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Experimental Therapeutics, Preclinical Pharmacology |
Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [R. B., P. C., I. P.], and Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710 [D. B.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our laboratory is focused on the development of immunotoxins for cancer therapy. Immunotoxins are made by fusing a targeting moiety, such as an antibody or a portion of an antibody, to a protein toxin such as Pseudomonas exotoxin.
PE is a potent bacterial toxin composed of three major domains (6) : (a) domain Ia (amino acids 1–252) is the cell binding domain; (b) domain II (amino acids 253–364) is responsible for translocation into the cytosol; and (c) domain III (amino acids 400–613) ADP-ribosylates elongation factor 2, arresting protein synthesis and causing cell death, and also contains the COOH-terminal sequence REDLK, which directs the endocytosed toxin to the ER. Domain Ib (amino acids 365–399) is a minor domain, and its function is unknown. PE38 is a modified form of PE in which all of domain Ia and amino acids 365–380 of domain Ib have been deleted.
In a previous study, we described the properties of immunotoxin MR1(Fv)-PE38, which was constructed by fusing the scFv of MR1 to PE38, a truncated form of PE (1) . Our laboratory has developed many different recombinant immunotoxins, several of which are now in clinical trials (7, 8, 9) . Generally, the scFv genes are joined with the PE38 gene by a short linker and cloned into a T7-based expression vector. The recombinant immunotoxins are expressed in Escherichia coli, where they accumulate in inclusion bodies. After the inclusion bodies are washed extensively, they are dissolved in guanidine hydrochloride, and the protein is renatured and purified by ion-exchange chromatography and gel filtration. The resulting molecules are active and are directed to a cell-specific antigen by the scFv. Cell death is caused by the activity of the toxin.
To be useful as therapeutic agents, immunotoxins should have a high affinity for the antigen, resulting in a high cytotoxicity toward cells expressing the antigen. It is also necessary that the immunotoxin be produced with a high yield. MR1(scFv)-PE38 falls short in both of these criteria, with a Kd of 8 nM and a yield of 2%.
Antibodies bind to antigens via residues in their CDRs. Consequently, CDR mutagenesis is a widely used technique for improving the affinity of Fab and Fv fragments of antibodies. There are a number of different approaches to CDR mutagenesis. Most of these approaches, such as codon-based mutagenesis (10) , CDR walking (11 , 12) , error prone replication (13) , and synthetic CDR construction (14) , require the construction of large libraries that are technically difficult to make and hard to handle. Lately, the trend in antibody affinity maturation has been toward the isolation of high-affinity binders from relatively smaller-sized libraries (15, 16, 17) . All of these approaches involve the construction of expression libraries of antibodies with mutations in the CDRs and selection for better binders. Of the several different approaches to achieve this, filamentous phage display technology is the best approach in terms of efficiency and speed.
Phage display technology has become a useful tool for screening large peptide or protein libraries (18, 19, 20) . scFvs can be expressed on phagemid vectors as fusions with M13 gene 3 protein using the pCANTAB5E or similar vectors (1) . The fusion proteins are expressed in E. coli and, in the presence of helper phage, are displayed on the tips of the M13 phage, which can be collected from culture media. Phages that display scFv fusion proteins and bind to specific antigens are selected by panning the phage libraries on cells expressing the antigen or on a surface to which the antigen is coupled, such as magnetic beads. Phages that do not bind are washed away. Bound phages are eluted and amplified by reinfecting E. coli. Several rounds of panning result in an enrichment of specific binders. By making the panning conditions more stringent, better binders can be separated more effectively from poor binders.
In the current study, a phage display library was made with MR1 scFv. Initially, we introduced random mutations in the heavy chain CDR (VHCDR3), an area that has a major role in antigen binding (21) . Panning on cells expressing EGFRvIII produced several mutants which, when used to construct immunotoxins, had improved affinity, cytotoxicity, and yield. Analysis of these variants revealed that they all had mutations localized to a region of the VHCDR3 that qualifies as a hot spot for hypermutation (22, 23, 24) . Hot spots are defined by the consensus sequence G/A-G-T/C-A/T or A-G-C/T. The latter contains serine codons found in the variable domain genes of antibodies (25) . Hot spots are regions that mutate at a high frequency during the maturation of antibodies in mice. Panning the VHCDR3 library did not yield any clones containing mutations outside the hot spots. This result confirms our previous data demonstrating that randomizing hot spots is more likely to produce mutants with improved affinity (17) . Therefore, we used the best binder obtained from panning the VHCDR3 library to make a phage display library randomizing a hot spot in the light chain CDR3. Panning this library produced several more mutant clones. One of these Fvs, when expressed as an immunotoxin, showed a further increase in binding to EGFRvIII peptide and increased cytotoxicity to cells expressing the mutant receptor.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The following
oligonucleotides were used: (a) CDR3Hb,
5'-CTTGGCCCCASNNSNNSNNAGAGGTACTAGAATAGCCTC TTGTGCA-3'; (b)
CDR3Hd, 5'-CTTGGCCCCACATAGCATASNNSNNSNNAGAATAGCCTCTTGTGCA-3';
(c) CDR3Hf, 5'-CTTGGCCCCACATAGCATAAGAGGTACTSNNSNNSNNTC
TTGTGCA-3'; (d) S1, 5'-CAACGTGAAAAAATTATTATTCGC-3'; and
(e) AMBN, 5'-GCTAAACAACTTTCAACAGTCTATGCGGGCAC-3'.
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Panning the VHCDR3 Library.
NR6M cells grown in DMEM containing 10% fetal bovine serum plus 750
µg/ml G418 were harvested using 0.02% EDTA (Sigma E-8008). Cells
(2 x 107) were pelleted, resuspended in 10
ml of cold blocking buffer (2% BSA and 0.02%
NaN3 in Dulbecco’s PBS), and rotated slowly for
1 h at 4°C. The cells were pelleted and resuspended in 5 ml of
cold blocking buffer. Phages (1 x 109) from
each of the heavy chain CDR3 libraries were added to the cell
suspension, and the mixture was rotated slowly at 4°C for 2 h.
The cells were washed twice with 10 ml of cold blocking buffer and
resuspended in 5 ml of cold blocking buffer. MR1dsFVPE38 was added (2
µM, final concentration) as a competitive inhibitor, and
the suspension was rotated slowly at 4°C for 2 h. The cells were
washed three times with 10 ml of cold blocking buffer. Bound phages
were eluted by resuspending the washed cells in 1.5 ml of ice-cold 50
mM HCl and incubated on ice for 10 min. The NR6M cells were
pelleted, and the eluted phages were transferred to a new tube
containing 200 µl of 1 M Tris (pH 8.0). The eluted phages
were titered to determine the number of phages captured. The eluted
phages (0.5 ml) were then amplified by reinfecting E. coli
TG1 for use in the next round of panning.
Construction of the VL Mutant Library.
Heavy chain CDR3 mutant (S98P-T99Y) was used as a template in a
two-step PCR that introduced randomizations in the hot spot located in
the light chain CDR3 (Fig. 1)
. In the first reaction, 50 pg of the
phagemid containing heavy chain mutant (S98P-T99Y) were mixed with 20
pmol of the DNA oligomers VLMUT (5'-GATTACTACTGTTTGCAANNSNNSAACGTGCC
TCTTACA-3') and AMBN in a 50-µl volume. The mixture was cycled using
the same profile used to generate the heavy chain CDR3 library. The
reactions generated 150-bp products containing randomization of the hot
spot in the light chain CDR3. After purification and quantitation, 2
pmol of the reaction products generated in the first PCR were used with
20 pmol of DNA oligomer S1 in a second PCR with 50 pg of phagemid DNA
containing the heavy chain mutant (S98P-T99Y) as the template. The
template and primers were mixed with two PCR Beads in a 50-µl volume
and cycled using the above-mentioned profile. The reactions generated a
876-bp library which contains the VHCDR3 mutation
(S98P-T99Y) and randomization of the hot spot in CDR3L. The PCR
products were digested with restriction enzymes SfiI and
NotI, purified, and ligated with the pCANTAB5E vector as
described for the heavy chain CDR3 libraries. The ligation was
desalted, and one-tenth (40 ng) of the reaction was used to transform
E. coli TG1. The phage library containing 3 x
105 clones was rescued as described in the heavy
chain CDR3 construction. One-fourth of it was amplified and used for
the first round of panning.
Panning the VLCDR3 Library.
NR6M cells were harvested as described in the
VHCDR3 panning procedure. All steps and washes
were done in cold blocking buffer, except as noted. Cells (5 x
106) were resuspended in 1
ml of blocking buffer and rotated for 1 h. The cells were pelleted
and resuspended, rescued phage was added, and the suspension was
rotated slowly at 4°C for 2 h. The cells were washed three times
and then resuspended in blocking buffer containing 2 µM
MR1 heavy chain mutant (S98P-T99Y) scFv-PE38 and rotated slowly at
4°C for 2 h. The cells were washed three times, and bound phages
were eluted and neutralized as described for the heavy chain CDR3
library. The eluted phages were titered and rescued for the next round
of panning.
Phage Rescue.
The phages captured after each round of panning were amplified for use
in the next round of panning. For rescue, E. coli TG1 was
grown in 10 ml of 2xYT containing 2% glucose incubated at 37°C with
shaking at 250 rpm. When the
A600 nm
reached 0.3, 0.5 ml of the captured phage was added to the culture, and
the incubation was continued. After 1 h, ampicillin (100 µg/ml,
final concentration) and 1 x 1010
plaque-forming units of M13KO7 helper phage were added, and incubation
continued for 1 h. The culture was then centrifuged, and the
pelleted bacteria were resuspended in 10 ml of fresh 2xYT media
containing ampicillin (100 µg/ml) and kanamycin (50 µg/ml) and
incubated at 37°C with shaking at 250 rpm for 16 h. The bacteria
were pelleted by centrifugation in a Sorvall SS34 rotor at 8,000 rpm
for 20 min. The phage-containing supernatants were filtered using a
0.45-µm syringe filter unit. The phages were then precipitated by
adding 2 ml of PEG/NaCl and incubated on ice for 30 min. The
precipitated phages were pelleted by centrifugation in a Sorvall SS34
rotor at 10,000 rpm for 20 min and then resuspended in 1 ml of NTE. The
rescued phage library was titered and stored at 4°C.
Detection of Positive Clones.
After the third round of panning, 48 clones from the heavy chain CDR3
library were analyzed for binding to EGFRvIII peptide
(LEEKKGNYVVTDHSGGK-biotin) using ELISA. Twenty-two clones that gave the
strongest signal were subjected to DNA sequencing and further analyzed.
After the fourth round of panning, 48 clones were analyzed by ELISA,
and the DNA sequence of the 10 clones with the strongest ELISA signal
was determined. For the light chain library, after the second round of
panning, 48 clones were analyzed by ELISA, and the 17 clones with the
strongest signal were sequenced. After the third round, 20 clones were
analyzed, and 10 clones were subjected to DNA sequencing. To rescue the
phage from the individual clones, single colonies were picked from the
final panning titer plate and inoculated into 150 µl of 2xYT with 2%
glucose and 100 µg/ml ampicillin in a 96-well culture dish. The dish
was incubated at 37°C with shaking at 150 rpm. After 3 h, 20
µl of the culture were transferred to the wells of a second dish
containing 100 µl of 2xYT with 2% glucose, 100 µg/ml ampicillin,
and 1 x 108 M13KO7 helper phage and
incubated for 2 h. The cultures were pelleted, resuspended in
fresh 2xYT plus ampicillin and kanamycin, and then grown for 16 h.
The cells were pelleted, and 50–100 µl of the phage-containing
supernatants were assayed in ELISA. Phage ELISA was performed as
described previously (1)
, except that 100 µl of
3,3',5,5'-tetramethyl benzidine (BM blue; Boehringer Mannheim)
were used as a substrate for detection. After blue/green color
developed, 100 µl of 2 M
H2SO4 were added to stop
color development. Absorption was measured at 450 nM.
DNA Sequencing.
DNA sequencing was preformed using the PE Applied Biosystems Rhodamine
Terminator Cycle Sequencing Kit. The samples were run and analyzed on a
PE Applied Biosystems Model 310 automated sequencer.
ScFv Immunotoxin Plasmid Construction, Expression, and
Purification.
ScFvs from selected phagemid clones were PCR-amplified using primers
that introduced NdeI and HindIII restriction
sites. The products were then digested and cloned into a T7-based
expression vector in which the scFv is fused to a truncated version of
PE. The plasmids were transformed into the expression host BL21
(
DE3). The MR1 mutant immunotoxins were expressed and prepared as
described previously (26)
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Surface Plasmon Resonance.
Binding kinetics were measured using a BIAcore 2000 Biosensor.
Steptavidin was bound to a CM5 research-grade sensor chip using amine
coupling reagents provided by BIAcore. Biotinylated EGFRvIII peptide
was bound to streptavidin by injecting 10 µl of a 10 nM
solution of the peptide over the chip. Immunotoxins were diluted to 25
µg/ml in HEPES-buffered saline. On and off rates were measured by
injecting 50 µl of the diluted immunotoxin over the chip surface at
10 µl/minute, and then allowing the bound material to disassociate
for 5 min or more. The remaining bound material was removed from the
EGFRvIII peptide by injecting 5 µl of 100 mM phosphoric
acid. Each immunotoxin was injected and analyzed at least three times.
Binding kinetics were analyzed using BIAevaluation 2.1 Software.
Cell Culture and Cytotoxicity Assays.
NR6M cells were cultured in DMEM plus 10% fetal bovine serum
supplemented with 750 µg/ml G418. Cytotoxicity assays measured the
inhibition of [3H]leucine incorporation
as described previously (27)
. The
IC50 of each of the immunotoxins was determined
using three wells for each point. Each immunotoxin was assayed at least
twice, and critical ones were assayed more frequently.
Bacteria and Cell Lines.
E. coli TG1 is a K12 strain with the genotype supE
thi1 
(lac-proAB)
(mcrB-hsdSM)5/F'[traD36 proAB
lacIqZM15]. E. coli BL21 (DE3) is a B
strain with the genotype F- ompT gal [dcm] [lon]
hsdSB(rB-mB-) with
DE3, a prophage carrying the T7 RNA polymerase gene. NR6 is a Swiss 3T3
mouse fibroblast variant cell line with no detectable EGFR. NR6M is the
NR6 cell line transfected with a cDNA for the mutant EGFRvIII receptor
under the control of the ß-actin promoter. The source of NR6 and NR6M
has been described previously (2)
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RESULTS |
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. A complete library in
which nine amino acid residues were randomized would require more than
3.5 x 1013 individual clones; instead, we
prepared three different libraries covering residues 95–97, 98–100,
and 101A-101C as described in "Materials and Methods." Library
H-CDR3 95–97 contained 1.6 x 106 clones,
library H-CDR3 98–100 contained 4 x 105
clones, and library H-CDR3 100A-100C contained 1 x
106 clones. To ensure the libraries were properly
made, five clones from each library were sequenced. As expected, each
clone had different amino acid combinations in the region targeted for
mutations. Because a completely diverse library randomizing three amino
acids would require only 33 x 103
independent clones, the size of the libraries ensures that all possible
DNA sequences are well represented.
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summarizes the number of phages
captured during each of the four rounds of panning. The enrichment
peaked at the third round and appeared to decrease at the fourth round.
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the only mutations
recovered were in positions 98 and 99. DNA sequence analysis showed
that the clones belong to five different groups: parental S98-T99 and
mutants P98-N99, P98-Y99, P98-F99, and P98-W99. We also examined phages
selected after the third round to see whether good binders were lost
between rounds 3 and 4. We analyzed 48 individual clones for binding to
the EGFRvIII peptide by ELISA, and 22 of these that gave the highest
signal were subjected to DNA sequencing. Again, the only mutations
recovered were in positions 98 and 99 (Table 3)
. A variety of amino
acids were recovered in these positions. The most frequent was S98-T99
(wild type), followed by P98-S99, P98-Y99, P98-N99, and A98-D99. The
fourth round of panning resulted in an enrichment of clones P98-N99 and
P98-Y99 over the wild type (ST) and a loss of several clones and also
produced a new clone containing P98-F99.
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; we find that compared with the
parental clone (S98-T99), which had an IC50 of
8.0 ng/ml, there were six clones that were more cytotoxic. These were
P98-Y99, which had an IC50 of 3.5 ng/ml, followed
by P98-N99, P98-W99, and P98-I99, which had an
IC50 of 4.5 ng/ml, P98-F99, which had an
IC50 of 6 ng/ml, and P98-V99, which had an
IC50 of 6.5 ng/ml. Four clones, P98-S99, W98-V99,
S98-W99, and P98-T99, had IC50s identical to the
parental clone.
Construction of the VLCDR3 Library.
Interestingly, all of the mutants recovered after panning the
VHCDR3 libraries were localized to positions 98
and 99. The DNA sequence of these two residues constitutes a hot spot,
which is a region that undergoes mutations during the in
vivo affinity maturation of an antibody. We therefore decided to
use the mutant with the highest cytotoxic activity from the
VH library and subject it to mutagenesis,
targeting only the hot spot in VLCDR3. The
sequence of VLCDR3 is shown in Table 1
. Our
library introduced randomizations in the hot spot located in residues
91 and 92. The VLCDR3 phage library was
constructed as described in "Materials and Methods" and contained
3 x 105 clones. Because a library of only
1024 clones is required to achieve all possible codons in the two
positions chosen for mutation, we assume that all possible DNA
sequences were abundant in this library.
Panning of the VLCDR3 Library and Analysis of Selected
Clones.
Three rounds of panning were carried out, and the phage captured at
each step is shown in Table 2
. For this library, we obtained more
enrichment in the second round of panning than in the third round.
After the second round of panning, 48 individual clones were rescued,
and binding to the peptide was measured by ELISA. The DNA sequences of
17 clones that gave the strongest signal were determined. As shown in
Table 4
, five different mutants were
obtained. All retained the wild-type serine residue at position 91 and
had mutations at residue 92. The most frequent mutation at residue
92 was F92W (6 of 17 clones) followed by F92R (3 of 17 clones),
F92S (2 of 17 clones), F92L (1 of 17 clones), and F92 M (1 of 17
clones). Of the 17 clones analyzed, 4 were found to be of the parental
type.
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. Twenty clones were picked at random, and
the phages were rescued and checked for binding to the peptide by
ELISA. Ten clones that gave the strongest signal were chosen for DNA
sequence analysis. The DNA sequence analysis revealed that there were
three different clones present, all of which were represented in round
2. Besides one parental clone, these clones were F92S (7 of 10 clones),
F92W (1 of 10 clones), and F92R (1 of 10 clones). There were no new
mutants found, and we did not find mutants F92L and F92M.
Cytotoxicity and Binding Properties of the VLCDR3
Mutants.
The different Fvs obtained were used to make immunotoxins, and their
cytotoxic activities and binding affinities were measured using the
purified recombinant proteins. Only one mutant, F92W, gave an
immunotoxin that was more active than the parent. Its
IC50 was 1.3 ng/ml, as compared with 3.5 ng/ml
for its parent (Table 4)
. The other mutants had lower activities. The
data in Table 4
also show that F92W has a higher affinity
(Kd = 3 nM) than
the parental clone F92 (Kd = 6
nM). One other mutant, F92L, had a slightly
increased affinity (Kd = 4
nM) but did not have an increase in cytotoxicity.
Fig. 2
shows a BIAcore sensorgram
comparing the binding properties of the parental clone with the most
active immunotoxin made from the Fv obtained from the
VHCDR3 library (VH
S98P-T99Y) and the most active clone obtained from the
VLCDR3 library (VH
S98P-T99Y VL F92W), now called MR1-1. The figure
shows that the two mutants have slower dissociation rates than the
parental Fv. The analysis of the immunotoxin [MR1-1(Fv)-PE38] with
both VH and VL mutations
showed that it had a decrease in koff
and a slight increase in kon resulting
in a Kd of 3 nM.
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shows two experiments in which
the cytotoxic activities of MR1(Fv)-PE38 were compared with the two
improved variants: MR1(Fv)(VH S98P-T99Y)-PE38 and
MR1-1(Fv)-PE38. In both experiments, MR1(Fv)-PE38 was the least active,
and MR1-1(Fv)-PE38 was the most active. The values shown in the table
are the means ± SD. Ps are < 0.01 when comparing
MR1(Fv)-PE38 with MR1-1(Fv)-PE38 and MR1(Fv)(VH
S98P-T99Y)-PE38 with MR1-1(Fv)-PE38 in experiment 1 and when comparing
MR1(Fv)-PE38 with MR1(Fv)(VH S98P-T99Y)-PE38, and
MR1(Fv)(VH S98P-T99Y)-PE38 with MR1-1(Fv)-PE38 in
experiment 2.
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4
show the yields
when 100 mg of inclusion body protein are subjected to purification. It
is evident that the yield of immunotoxin is greatly affected by the
mutations in the CDRs and increased from 2% with the parental
MR1(Fv)-PE38 to as much as 10–17.5% with many of the mutants.
MR1(Fv)-PE38 has been prepared on at least eight different occasions,
and the yield was never more than 2%. Each of the mutant molecules was
only prepared one time because the yields were very high, and there was
sufficient material for all of the experiments that were performed. |
DISCUSSION |
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Small libraries can be constructed by separately randomizing every amino acid residue of each CDR with the corresponding number of oligonucleotides and an equivalent number of transformations (10) . Making a library with 20 variants of a single residue and making one with a size requirement of 33,000 clones requires almost the same effort. Our approach of randomizing three residues at a time decreased the number of oligonucleotides needed, as well as the number of libraries that needed to be handled. By constructing and panning a VHCDR3 library made in this way, we isolated a variant that improved the cytotoxicity by more than 50% (IC50 was reduced from 8 to 3.5 ng/ml). Analysis of the clones isolated from this library and our previous study (17) showed that it should be sufficient to target mutations only to particular amino acids of the CDRs rather than trying to mutate each one. These residues are coded by DNA sequences called hot spots, which are prone to mutation during affinity maturation of antibodies (22) . We used this knowledge to focus the random mutations to the hot spot in VLCDR3 that involved only two residues. Thus, this library had a size requirement of only 1,024 clones. This library was made using a single degenerate oligonucleotide and one transformation. It yielded a clone that, when used for making an immunotoxin, had another 65% increase in cytotoxicity (from 3.5 to 1.3 ng/ml). Thus, we demonstrate that it is possible and relatively easy to make small libraries to fish out higher-affinity variants. This approach is not restricted to MR1 but has been found to work in two other Fvs we are studying (17) .3
Panning and Enrichment of the Libraries.
As stated previously, our aim was to isolate a variant of MR1 that
would bind to EGFRvIII with an improved affinity. Because we were
trying to substitute all of the positions with all 20 amino acids, it
is likely that the library would contain wild-type MR1. To try and
decrease the reisolation of these clones, we did our panning in the
presence of MR1(Fv)-PE38, a protein that should compete for wild-type
MR1 phage binding. As shown in Table 3
, we could not completely
eliminate MR1 interference. A probable reason for this is that various
Fvs are displayed to a different extent on the surface of phages, and
none of them had a much greater affinity than the parental MR1.
The pattern of enrichment (Table 2)
obtained with each of the two
libraries is very different. In the case of the
VH library, there is slow enrichment at the
beginning that peaks at round 3. With the VL
library, enrichment peaks at the beginning. We believe this is due to
of the nature of the two libraries. In the case of the
VH library there were no binders recovered from
the libraries that randomized residues 95–97 and 100A-100C. This
indicates that more than two-thirds of the initial mixture made by
combining the three libraries contained nonbinders. Because the few
binders restricted to residues 98–100 were being slowly selected from
a large population of nonbinders, the initial panning led to poor
enrichment. In the third round, enrichment was greater because the
population of input phage was dominated by the binders. The appearance
of a new mutant (P98-F99) despite a lack of enrichment after the fourth
round of panning is interesting. We see from our cytotoxicity and
BIACore analysis with purified monomeric protein that this mutant has
low affinity and cytotoxicity.
The VL library introduced randomizations into a hot spot region that is apparently tolerant to mutagenesis. This library had fewer nonbinders; hence, it was possible to enrich the binders after only two rounds of panning. Once these binders were selected, there was no further enrichment as observed in the larger VH library. These results suggest that the best time to analyze clones is early in the process. Panning after the enrichment peaks can be deleterious because of the risk of losing clones. It is possible that Fvs with low affinity but high expression may be preferentially enriched, whereas good binders may be lost. Evidence supporting this was observed while panning the light chain CDR3 libraries; mutant F92S with a low affinity (Kd = 22 nM) was found in 7 of 10 clones examined after the third round, whereas the best binder (F92W) was present only once. In contrast, in the second round, F92S was found in only 2 of 17 clones, whereas F92W was present in 6 of 17 clones.
Analysis of the mutant clones obtained from the three VHCDR3 libraries revealed that we never obtained any mutants from regions outside the hot spot that had any binding ability. This suggests that hot spots are regions that tolerate mutations and, at the same time, influence affinity. An analysis of many mutants that arise during in vivo antibody maturation reveals that many of these localize to the hot spots (22 , 28) .
Cytotoxicity and Affinity of Immunotoxins Made with Mutated MR1
Fvs.
Because our goal was to isolate a Fv with a higher cytotoxic activity,
we first measured cytotoxic activity and then measured affinity using
the BIAcore method. We found that increased cytotoxic activity did not
necessarily correlate with increased affinity (Tables 3
4)
. There
is no obvious explanation for this lack of correlation. Besides binding
affinity, which is measured at 22°C, there are many steps in the
intoxication process that could be affected by these mutations
including stability at 37°C, rate of internalization, proteolytic
processing, and transfer to the compartment required for translocation.
It is possible that one or more of these steps is affected.
One very striking difference observed among the CDR mutants is the
final yield of active monomeric protein. Recombinant toxins accumulate
in inclusion bodies as insoluble aggregated protein (immunotoxin).
Active monomers are produced by dissolving the inclusion bodies in 6
M guanidine-HCl, followed by controlled renaturation in a
redox system and separation of monomers from multimers and aggregates.
Our data show that mutations in one or two amino acids in the CDRs can
greatly increase yields (Table 3)
. The yield of MR1(Fv)-PE38 is only
2%, but it was dramatically increased to 17% with heavy chain CDR3
S98P-T99S mutations. Presumably, these mutations have a profound effect
on the folding pathway. In general, all of the heavy chain mutants
isolated in the initial mutagenesis of the heavy chain had a better
yield than the parental MR1, suggesting that the heavy chain of CDR3 is
very important for proper folding and that the phage expression system
may select in some way for proteins that fold more efficiently.
Consequently, phages containing better-folding Fvs would be present in
larger numbers and would be preferentially enriched during panning on
antigen. Thus, phage display may be useful for selecting Fvs with an
increased production yield.
In summary, we have produced a recombinant immunotoxin with increased cytotoxic activity toward a cell line expressing a mutant form of the EGFR by mutagenizing CDR3 of the heavy and light chains. The mutations obtained were all located in typical hot spots that have the DNA sequence Pu G Py A/T. This sequence is present in other CDRs of the MR1(Fv), and mutations in these regions may lead to further increases in cytotoxic activity.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Laboratory of Molecular Biology, National Cancer
Institute, NIH, Bethesda, Maryland 20892. 
2 The abbreviations used are: scFv, single-chain
antibody variable domain; EGFR, epidermal growth factor receptor; CDR,
complementarity determining region; PE, Pseudomonas
exotoxin A; PEG, polyethylene glycol; VL, variable
region of antibody light chain; VH, variable region of
antibody heavy chain; 2xYT, 16 g of bacto-tryptone, 10 g of
bacto-yeast extract, and 5 g NaCl per liter in H2O.
3 G. Salvatore and I. Pastan,
unpublished results.
Received 12/21/99; revised 4/ 7/00; accepted 4/13/00.
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