Note: Descriptions are shown in the official language in which they were submitted.
CA 02424255 2003-03-26
Immunotoxin
FIELD OF THE INVENTION
The present invention relates to the field of cancer therapy and particularly
to targeted therapy
using immunotoxms.
BACKGROUND OF THE INVENTION
The lack of significant advances in the treatment of metastatic or refractory
cancers has
stimulated the design of novel approaches to targeted cancer therapy such as
the use of antibody~~
based cancer therapeutics
Despite favorable initial responses, most advanced solid tumors develop
resistance to standard
treatments and relapse as incurable metastatic diseases ( 1 ). Since
increasing the dose of
conventional anticancer agents results in unacceptable side effects, the
design of novel therapies
based on the use of tumor selective targeting ligands and effector domains
employing different
mechanisms of action is of great rmportance. Antibodres targeting tumor-
associated antigens and
equipped with intrinsic cytotoxic or rmmunostimulatory effector functions have
shown promising
antitumor activity in preclinical and clinical studies (2-4).
Because of their different mechanism of action and especially because of their
built-in targeting
function that conventional anti-cancer agents do not have, immunotoxins may
add new options
for the treatment of malignancies resistant to conventional treatments
(26,28). ETA and its
homologues irreversibly block protein synthesis in cells by ADP-ribosylating a
posttranslationally
modified histidine residue of elongation factor 2, called diphthamide, which
ultimately triggers
apoptosis (48). Although resistance of°cells to ET'A was described as a
consequence of the
mutation of the crucial histidine residue or loss of enzyme activity required
for diphthamide
synthesis (49,5(1), this is a rather uncommon event and has not been confirmed
in other cell
systems. Nevertheless, the loss of the tumor antigen used for uptake of the
antibody-toxin tizsion
protein by the tumor cell is another conceivable mechanism of resistance.
An immunotoxin is a chimeric protein in which a toxin moiety is chemically or
genetically linked
to a monoclonal or recombinant antibody Antibodies with speciticities for
various tumor-
associated antigens have been investigated as carriers of"toxins, and the
majority of those that
target solid tumors have employed the effector function ot'truncated ET'A
(.ETA) which lacks the
cell binding domain I (aa 1-252) (20), F.T A irreversibly inhibits protein
synthesis by ADP-
ribosylation caf elongation factor 2 and therefore has to gain access to its
intracellular target in tire
CA 02424255 2003-03-26
Immunotoxin
cytoplasm (20,21 ). Thus, the most promising antigens for immunotoxin therapy
are those that are
efficiently internalized into tumor cells upon antibody binding by receptor-
mediated endocytosis
(22-25 ).
A number of chemical and recombinant immunotoxins that employ either plant or
bacterial toxins
as eff'eetor domains and that target distinct cell surface antigens associated
with tumor cells have
been shown to be potent and selective anti-cancer agents in preclinical
studies (28). However,
only few of them proved to be promising candidates for clinical use.
Several immunotoxins, either as chemically~linked first generation or
recombinant second
generation formats, have been tested in animal models and in patients with
advanced solid tumors
(20,26). Although early clinical data using imrnunotoxrn therapy for
refractory tumors remains
promising, the induction of neutralizing antibodies and dose-limiting side
effects associated with
VL,S or liver toxicity still remain obstacles to effective therapy (27-29). To
overcome these
limitations, more promising tumor-associated antigens have to be validated as
targets for nc:w
irnmunotoxin generations which are equipped with rationally engineered
effector functions
(2,30,31 ).
Mayor responses have been reported so far only for leukemias. In phase I
studies using LMI3-2, an
FTA based scFv immunotoxin which targets lL-2 receptor p-chain, responses have
been observed
treating hematologic malignancies (51,52). Moreover, two thirds of the
patients with refractory
hairy-cell leukemia involved in a phase I study showed complete remission
after treatment with
81.22, a recombinant F.'fA-based dsFv immunotoxin specific for the CD22
surface antigen (53)0
Both immunotoxins showed minor or reversible toxic side effects and thus
merited to be involved
in phase I studies for which currently a 'larger number of patients are being
recruited.
T wo recombinant F;TA-based dsfv immunotoxins are also currently being
evaluated for the
treatment of advanced solid tumors. The immunotoxin SSI(dsFv)-PE3R is directed
against cells
expressing mesothelin, a protein normally produced by mesothelial cells and
expressed also on
malignant mesotheliomas and ovarian carcinomas (54). The immunotoxin LMB-9 has
been
derived from the monoclonal antibody B3 that targets the LewisY antigen (55),
which is also
widely expressed in epithelial tissues, a fact that contributes to safety
concerns. LMB-9 and
SS I (dsfv)-PE3R are currently being tested in phase 1 clinical trials in
patients with advanced
solid tumors and first results are eagerly awaited. In a previous study with
3H patients suffering
from advanced carcinomas the chemical conjugate of~monoclonal antibody B3 and
ETA induced
one complete and one partial response (S6). In these studies vascular leakage
due to capillary
CA 02424255 2003-03-26
lmmunotoxin 4
damage was found to be dose-limiting, and subserauent preclinical
investigations revealed
significant binding of the B3 antibody to LewisY expressed on endothelial
cells (57).
In addition to HER-2/neu (25), Ep-CAM represents another more promising target
for antibody-
based therapy of solid tumors due to its abundant expression in many
carcinomas and its limited
distribution in normal epithelial tissues (5). Although, Ep-C.AM expression is
not exclusively
restricted to tumor cells, Riethmuller and co-workers found that application
of the anti-Ep-CAM
monoclonal antibody 17-1 A in patients with resected colorectal carcinoma or
minimal residual
disease reduced the overall mortality by 32%, decreased the recurrence rate by
23% (1), and
reduced the number of distant metastases (8).
Ep-CAM t is a 40 kDa transmembrane protein overexpressed in many solid tumors
including
carcinomas of the lung, breast, ovary, colorectum and squamous cell carcinoma
of the head and
neck (5). The limited expression of Ep-c"AM in normal epithelial tissues (5,6)
makes this antigen
an attractive target for cellular and antibody-based immunotherapy (7-9).
Recently, a transgenic
mouse model mimicking the Ep-(:SAM expression pattern in humans further
validated the
suitability of this target for imrnunotherapy by showing no localization of
the monoclonal
antibody MfW'31 in Ep-CAM-positive ruormal tissues ( 10).
The role of Ep-('AM in carcinogenesis and malignant progression is still
unclear, but there is
increasing evidence that it modulates cell-cell interactions ( 1 1 ) and that
its expression correlates
with the rate of cell proliferation ( 12) In addition, a promoting role of Ep-
CAM in tissue invasion
and metastasis has been suggested ( 13), and a strong correlation between Ep-
CAM expression
and tumor progression has been found in patients with squamous cell carcinoma
of the head and
neck'. Ep-C.'AM-specific antibodies have been used in imaging studies to
detect primary tumors
and localize distant. metastases in patients mth SCL,C (14) and NS("LC (15),
to trigger antitumor
immune responses ( 16), and to deliver cytotoxic effector molecules to tumors
in preclinical
models ( 17,18) and in patients ( 19).
~ The abbreviations used are: Ep-C'AM, epithelial cell adhesion molecule;
S('LC, ,mall cell lung cancer; NSC'L.C, non
small cell lung cancer; ETA, P.ceudvmona.v aemn,s;lnacu exotoxin A; VL..S,
vascular lean syndrome; seFv, single chain
antibody fragment: dsFv, disulfide-stabilized ,ingle-chain antibody fragment;
FBS, fetal bovine serum; IMA(',
innnobilized ion-metal affinity chromatography.; PAGE, polyacryiamide gel
electrophoresis; PBS, phosphate buffered
saline; MT'f, i-14,5-dimethylthiazol-?-yl]-?,5-etiphenyltetrazolium bromide;
F1TC' fluorescein isothiocyanate, MFI,
mean fluorescence intensity; E(.iF, epithelial growth factor AI.T, alanme
aminotransferase; AST, aspartate
aminotransferase; TNF, tumor necrosis factor
D. Tschudi, unpubLshed observation.
CA 02424255 2003-03-26
lmmunotoxin
Although in a previous study we reported the ability of the chemically
conjugated immunotoxin
lYIO('31-ET'A to eradicate small tumor xenografts in mice as well as its
failure to delay the
growth of larger tumors ( 18), we concluded that, due to its relatively large
size of 200 kDa, the
immunotoxin was unable to homogeneously distribute wrthin the tumor mass and
thus could only
affect an insufficiently small proportion of clonogenic tumor cells. Support
for this hypothesis is
promded by others who reported an inverse correlation between immunotoxin size
and efficacy
(32j. The tumor targeting and tissue distribution properties of ammunotoxins
can be substantially
improved by using small scFv as targeting ligands (22,2ni,25,3:3-37). We have
recently described
the enhanced tumor localization of scFv 4DSM0('.B, which was derived by
grafting the
hypervariable loops of monoclonal antibody MO('3 l onto the humanized
framework of'the anti-
HF:R-2ineu scl~'v 4DS and by additionally changing eight critical care
residues to obtain a high
molecule stability (3H). We have now teound that the development ofa fully
recombinant Ep-
t."AM-specific single-chain immunotoxin based on 4DSMOCB, can be employed to
achieve
favorable tumor localization and potent antitumor activity against carcinomas
of diverse
histologrcal ongins in vivo.
SUMMARY OF THE INVENTION
The present invention is predicated on the finding that 4DSMO CB-ETA exerts
significant growth
inhibition upon systemic administration to mice bearing large established
tumor xenografts ( 160
mm;' from colorectal, small cell lung or squamous cell carcinoma of the head
and neck. The
present invention reports on two dose schedules that were well-tolerated and
proved to be very
effective in inhibiting tumor growth. The three-week treatments with a total
dose of 45 trg
eradicated a significant fraction of°the tumors, and some mrce remained
tumor-free during the
whole study. However, in contrast, after completion of the shorter one week
treatment with a total
dose of 30 lrg 4DSMOCB-ETA, HT29 tr.unors rapidly resumed their growth.
BRIEF DESCRIPTION OF THE DRAWINGS
Thz present invention is illustrated but not limited by the attached drawings
of which:
Frgure l A is a schematic representation of the scFv-toxin fusion protein
precursor, which
includes the ompA signal sequence for periplasmic expression. 'The scFv
antibody fragment
~4D~M()(~B is fused to the Pseudomonas exotoxin A (ET'A;52_~os) by the linker
shown. The
protern is tlanked by two hexahiscidine tags, the C.'-terminal of' which
precedes the ER retention
signal 1CDEL.
CA 02424255 2003-03-26
lmmunotoxin 6
Figure ( B is a three-dimensional model of the mature 4DSMOCB-ETA., The
structure of the scFv
(VL in red, VH in orange), of ET'A;52..~r,x (domain II in light-blue, domain
Ib in green and domain
III in violet) and of the linking peptide (green) are shown. Both
hexahistidine tags are indicated in
yellow.
Figure 1 (.' is a copy of a chromatogram. 'Total extract of SB536 bacterial
culture samples before
(-) and after (+) IPTG induction and 10 p,g of 4DSMOCB-ETA immunotoxin
purified by Ni2+-
IDA and anion exchange affinity chromatography columns coupled in series, were
analyzed on
10% SDS-PAGE under reducing conditions
Figure I D shows the immunotoxm proteins present in the same samples as
visualized on a
Western blot using a HRP-conjugated anti-tetrahistidine antibody. Markers are
shown in lane M:
myosin ( M~ 20H,000), w-galactosidase (Mr 1 19,000), bovine serum albumin (M,.
94,000),
ovalbumm (Mr 51,100), carbonic anhydrase (Mr 35,400) and soyabean trypsin
inhibitor (M~
2H,so0)
Figure 2 shows the chromatograms before (0 h) and after 2 h, 4 h, H h, 10 h
and 20 h incubation
were recc'~rded at 2H0 nm. The monomers eluted at approximately l.4 ml as
verified by calibration
with the molecular weight standards alcohol dehydrogenase (A, M~ I 50,000),
bovine serum
albumin (B, M, H6,000) and carbonic anhydrase (C', M,29,000). which eluted at
1.31 ml, 1.38 ml
and 1.54 ml, respectively (retentiun volumes shown by arrows). The stability
of 4D5MOCB-
ETA was assessed by size exclusion chromatography. 'The immunotoxin was
incubated at a
concentration of 200 ugiml at 37"C: in PBS and samples were analysed by gel
filtration at
different time points for comparison.
Figure 3 is a graphic depiction of cell growth measured in MTT assays as
described in the
"Material anc~ Methods" section. Data represent mean values of at least six
independent
determinations each carried out in quadruplicates (overall SD<5'%). Four Ep-
(:AM-positive
tumor cell lines were incubated for 72 h with 4DSMO('B-ETA at concentrations
ranging from
0.0001 to 100 pM.
Figure 4 is a graphic representation of impairment of liver function upon
treatment with
4DSMOC'B-ETA. C'S7BL/6 mice were treated every other day with escalating doses
of
4D5MOC'B-ETA Two groups received 5 pg (?50 pg.kg-l ) or 10 pg (500 pg.kg-1 )
doses for three
cycles, while another group was treated twice with 20 yg ( 1000 ltg.kg-1 ).
Twenty-four hours after
CA 02424255 2003-03-26
Immunotoxin
the last challenge the activities of plasma transaminases were determined and
compared to mice
treated with PBS (0 pg.kg-1 ). The transaminase activities of mice treated
with a single lethal dose
of wild-type E'TA- (85 pg.kg-1), as described by Schumann et al. (47)., are
also shown. Data are
expressed as the mean t SD (n = 3).
Figure 5 is a graphic representation of tumor growth illustrating the
antitumor effect of
~DSMOCB-ETA in mice. Athymic mice bearing large tumor xenograf'ts ( 160 mm; in
average)
derived from the Ep-CAM-positive cell lines HT29, SW2 and (.'AC.27 remained
untreated ( ? -)
or were treated by i.v. injections every second day with either nine doses of
5 pg 4DSMOCB-
ETA each for three weeks (-; -), or with three doses of 10 tig each (-'? -).
In a control experiment,
mice bearing Ep-CAM-negative (.'OLO320 xenografts were also treated with
4DSMOCB-E.TA
according to the dose schedules mentioned above. 'The tumor size is given
relative to the initial
median tumor size of 160 mm3 at the start of treatment and data represent the
mean values pSD
of'the various groups (n - 7).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The immunotoxin 4DSMOC:B-ETA was developed by fusing the highly stable
humanized scFv
4DSMO('B (38) to a truncated form of hTA comprising amino acids 2.52-608 and
the C-terminal
eukaryotic ER retention sequence KDEI.. The 4DSMOCB-ETA is extremely potent in
the
femtomolar range and potently inhibits the growth of°carcinoma cells of
diverse histological
origins in a highly antigen-specific manner as demonstrated by an increase in
the cytotoxic
potency by more than four orders of magnitude, compared to antigen-negative
cells. Moreover,
the antigen-specific action of 4DSMOCE~-ETA was further corroborated in
competition assays
using an excess of scFv 4D5M0('B. Similar to the findings reported from
immunotoxins
targeting other tumor-associated antigens including HER-2/neu and E(iF
receptor (25,59), the
cytotoxic activity of 4D5M0(:B-f=TA did not correlate with the amount of
target antigen
expressed on the tumor cell surface. Thus, it is likely that other cell type-
specific parameters, such
as rate of internalization, intracellular trafficking and fate of the enzyme
domain are further
determinants of immunotoxin efficacy. In terms of its in vitro cytotoxicity
4DSMOCB-ETA is the
most potent Ep-C'AM specific iinmunotoxin that has been reported in the
literature and was 1000-
fold more potent than the chemical immunotoxin conjugate MOC'31-E:TA ( 18).
~, prerequisite for the optimal binding of antibody-based therapeutics to
target antigens expressed
an the surface of tumor cells and for efficient tumor localization is protein
stability under
physiological conditions. The extremely potent cytotoxicity shown by 4D5MOCB-
ETA may at
CA 02424255 2003-03-26
lmmunotoxin
least partly be due to the stability of the targeting scF'v (38). The
immunotoxin itself was obtained
to more than 90% from the soluble fraction of°ter bacterial lysis, was
monomeric and could be
expressed and purified with a yield of approximately 0.5 mg per liter
bacterial culture from
simple shake flasks. These are excellent prospects for scale-up by high cell
density fermentation
(60). The high stability of the immunotoxin was confirmed by the large
proportion of molecules
that eluted in monomeric form after 20 h incubation at 37°C in PBS, a
result that was also
obtained by incubating the radioactive-labeled immunotoxin in serum. Addition
of protease
inhibitors prolonged the stability for more than 48 h, indicating that protein
degradation was not
due to intrinsic molecule instability, but rather was a side effect occurring
during purification.
The addition of ETA and a second hexahistidine tag at the N-terminal end of
the scFv did not
interfere with the binding properties of this ligand.
In viva, the systemically administered 4DSMOCB-ETA was cleared from the blood
with slightly
slower kinetics when compared to the sc:Fv, probably as a consequence of its
increased molecular
size. The blood clearance rate inversely correlated with the amount
of'radioactivity in the kidney
which was lower for the immunotoxin than for the scFv (ID/g tissue 22.79% vs.
300%).
Although all doses of immunotoxin were well-tolerated and mice did not show
any signs o.f
illness such as weight loss, the accumulation of 4I)5M0C'B-ETA in liver,
spleen and bone raised
the issue of potential toxicity to these tissues. The inhibitory effect of ETA
on protein synthesis is
known t<> induce severe hepatotoxicity by sensitizing hepatocytes to the
action of TNF, which is
released by Kupffer cells upon E'fA mediated T cell stimulation and induces
liver cell necrosis
(46,47 ). 'To assess the degree of liver damage upon 4DSMOCB-ETA treatment,
immunocompetent mice received repeated doses of 5 pg (250 p,g.kg-I ) or 10 pg
(500 pg.kg-1 )
immunotoxin every other day for three cycles. In both cases, the level of
AC,TiAST activity in the
plasma of~ treated mice did not change significantly compared to untreated
controls. First signs of
impaired liver function only appeared after treatment with a 20 pg ( 1 mg.kg-1
) dose given twice
every other day (7.5-fold increase over control). In line with these findings,
histological analysis
of liver specimens did not reveal any signs of E'TA-induced pathological
changes, except for the
two 20 pg treatments, which induced moderate hepatocyte necrosis. A recent
report has shown
that E'T'A-induced hepatotoxicity and VLS can be circumvented and enlarge the
therapeutic
window by pre-treatment with anti-inflammatory agents, such as indomethacin or
soluble TNF
receptor and non-steroidal drugs, respectively (27,29). 'The lack of
correlation between organ
specific accumulation and toxicity of thc: tmmunotoxin strongly suggests that
the radioactivity in
these highly perfused organs simply reflects the presence o1'non-cell bound
and noninternalized
~mmunotoxin in the large blood pool and capillary network. On the other hand,
it is unclear
CA 02424255 2003-03-26
tmmunotoxin 9
whether in specialized phagocytes such as Kupffer cells of the liver,
internalized proteins <;an
escape into the cytoplasm, e.g. to interact with the elongation factor-2, or
are rapidly directed to
lysosomal degradation (61). In addition, it remains to be determined whether
hexahistidine tags
can affect the biodistribution behaviour of recombinant proteins in viva. In
the kidney the level of
radioactivity is always higher with metal-labeled than with iodinated proteins
which are prone to
dehalogenation (6~,). Therefore, metal-labeled proteins probably more
accurately reflect the real
picture of in viva biodistribution (63).
(,onstruction and purification of 4DSM(.)(."B-ETA
To make the Ep-CAM-specific scFv 4DSMOCB into an immunotoxin, this scFv
antibody was
fused to a truncated ETA (ETAZsz_~,>H) by means of a ~0 amino acid long
peptide linker (Figure
1 A). The C-terminal original ER retention sequence REDLK of wild-type ETA (aa
609-613) was
replaced by the mammalian counterpart KDEL which increases the cytotoxic
potency of the toxin
m tumor cells (38, 45). Furthermore, we added a second hexahistidine sequence
at the N-terminus
of 4DSM()('B to increase the efficiency of purification by Ni2+-1DA affinity
chromatography
(Figure l A) The final construct encoded a protein of 64R amino acids with a
theoretical
isoelectric point of 5.9. figure lB show; a computer model of the mature
4DSM0C.'B-ETA
immunotoxin molecule.
During 1P'f(~ induction, more than 90% of the total immunotoxin detected by
Western blot was
found in the periplasmic soluble fraction of E. coli and was released upon
cell fractionation. The
Bnal product yield was 0.5 mg of a 95°/.. pure immunotoxin preparation
per liter bacterial culture
in standard shake t7asks. The product mtgrated at the expected size of
approximately 70 kDa on
SDS-PAGE (Figure 1C) and the theoretical M~ of 69,737 Da was verified by mass
spectrometry.
I'he absence oi' proteolytic degradation was conftrmed by Western blot
analysis (Figure l D).
lmmunoreactivity and stability of 4DSMOCB-ETA
Thermal stability and resistance to protease degradation of an nnmunotoxin is
of paramount
importance for its tumor targeting properties, and thus for therapeutic
efficacy. To investigate the
stability of 4DSMOC:B-E'TA, the fusion protein was incubated in PBS for
different time periods
at 37°(.' and the rate of degradation was analyzed by gel filtration
essentially as described (38). As
shown in Figure 2, upon a 4 h incubation at 37°C, 91°i~ of the
immunotoxin molecules still eluted
as monomers at the retention volume of 1.4 ml, corresponding to a M~ of
approximately 66 kDa.
The amount of 4DSMOC B-ETA only slowly decreased with time and approximately
47% of the
CA 02424255 2003-03-26
Immunotoxin 10
initial protein still eluted in monomeric form after 20 h at 37°C.
Similar results were obtained
upon incubation of ''''"'Tc-labeled 4DSMOCB-ETA in human serum, further
corroborating the
suitability of the immunotoxin for in vivo application.
To assess the effect of the additional N-terminal hexahistidine tag on the
antigen binding affinity.,
we determined the amount of immunoreactive immunotoxin in a binding assay as
described (43).
Upon a 1 h incubation at 37°C, the y''"'TC-triearbonyl quantitatively
bound to the histidine t;~gs of
the immunotoxin. As determined in cell binding assays, g0-90% of the
immunotoxin retained its
binding activity for Ep-(."AM after the labeling procedure. The K" of the
immunotoxin to Ep-
C.'AM expressed on SW2 cells was determined to be 4 nM, which was essentially
the same as
observed for the scFv 4DSMOCB assessed in a similar test system (3H). The low
level of
immunotoxin degradation could he completely prevented by the addition of
protease inhibitors
even after a 48 h incubation at 37°C in PBS (data not shown). Thus, the
immunotoxin
4DSM0<.'B-ETA retained all the favorable biophysical properties of the
parental scFv.
Ep-CAM expression on tumor cell lines
Ep-CAM is overexpressed in many solid tumors of diverse histological origins
(5). As shown in
Table t, the highest level of Ep-C'AM was expressed on HT29 cells (MFI 696.1
), followed by
MC'F7 (MFi 419.5), CAL27 (MF1 415.3) and SW2 cells (MFI 372.4). The cell tines
RL and
COLO320 cells did not express Ep-CAM and were used as antigen-negative
controls.
TABLE 1
Tumor cell line Tissue of origin Staining Controlb
MFI"
Tumor cell line Tissue of' origin Staining Control'
SW2 lung 372.4 t 4.0 3.4 ~ 0.5
HT29 colon 696.1 t 1 1.4 5.4 f 0.4
C AL27 tongue 415.3 t 4.8 5.6 t 0.3
MCF7 breast 419.5 ~ 1.1 4.5 t 0.6
RL B lymphoblast 4.2 t 0.2 4.0 t 0.2
COL0320 colon 5.5 t 0.1 5.1 t 0.1
" The values are expressed as mean fluorescence intensities (MFI t SD) of
three
independent FAC S analyses of tumor cell lines stained with the Ep-CAM-
specific
antibody KS 1 ~4.
~' lJnspecitic staining was assessed by incubation of cells with the FITC-
conjugated
secondary antibody alone.
CA 02424255 2003-03-26
Immunotoxin 1 1
C.'ytotoxicity of 4DSMOCB-ETA against tumor cells in vitro
To determine the ability of 4DSMOCBI?TA to specifically inhibit the growth of
Ep-CAM-
positive tumor cells, MTT assays were performed. The immunotoxm was
specifically cytotoxic
against Ep-CAM-positive cell lines and did not affect the growth of'the Ep-
(:AM-negative cells
RL and C OLO320 in the broad range of concentrations tested (Figure 3). SW2,
CAL2'7 and
MCF7 cells were found to be equally sensitive to the cytotoxic effect of
4D5MOGB-ETA and
their proliferation was inhibited with an IC50 of only 0.00 pM. Despite the
highest level of Ep-
CAM expression (Table 1 ), HT29 cells were found to be the least sensitive
(IC50 of 0.2 pM). In
the range of concentrations tested, the cyt.otoxicity of the immunotoxin was
completely blocked
by an excess of scFv 4DSMOC.B (data not shown)..
As determined in [3H]leucine incorporation assays (data not shown), treatment
of SW2 cells with
4DSMOCB-ETA inhibited protein synthesis with an IC50 of 0.01 pM, and this
effect showed the
same doseresponse relationship as measured in the cell viability assays
described above. Protein
synthesis was not inhibited in the antigen-negative control cell line RL.
Tumor localization of 4DSMOCB-ETA in mice
(n order to spare normal tissues from cytotoxic damage and employ the full
cytotoxic potential of
4DSMO('B-ET'A demonstrated ire vitro for targeted cancer therapy, the
selective and preferential
localization of the immunotoxin to Ep-(."AM-positive tumors is a prerequisite.
We assessed the
tumor localization properties of 4DSMOC'B-ETA in a biodistr~bution experiment
in mice bearing
established Ep-CAM-positive SW2 and Ep-C'AM-negative COLO320 xenografts at the
contralateral flanks As shown in Table ?, the maximum dose of radiolabeled
4DSMOCB-E,TA
detected in SW2 tumors was 2.93'% ID/g after 4 h, which then gradually
decreased to 1.95°~o ID/g
after 24 h. After 48 h radioactivity was still 1.13°/> ID~g tumor
tissue. In COL,O320 control
tumors 4DSM0(~B-ETA localized with a maximum dose of 1.65% ID/g after 30 min,
which then
rapidly declined to 1.06% II)ig after 4 h and showed only background levels
after 48 h. As
expected from its larger size, 4DSMOCB-FT'A showed a slower blood clearance
than the parental
scFv 4DSMOCB (data shown in Table 2 for comparison).. After 24 h the total
dose of
4DSM()CB-ETAin the blood was 0.42°r« ID~g, which was l.~-fold more than
measured for the
scFv (0.28% ID/g). Moreover, localization of the immunotoxin in SW2 tumors was
also delayed
compared to the scF'v, and the distribution of 4DSMOCB-ETA revealed a
tumor:blood ratio of
5.38 after 48 h, which was comparable tc~ the ratio obtained with the scFv
after 24 h.
CA 02424255 2003-03-26
Immunotoxin I 2
At each time point 4DSMOCB-ETA preferentially accumulated in Ep-CAM-positive
SW2
tumors compared to COLO320 control tumor with a SW2:COL0320 ratio varying
between 1.28
and 2.95. This indicates that. 4DSMOCB-ET'A was retained in Ep-CAM-positive
tumors by
specitic antibody-antigen interactions and cellular uptake, and suggests that
its marginal
accumulation in COLO320 control tumors may be do to the increase in vascular
permeability
often found in tumors. Analysis of normal tissues revealed that 4DSMOCB-ETA
localized also in
the kidney, spleen, liver and to a lower extent in the bone tissue of the
femur.
TABLE 2
Biodistribution of f''~~"TcJ-labeled 41)SMOC'B-ETA in micc3 bearing SW2 and
COL0320 tumor xenografts
4DSMOCB-ETA 4DSMOCB
immunotoxin scFv '
1l.) 30 min 1 h 4 h 16 h 24 h 48 h 24 h
min
(~~-;) (n=3) (n-:..3)(n=-3) (n3) (n:r-3) (n=3) (n=3)
~issw ~uID/g<'iolD/g %ID/g oID/g %IDlg ~olD/g %ID/g %ID/g
Blood 27.21 17.46 10.08 2.23 f 0.57 0.42 0.21 f 0.28
f 4.26 t 3.28 t 2.89 0.:32 f (1.14 t 0.04 0.02 t 0.06
Heart 10.84 6.69 6.20 2.23 f 1.14 0.64 0.52 t 0.28
t 1.96 f 3.88 +. 1.10 0. I f 0.04 t 0.47 0 05 t 0.09
8
Lung t 1.56 8.38 6.21 2.59 f 1.25 0.97 0.77 t i..14
f 1.66 t ) r 0.71 0.26 f 0.13 ~ 0.12 0.22 t 0.60
77
Spleen 735 f 12.17 I 1.64 7.70 f 8.81 6.24 4.07 f 0.70
1.50 f 1.70 t 1.74 5.65 ~ 1.26 t 0.68 1.69 f 0.13
Kidney 22.49 32.68 33.50 42.54 32.98 22.79 16.54 300.(1
f: 8.28 f. 1.49 t 2.32 f 6.(11 .t 1.14 f 1.76 f 0.39 t 85
0
Stomach 0.92 1.2 3 2.72 0.85 r I .15 0.63 0.45 t 0.24
f ().30 f ().40 + I 0. I ~ ().69 ~ 0.09 0.09 f_
.3 3 3 0.24
Intestine 1.78 2. I 2.31 2.30 t 1.21 0.90 0.58 t 0.30
~ (1.52 9 t i: 0.5'21.09 t f).22 t 0.09 0.06 ~ 0.07
(1.05
Liver I 5.47 20.44 I 9.97 20.2() 16.28 ( 3.70 8.44 f 2.38
t 3.24 f 0.70 t 3.77 f 1.26 t 2.51 f 1.83 0.49 t 0.52
Muscle 0.63 0.8() 0.57 0.58 ~ 0.37 0.26 0.16 f 0.10
t 1).14 ~ (1.15 i 0.07 0.19 ~ f1,()3~ 0.05 0.02 t 0.02
Bone (femur) 3.91 6.24 3.96 3.76 t 3.(15 2.85 I .08 0.06
f 0.97 t (1.96 .z 0.23 0.43 ~ ().37 t 0.49 ~ 0.20 t (').05
COL0320 tumor0.79 I .65 1.26 I .19 1.06 0.66 0 55 f ND
t 0.10 f (1.33 t: 0. t. 0.05 ~- 0.20 f 0.03 0.14
I 1
SW2 tumor 1.01 2.45 2.45 2.93 t 2.26 I .95 I 13 t 1.47
t 0.22 t 0.3:3 -r. 0.80 ~ 0.53 t 0.37 0.08 t 0.32
0.47
Ratios
COL0321) tumor/blood(1.03 0. l0 0.13 0.48 2.09 1.57 2 61
ND
SW2 tumor/blood(1.04 0.14 0.24 1.31 3.97 4.(i4 5-38 5.25
SW2 tumor/COL.03201.28 1.48 1.94 2.76 1.9(l 2.95 2 05 ND
tumor
" Biodistribution of9g"'Tc-labeled 4DSMOCB-ETA immunotoxin was determined
following i.v
injection in mice bearing SW2 and COL0320 tumor xenografts at contralateral
site. Data
represent the percentages of injected dose (ID t SD) per gram tissue. ND, not
determined.
'' Ratios presented were calculated from averages of tumor: blood or
tumoraumor ratios of
Individual mice.
Taken from Willuda et al~ ( 1999) for comparison.
CA 02424255 2003-03-26
~mmunotoxin I :i
Toxicity of 4DSMOCB-ETA in mice
'The unexpected high localization of radioactivity in liver., spleen and bone
raised the issue of
potential toxicity to these tissues under immunotoxin therapy. To assess the
toxicity of escalating
doses of 4DSMOCB-ETA, C57BL/6 mice were used as immunocompetent hosts which,
in
contrast to athymic mice, are more sensitive to wild-type E'TA-mediated liver
damage (46,47).
Interestingly, the determination of ALT AST levels in the plasma of C57Bt./6
mice 24 h after
treatment with three cycles of either 5 lig or i 0 pg immunotoxin given every
other day, did not
reveal immunotoxin mediated impairment of liver function (Figure 4). Elevated
transaminase
activity was only observed upon administration of two 20 pg doses of
immunotoxin which
equaled the activity measured in control mice upon administration of a toxic
dose of wild-type
ETA (46). In line with these results, few sites with necroriC hepatocytes were
only found upon
treatment with the highest immunotoxin dose. At all doses tested, analysis of
the spleen and the
cellular components of whole blood samples did not show any signs of
histopathological changes
or myelosuppression, respectively (data not shown). Thus, the accumulation of
radioactivity in
liver, spleen and bone does not reflect the amount of cell-bound or
internalized immunotoxin in
khese tissues
Antitumor activity of 4DSMOCB-ETA
An essential requirement for the clinical use of anti-cancer agents is a
significant antitumor
activity demonstrated in animal models of human tumor xenografts. To
investigate whether the
potent in vitro cytotoxicity and the favorable tumor localization properties
of 4DSMOCB-E:TA
translate into antitumor activity, mice bearing large established SW2, HT29 or
CAL27 tumor
xenogralts were treated by i.v. injection of the immunotoxin with two
different dose schedules: (i)
45 pg total, with .5 p.g given every second day for B weeks, (ii) ~i0 pg
total, with 10 lig given every
second day for I week. Mice bearing Ep--CAM-negative ('O'L.O320 tumor
xenogratts were used
as controls. All treatment doses were well tolerated and mice did not show any
signs of toxicity
such as weight loss or impaired liver function (Figure 4).
As depicted in Figure 5, a significant inhibition of the growth of all Ep-CAM-
positive tumors was
achieved by treating mice with either the 5 p.g or the 10 wg dose schedule.
Treatment of mice
bearing SW2 xenografts resulted m a shrinkage of the tumor volume to maximal
20% of' the
initial size and a slight resumption of growth to a final 2.6-fold size
increase at the end of the
monitored period. A similar effect was achieved upon treatment of ('AL27
tumors, which were
reduced to maximal 60% of the initial volume. Fifty days after start
ot'treatment the median
CA 02424255 2003-03-26
tmmunotoxin 14
tumor volume did not exceed 1.4-fold the initial size. 'Two mice out of 7
treated with the S pg
dose showed complete tumor regression and remained tumor free. Neither CAL27
nor SW2
tumors showed a significant difference Gn their tumor response to the two
treatment schedules.
For HT29 tumors strong growth inhibition was achieved with the 5 pg dose given
for 3 weeks
when sizes decreased to 0.7-fold of the initial volume. As already observed
for CAL27 tumors, ~
out of 7 mice showed complete regression of their HT29 tumors. Unexpectedly,
the efficacy of
the 10 ltg schedule was comparatively luwer, indicating that for these tumors
a long-term
treatment is more effective. No antitumor effect ot'4DSMOCB-ETA was seen in
mice bearing
Ep-CAM-negative COLO320 control tumors (Figure 5).
EXAMPLE 1
Tumor cell lines
The colorectal carcinoma cell lines HT29 (HTB-38), COL0320 (CL-220), the
breast
adenocarcinoma cell line MCF7 (HTB-22) and the non-Hodgkin"s lymphoma cell
line RL (CRL,-
2261 ) were obtained from the American Type Culture Collection (ATCC,
Rockville, MD). The
squamc.~us cell carcinoma cell line' of the tongue CAL27 was kindly provided
by Dr. S. D. Bernal.,
Dana-farber C.."ancer Institute, Boston, MA The small cell lung carcinoma cell
line SW2 was
raised in our laboratory. Except for C'Af.27, which was maintained in
Dulbecco's Modified Eagle
Medium (Life 'Technologies, Grand Island, NY), cell lines were grown in RPMI-
1640 (Life
Technologies Inc., Grand Island, NY). Both media were supplemented with 10%
FBS (Hyclone,
Europe Ltd. ), 2 mM L-glutamine., 50 IU~ ml penicillin and 50 pg/ml
streptomycin. Cell cultures
were maintained at 37°C in a humidified atmosphere containing
5°~~ C02.
EXAMPLE 2
Construction of the 4DSMOCB-ETA expression vector
'The sequence encoding a truncated form of ETA ('E'TA~s~.~,"H) was amplified
by PCR from
plasmid pSW200 (25) and cloned as an 1 164 by EcoRI-Hinc~III fragment
downstream of the Ep-
C.'AM-binding 4DSMOC'B scFv sequence present in the pIG6-based (39) 4I)SMOCB
scFv
expression vector (38) The primers (Tox 1 ~
('TCGGAATT('GOTG(~(.'GCGGCGGAGTTCCCG
AAACCGTC'C: ACC'C.'CCJCC GGGTTCTTC: TGGT'TTA; Tox2: GTC'AAGCTTC.'TAC.'AGTTCGT
CTTTATGGTGATGCiTGGTGATCiCG(.'C"GGTT'TC('C'CK,GC"TG) introduced an EcoRI
CA 02424255 2003-03-26
lmmunotoxin ] 5
restriction site between scFv and toxin and a C-terminal hexahistidine tag
followed by the
endoplasmic reticulum ( ER) retention signal KDEL, a stop colon and a HindIII
restriction site.
To improve purity and yield during IMAC, a second hexahistidine tag was added
at the N-
terminus between the periplasmic signal sequence and the 4D5MOC'B coding
region. To this end,
two pairs of oligonucleotides (Xbal 5': ('TAGATAACG.AGGC~CAAAAAATGAAAAAGACAG
C'TA'TCGCGA'TTGCAGTGGCACTGGC'TGGTTTCGCTACCGT: Xbal 3': GCCACTGC'AAT
CGCGATAG("TGTCTTTTTCATTTT TTGCCCTCGT'fA'T; and EcoRV 5': AGCGCAG(~CCG
AC.CACCATC ATCACCATCA("CAT; ~;coRV 3': 'T("GTGATGGTGATGATGGTGGTCGGCC.
TGCGC".TACGGTAGCGAAACCAGCCAGT) were heated to 80°(', allowed to
anneal by
gradually cooling to room temperature and then ligated between the Xbal and
EcoRV sites of
pIG6-4DSM0('BETAH6KDEL. The sequence was experimentally confirmed.
F',XAMPLE 3
Expression and purification of 4D5MO('B-ETA
F'or periplasmic; expression of 4D5MOC'B-ETA the vector pIG6 was used, which
places the gene
under lac promoter control in SB536, an ~'. coli strain devoid of the
periplasmic proteases HhoA
and HhoB (40). Five ml 2YT medium containing ampicillin (100 pgiml) were
inoculated with a
single bacterial colony containing the 41)SMOCB-E'TA expression plasmid and
grown overnight
at 25°C. The bacteria were diluted in one liter of 2YT' medium
supplemented with 0.5% glucose
and ampicillin ( 100 pg/ml) to reach an fi5s~, nm between 0~ 1 and 0.2 and
transferred to 3-liter
baffled shake flasks. The culture was further grown at 25°(' to an A~5"
nm of 0.5 and
immunotoxin production was induced for 4 h by adding a final Concentration of
1 mM isopropyl-
p-D-thiogalactopyranoside (IPTG, Sigma). The harvested pellet derived from a
bacterial culture
with a 6na1 ,A55~ nm of 6 was stored at -80°(' For purification, the
pellet obtained from a one liter
culture was resuspended in 25 ml lysis buffer, containing SO mM Tris-HC1 (pH
7.5), 300 mM
NaCI, 2 mM MgSO4 and supplemented with EDTA-free protease inhibitor cocktail
(Roche
Diagnostics, Mannheim, Germany) and DNase I. The bacterial suspension was
lysed with two
cycles m a French Pressure Cell press (SL.S Instruments, Urbana, IL),
centrifuged at 48'000 g in a
SS-:34 rotor for 30 min at 4°C" and subsequently filter-sterilized
(0.22 pin). The immunotoxxn
present in the cleared supernatant was pin°i~ed by chromatography using
a BIOCAD-System
(Perceptive BioSystems) with a NiZ+-iminodiacetic (IDA) column and a HQ/M-
anion-exch;~nge
column coupled in-line as described (41 ). Before the lysate was loaded, the
Niz+-IDA column
was equilibrated with 20 mM Tras (pH 7.5), 300 mM NaC:I_ After loading, the
column was
washed three times with different salt solutions, all buffered with 20 mM Tris
(pH 7.5), in the
CA 02424255 2003-03-26
lmmunotoxin t 6
order 300 mM, 510 mM and 90 mM NaC'1. Subsequently, the column was washed with
20 mM
Tris (pH 7.5), l0 mM imidazole, 90 mM NaCI, before the bound immunotoxin was
eluted with
the same solution containing 200 mM imidazole (pH 7.5). 'The eluate was
directly loaded onto the
HQ/M-anion-exchange column and the bound immunotoxin was eluted with a salt
gradient of 90a
1000 mM NaCI, buffered with 20 mM Tris (pH 7.5). The fractions containing
4D5MOCB-ETA
were collected and concentrated using a 10 kDa cutoff filter by centrifugation
at 2000 g and 4°C
(Ultrafree-MC low protein binding, Millipore). The quality of purified 4DSMOCB-
ETA was
analyzed by a l0% SDS-polyacrylamide gel and Western blotting using a
horseradish peroxidase
(HRP)-conjugated anti-tetrahistidine antibody (QIAGEN, Elilden, Germany)
diluted 1:5000
according to the manufacturer's recommendations.
EXAMPLE 4
Analytical gel tiltration and determination of thermal stability
Ten micrograms of purified 4DSMOCB-EVTA were diluted in 50 pl PBS pH 7.4
containing
0.005°~o T'ween-20 and subsequently incubated at 37°C. Samples
were analyzed at different time
points (after 0 h, 2 h, 4 h, 8 h, 10 h and 20 h) by gel Eltration using the
Smart system (Pharmaczaj
Uppsala) with a Superose-12 PC3.2!30 column. The column was calibrated in the
same buffer
with three protein standards: alcohol dehydrogenase (M~ 150,000), bovine serum
albumin ( M,
66,000) and carbonic anhydrase (M,-29,000). 'The same analytical setting was
used to assess the
thermal stability of the '''''"Tc-labeled immunotoxin after a 20 h incubation
at 37°C in human
serum. The amount of immunotoxin monomers was determined by ~-scintillation
counting of the
eluted fractions.
EXAMPL.,E S
Radiolabeling and determination of antigen binding affinity
4DSMOCB-ETA was radioactively labeled by stable site-specific coordination of
''~"'Tc-
tricarbonyl trihydrate to the hexahistidine tags present in the protein
sequence (42); This
spontaneous reaction was induced by mixing 30 pl of immunotoxin solution ( 1
mg/ml) with one
third volume of 1 M 2-[N-morpholinojethanesulfomc acid (MES) pH 6.H and one
third volume of
freshly synthesized ''''"'Tc-tricarbonyl compound. The mixture was incubated
for 1 h at 37°C and
the reaction was stopped by desalting over a Biospin-6 column (BioRad,
Hercules, CA)
equilibrated with PBS containing O.OOS°,~~~ Tween-20q according to the
manufacturer's
CA 02424255 2003-03-26
Immunotoxm I 7
recommendation. The percentage of immunoreactive immunotoxin was assessed as
described by
Lindmo and co-workers (43). The binding affinity of the ''y"'Tc-labeled
immunotoxin was
determined on SW2 cells in a radio-immunoassay (R1A), essentially as described
for the scFv
4DSMOC'B (3H).
EXAMPLE 6
Cell growth assay
Inhibition of cell growth upon treatment with 4DSMOCB-ETA was determined in
standard MTT
assays based on the reduction of tetrazolium salt to formazan by the enzymes
from viable cells
(44). Briefly, 5,000 tumor cells were seeded in 9fi-well ELISA microplates in
a total volume of
50 gl culture medium per well Immunotoxin concentrations ranging from 0.0001-
100 pM were
added m a total volume of 100 pl per well and cells were incubated fc7r 72 h
under standard cell
culture conditions. Ten microliters of a 10 mg%ml MTT (Fluka) solution were
added to each well
and the plates were incubated for further 90 min at 37°C. Cell lysis
and formazan solubilization
were achieved by addition of l00 pl lysis butter containing 20% SDS in 50%
dimethylformamide
(pH 4.7 adjusted with a solution consisting of 80% acetate, 20% I M HCl) and
the released
formazan crystals were allowed to dissolve overnight at 37°C.
Absorption was quantified at 590
nm using a SPE.,(.'TRAmax 340 rnicroplate reader (Molecular Devices,
Sunnyvale, CA). To
demonstrate that the cytotoxicity of4D5MOCK-E'fA was due to inhibition of
protein synthesis in
oells, [3H]leuc~ne incorporation assays were performed as described ( 18).
Briefly, 2 x 104 <:ells
per well in leucine-free cell culture medmm were seeded into 96-well plates
and incubated with
increasing concentrations of 4DSMOC.B-ETA diluted in leucine-free medium to a
final volume of
200 pl. Cells incubated in leucine-free medium without immunotoxin were used
as control. Upon
a 24 h incubation at 37°(. under standard cell culture conditions,
cells were pulsed with 10 p.l
medium containing 1 yCi [4,5-;3HJleucine (specific radioactivity 5 TBq/mmol)
per well for 6 h
and harvested onto glass-fiber filters using a Harvester 96 (Tomtec, Hamden,
CT). The
radioactivity incorporated into cells was quantified m a Trilux 1450 Icquid
scintillation MicroBeta
counter (PerkinElmer Life Sciences, Wellesley, MA) and expressed as
percentages relative to
untreated controls.
CA 02424255 2003-03-26
~mmunotoxin I H
EXAMPLE 7
Flow cytometry
Cell surface expression of Ep-CAM was quantified by flow cytometry using the
mouse IgC~~a
KS114 (BD PharMingen, San Diego, ('A). As secondary antibody a FITC-conjugated
goat anti-
mouse F(ab')2 IgG (H+L) (Zymed Laboratories, San Francisco, CA) was used. All
staining steps
were performed in a staining buffer consisting of PBS supplemented with 1 %
(w/v) BSA and
0.04% (w/v) sodium azide. Cells (5 x 105) were harvested, washed twice with
ice-cold staining
buffer and incubated on ice for 45 min ire a total volume of l00 lrl staining
buffer containing l Irg
of the first antibody. Cells were washed and further incubated with 400 ng of
FITC-labeled
antibody in a final volume of 100 pl. After 30 min on ice, cells were washed
and resuspended in
300 p.l staining buffer for analysis. Fluorescence intensity was measured at
430 nm using a
FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA) and
quantified using the
CellQuestPro software (Becton Dickinsc7n, San Jose, CA ).
EXAMPLE 8
Mice
Six to R weeks old female CD-1 (ICR nu/nu) mice (Charles River Laboratories,
Sulzfeld,
Germany) were used. They were kept under specific pathogen-free conditions
according to the
guidelines of the Veterinary Office of'the Kanton Zurich. Tumors were raised
at the lateral flank
by s.c. injection of 107 cells and randomized to constitute groups with an
average tumor si~:e of
l60 mm3 Ten weeks old female (.'S7Bl.,i6 mice (Janvier, Saint Isle. France)
were used to
determine immunotoxin-specific toxicity in immunocompetent animals, which are
more sensitive
to the f?TA-mediated T cell stimulation that results in the production of TNF
by Kupffer cells and
perform by cytotoxic T cells.
EXAMPLE 9
Biodistribution study of 4DSMOC'B-ETA
To investigate the distribution of 4DSMO CB-ETA m mice, 6 wg yy'"Te-labeled
4DSMOCB-ETA
(specific radioactivity of 98.y TBq/mmo() were diluted in a total volume of
150 pl PBS and were
injected r.v.. mto mice bearing established SW2 and ("010320 tumor xenografts
at the contralateral
CA 02424255 2003-03-26
lmmunotoxin 19
flanks. Mice were sacrificed at different time points ( 10 min, 30 mm, 1 h,
4h, 16h, 24h and 48 h)
after treatment and organs were removed to measure the accumulated
radioactivity using a p-
counter. 'The amount of radioactivity per gram organ was given as percentage
of the total injected
dose which was arbitrarily set to l00%,
EXAMPLE 10
Cn viva toxicity of 4DSMOC'B-ET'A
Toxicity of the immunotoxin was determined in C'S7BL/6 mice by measuring
ALT/AST acaivity
in plasma upon repeated injections of escalating doses of 4D5MOC'B-ETA given
every other day
for three cycles (S Itg and 10 pg dose) or for two cycles (20 p,g dose). Whole
blood samples were
taken to assess the degree of myelosuppression based on alterations of'
cellular components. In
addition, tissue specimens from the livers and spleens of immunotoxin treated
mice were
analyzed for hcstopathological changes upon hematoxylin/eosin staining.
EXAMPLE 11
Antitumor activity of 4DSMOCB-ETA
Mice bearing tumor xenografts derived from the Ep-("AM-positive cell lines
C'AL27, HT29 and
SW2, and the Ep-C'AM-negative cell line COL0320 were treated i.v. every second
day with
either S ltg 4DSM()CB-ETA for a total of 9 applications (total dose 4S ltg),
or with l0 lug every
second day for a total of a applications (total dose 30 pg) in a volume of l00
pl PBS. Tumor
xenografts from untreated mice were used as control Tumor size was calculated
by measurement
of the shortest and longest perpendicular diameter using digital calipers
according to the formula
(short diameter)' x (long diameter) x 0.s,
CA 02424255 2003-03-26
lmmunotoxin
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CA 02424255 2003-03-26
Immunotoxin 21
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