibody-PE40 chemical
2b). An scFv gene for an anti-TAC conjugate. This suggests that genetic
monoclonal antibody has been con- fusions of antigen-binding mol-
structed and fused to a segment of ecules and toxin genes may, in some
DNA encoding amino acid residues instances, have greater biological
253-613 of Pseudomonas exotoxin activity than chemical conjugates
PE40 la, thus generating an scFv where the methods of chemical con-
immunotoxin. The anti-TAC anti- jugation inactivate the binding pro-
body binds to the low-affinity IL-2 tein. In addition, in fusion proteins,
receptor expressed in large amounts the toxin is always linked to the
by HUT-102 cells. This type of C-terminus, whereas in chemical
receptor-binding antibody may be conjugates it may be linked randomly
useful in immunomodulation. The to various sites.
protein produced by this fusion The anti-TAC-PE40 scFv has also
gene, anti-TAC-PE40, has the anti- been used to destroy cultured malig-
gen binding specificity and a bind- nant cells in blood samples from
ing affinity estimated to be within a patients with adult T-cell leukemia 31.
factor of three of that of the starting These cell frequently express high
monoclonal antibody (Table 1). The levels of the IL-2 receptor recognized
binding affinity was estimated from by the anti-TAC scFv construction;
competition experiments where the this receptor is not expressed at such
binding of radiolabeled anti-TAC high levels in normal cells. Treat-
antibody to TAC antigen on HUT- ment of patients with adult T-cell
102 cells was in competition with leukemia with the anti-TAC-PE40
either anti-TAC antibody or scFv scFv may prove possible in the
anti-TAC-PE40 protein. The anti- future.
136
--Table I genes for enzymes such as bacterial
Affinities of scFvs for antigen
Antibody Antigen
3C2 Bovine growth
hormone
4-4-20 Fluorescein
26-10 Digoxin
anti-TAC IL-2 receptor
MCPC603 Phosphoryl-
choline
B6.2 Antigen
expressed on
LS174T cells
a Relative m~3surement by competition.
Possible applications ofscFvs
An scFv is monovalent (i.e. it has
one antigen-binding site) and has a
molecular weight of ~-26 kDa. Such
a small antigen-binding molecule
may be useful for (1) in vivo diag-
nosis and treatment of diseases such
as cancer or cardiovascular disease,
(2) in vitro diagnostics, (3) separ-
ations, (4) biosensors, or (5) catalytic
antibodies. The properties of scFvs
offer potential advantages in each of
the applications.
• Diagnosis and treatment of disease
The B6.2 scFv can target human
tumor xenografts in mice 29 and the
anti-TAC--PE40 scFv can destroy
malignant cells from patients with
adult T-cell leukemia 31. Although
this is encouraging, the usefulness of
scFvs to treat cancer in vivo has yet
to be demonstrated.
Antibodies can be used to deliver
toxic substances to tumors. (The aim
is to deliver as much toxic material
as possible to the tumor sites while
any unbound material clears from
the body, resulting in destruction of
the tumor without toxic side effects.)
As yet, experiments to test the thera-
peutic efficacy of these carrier
scFvs proteins in animal model sys-
tems have not been performed. How-
ever, since the localization of scFvs
to tumor is rapid these proteins
could be used to deliver oc-emitting
radionuclides with a short half-life
(~.~. astatine and bismuth), or highly
toxic drugs or toxins. The advan-
tages of scFvs as carriers is that they
would either bind to the target tumor
or clear quickly from the body,
TIBTECH-APRIL 1991[Vol.9]
alkaline phosphatase or horseradish
peroxidase which have already
proven useful in immunoassays.
Affinity (Ka) M -1 Producing the antigen-binding pro-
Fab scFv Ref. tein with the enzyme as a fusion
eliminates the need for enzyme con-
Ia 0.25-0.50 1 jugation. This may increase sensi-
tivity, as was seen for the anti-TAC-
1.7 X 10 l° 1.1 X 109 1 PE40 fusion la.
2-3 x 10 e 3-5 X 107 2
Another interesting possibility
9.7 X 109 3.5 X 10 g 18
1.6 x 105 1.3 × 10 s 17
would be to fuse two scFv genes to
produce a molecule of twice the size
2.6 × 10 e 3.2 x 10 a 29 that would contain two antigen-
binding sites. If the two fused genes
were identical, the product would be
a divalent antigen-binding protein,
Alternatively, if the two fused genes
had different specificities the prod-
uct would be a bispecific antigen-
binding protein. The production and
thereby reducing the toxic side correct refolding of these more com-
effects. plex molecules might prove more
If the clearance of scFvs from the difficult; though as more is learned
body is so rapid that too few of the about folding of recombinant pro-
proteins bind to the tumor to be teins this should be feasible.
effective as therapeutics, it may be
possible to engineer a change in • Other applications
them to increase the clearance time. The other applications for scFvs
For example, the clearance and bio- are biosensors, bioseparations and
distribution may be affected by the catalytic antibodies. The advantage
net charge on the molecule. A series of scFvs in these applications is the
of scFvs varying in pI could be made, ease of making alterations in the
and their serum clearance and bio- amino acid sequence and examining
distribution studied as charge is the effect on structure and function
varied. These small antigen-binding of the proteins.
proteins offer an excellent oppor-
tunity to study systematically the Future directions
behavior in rive of a protein that In addition to the potential medi-
has been altered but maintains cor- cal and commercial applications of
rect antigen binding. scFvs, scFvs provide a valuable tool
for studying antibody-antigen inter-
• In vitro diagnostics actions in detail. A good example
Fusion proteins comprising anti- is the anti-fluorescein scFv 1 (see
body chains and enzymes have been above) where a three-dimensional
produced successfully a2.a3. The first structure of the Fat) bound to fluor-
fusion was of the variable region and escein has been determined 34. The
first constant region of a mouse l~- structure provides a starting point
chain with the gene for Staphylo- for testing amino acid substitutions
coccus aureus nuclease 32. When the in the scFv which enhance or reduce
fusion was introduced into a my- affinity for fluorescein. Because the
eloma cell line producing ~.1 light genetic constructs and altered ver-
chain, the cell line produced an sions of the proteins are so easily
F(ab')2 molecule with nuclease ac- produced in large quantities, this
tivity 32. A second fusion was pro- type of iterative study is now
duced between the same heavy possible.
chain (~), and the gene coding for
E. colt DNA polymerase Klenow Conclusion
fragment: introduction of this fusion Since antibody fragments can be
into the myeloma cell line produced made by protease treatment of whole
active Fab fragments having DNA antibodies, it came as no surprise
polymerase activity 33. that F(ab')2 and Fab fragments could
These two examples suggest that be synthesized as recombinant pro-
scFv genes could be fused to the teins. However, that the two chains
TIBTECH- APRIL 1991[Vol.9]
of the variable region of an antibody
could be connected with an oligo-
peptide, the gene to synthesize the
designed protein constructed, and
an active single-chain, antigen-bind-
ing protein produced, was by no
means obvious. The end result is an
experimental system for reproducing
the antigen binding of an antibody in
E. coli or Bacillus subtilis where
=products can be produced and the
antibody-antigen interactions stud-
ied in great detail. The ultimate
result of this technology will be the
selection of new antigen-binding
specificities and as Winter and
colleagues have set out to do by ex-
pressing an scFv gene on the sur-
face of bacteriophage fd by fusion of
an scFv gene with gene lII of the
phage 3s. New specificities can be
selected by the insertion of new
variable regions into the scFv gene
and the phage containing the
n e w binding specificity fished out
of solution by chromatographic
methods.
References
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Lee, S-M., Lee, T., Pope, S. H.,
Riordan, G. S. and Whitlow, M.
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2 Huston, J. S., Levinson, D., Mudgett-
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Reid-Miller, M. and Perry, H. M.
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(1987) Sequences of Proteins of
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6 Padlan, E. A. (1977) Q. Rm,. Biophys.
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7 De Prevdl, C. and Fougereau, M.
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