Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF FRAGMENTING CERTAIN MONOCLONAL ANTIBODIES
Field of the Invention
; This invention relates to a method of
fragmenting monoclonal antibodies and to Fab'
monoclonal antibody fragments.
BACKGROUND OF T~E INVENTION
A number of monoclonal antibodies have found
' use in targeting tumor cells. B72.3 is a mouse
¦ monoclonal antibody prepared from immunizations using
10 a membrane enriched fraction of breast tumor ~ ~
metastases to the liver (ATCC HB8108). It reacts ~-
with 50% of breast carcinomas and 80% of colon ~ ~-
carcinomas. ~owever, it has no significant
reactivity with normal human tissues in vitro.
Therefore, it is clinically useful for the detection
of colon carcinoma lesions in situ.
Monoclonal anti-tumor antibodies have great
promise for radioimmunodetection and localization of
tumors. B72.3 detects a high molecular weight human
tumor associated glycoprotein TAG-72, which has been
purified and characterized. This antigen i8 obtained
from LS174T cells. LS174T is a cell line originally 1
derived from a human colon carcinoma which expresses
TAG-72 on its surface and in its cytoplasm.
Expression of this antigen is increased significantly
in xenografts of LS174T cells in nude mice compared
to LS174T cells maintained in tissue cell culture.
In humans, TAG-72 is a shed antigen 80 that B72.3 has
been u~ed as the basis of an assay for serum levels
of this molecule. A correlation is claimed between
serum.levels and the presence of certain cancer types.
In radioimaging studies using a murine
monoclonal antibody to carcinoembryonic antigen in a
human colon carcinoma xenografted into hamsters,
F(ab')2 fragments were shown superior to Fab
fragments and the intact antibody for
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scintiscanning. In double-label experiments with
anti-CEA antibody and control monoclonal IgG,
F(ab~)2 fragments were found to give better and
more rapid specific tumor localization than intact
antibody or Fab fragments.
F(ab')2 fragments also offer significant
promise for tumor imaging and possibly therapy
(J. Nucl. Med. 24: 316-326, 1983). For a number of
reasons it is better to use fragment8 of antibodies
[F(ab')2 and Fab] rather than intact antibodies for
immunoscintograph. Because of their smaller size, it
is easier for antibody fragments to penetrate
tumors. Fragments are also cleared from the
circulatory system faster than intact antibodies
thereby lowering the background signal in tissues
perfused with blood. More rapid clearing of
fragments would allow the use of radionuclides with
shorter half lives and thus reduce the radioactive
burden a patient receives. Fragments lacking the Fc
region of the intact antibody would not interact with
the tissues that have Fc receptors as part of their
normal structure. This would reduce non-specific
binding (background).
PROBL~M OF THE INVENTION
Certain monoclonal antibodies such as B72.3
are difficult to fragment with pepsin and papain.
These are the proteolytic enzymes conventionally used
for fragmentation. See On The Fragmentation of
Monoclon~al IgGl. IgG2a and Ig~2b BALB/c Mice, Parham
et al, Journal of Immunology, 131, December 1963 and
Preparation of F(abl)2 Fragmen~s From Mouse IgG of
Various Subclasses, Lamoyi et al, Journal of
Immunological Method 56 (1983) 235-243. Bromelain is
also ineffective in that the yield of F(ab')2
fragments is low. Also, bromelain fragmentation
produces additional fragments having molecular
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weights and structure similar to B72.3 F(ab')2
fragments thereby increasing the difficulty of
purifying B72.3 F(ab~)2 fragments.
~ SUMMARY OF THE INVENTION -
'Z 5 A convenient method for making F(ab')
Z fragments from certain monoclonal antibodies has been
found. The method comprises the steps of:
a) selecting a monoclonal antibody from the
group consisting of Kallestad W0214, Kallestad W4507,
10 DAS5, Phe 1.9, B72.3 and GAC 39; and ;~
b) subjecting the monoclonal antiboty to
chymopapain digestion, thereby fragmenting the
~ antibody to form F(ab')2 fragments.
'~ The fragments can be purified with anion
exchange chromatography.
Cleavage of B72.3 with chymopapain produced
F(ab~)2 fragments in good yields. The fragments -
were easy to purify using anion exchange
chromatography. Moreover, Fab' fragments can be
- 20 prepared from B72.3 F(ab')2 fragments by selective ~ -~
reduction of the inter-heavy chain disulfide bonds of
the F(ab')2 fragments with dithiothreitol (DTT).
The F(ab~)2 and Fab' fragments retained the ability
Z to bind to TAG-72, the tumor associated glycoprotein
25 recognized by the intact antibody.
DETAILS OF THE INVENTION
Chymopapain is a proteolytic enzyme from
papaya latex. Experiments were performed to study
the effect of various concentrations of chymopapain
30 and various times of proteolysis on the fragmentation
of B72.3. In general at least 4% up to about 10% by
I weight (compared to weight of B72.3) of chymopapain
is required at temperatures as low as 25C for
several hours. The preferred range of conditions for
35 giving primarily F(ab'~2 fragments with very few
¦ side products are 4 to 8% chymopapain (percent by
~ weight
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compared to weight of B72.3) for 1 to 3 hours at
about 37C.
Procedures used in carrying out the
following examples to confirm that the method of this
invention i9 operative were as follows.
ELISA
ELISA determination of binding activity of
B72.3 and its fragments was carried out as follows.
The antibody or its fragments were diluted to 3.16 x
10 7 M in 0.5% BSA-PBS. BSA-PBS is 0.010 M sodium -
phosphate, pH 7.9, containing 0.15 M sodium chloride
and 5 mg/ml bovine serum albumin. Further dilutions ---
(square root of 10) were made in the same buffer. '~`
One hundred ~L of each dilution was added to the
appropriate wells of microtiter plates that contained
antigen (and had been blocked by the addition of 200
~1 of 0.5% BSA-PBS for 1 hour at 37C) to allow the
antibody-antigen complex to form. The wells were
then washed 3 times with PBS solution and 100 ~L of
a 1:5000 dilution of an anti-mouse F(ab~)2-peroxi-
dase (HRP) conjugate was added to each well. The
plates were incubated for 1 hour at room temperature
with shaking. The plates were then washed six times
with PBS solution. Color was developed in each well
by adding 100 ~1 of peroxidase (HRP) substrate
buffer (9.0 mL nanopure water; 1.0 mL of 170 mM ~ -
citric acid, 650 mM sodium phosphate, 0.1%
merthiolate, pH 6.3; 5 ~L 30% ~22 and 2
tablets of orthophenylene diamine tablets). Color
30 development was stopped by the addition of 100 ~L ~
of 4.5 N sulfuric acid to each well, after the ,-
desired color density was reached. The plates were
read colorimetrically using a 492 nm filter.
Sla~ Gel Electrophoresis - Sodium Dodecyl
35 Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) -
was carried out using either a ~oefer Protean
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Apparatus (~oefer Scientific Instruments, San ~ -~
Franciaco, CA) or a Pharmacia Phastgel System
J (Pharmacia Biotechnology, Inc., Piscataway, NJ). Ten
percent polyacrylamide gels (1.5 mM thickness) were -
5 prepared for use as the resolving gel with the ~oefer
apparatus. Pharmacia 10-15% gradient gels werç used
with the Phastgel System. Manufacturer~s - -
instructions were followed for the respective systems. ~ :
f
Western Blots
Electrophoretic separation (SDS-PAGE) was
done using the ~oefer apparatus under both reducing
and non-reducing conditions. The gels were run at 50
volts (constant) overnight with cooling.
Fifteen ~g of B72.3 fragments in 100 ~L were
placed in the sample wells.
The electrophoresed samples were transferred
I from the acrylamide gel to nitrocellulose paper
f (BioRad Trans-Blot cell). A transfer "sandwich" was
20 formed according to conventional procedures. The
f sandwich was immersed in transfer buffer (240 mL
! "lOX" electrode buffer, 60 g Tris, 280 g glycine in
¦ 2 L distilled water), 1600 mL chilled methanol, 24 mL
1 10% SDS, and 2136 mL chilled distilled water in the
~ 25 transfer unit and transferred for 1.5 hours, 70
¦ volts, with cooling.
¦ One group of the samples was stained for
f protein by immersion in a solution of 0.1% Naphthol
f Blue Black in 45% methanol, 10% acetic acid and 45%
30 water for about 10 minutes or until protein bands
were visible. The rest of the nitrocellulose paper -~
was blocked with 1% BSA-PBS for 12 hours before being
scored. For F(ab')2 scoring, either a rabbit -~
anti-mouse F(abl)2-peroxidase conjugate (Jackson ~ -~
Labs, 315-3547, 1:1250 dilution in PBS containing
0.05% Tween 20 and 0.01% merthiolate), or a goat
,:
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anti-mouse F(ab~)2-peroxidase conjugate (Bohringer
Mannheim, 60530, diluted according to manufacturer~s
instructions in PBS containing 0.05% Tween 20 and
0.01% merthiolate> was used.
The blocked nitrocellulose paper was a)
incubated with the selected diluted conjugate for 1
hour with gentle shaking; b) washed three times
with PBS containing 0.05% Tween 20 and 0.01%
merthiolate; c) washed once with substrate buffer
10 (17 mM citric acid, 65 mM phosphate, 0.01%
merthiolate, pH 6.3); and d) incubated with substrate
solution (60 mg of 4-chloro-1-naphthol in 20 mL
methanol at -20C plus 80 mL of 17 mM citric acid,
65 mM phosphate, p~ 6.3; 60 ~L of 30% H202)
with shaking until the desired amount of color formed
~ (about 5-20 minutes). The paper was washed with
¦ distilled water to stop the color development. For
Fc scoring, the same procedure was used except the
scoring reagent was a rabbit anti-mouse Fc-peroxidase
! 20 conjugate (Jackson Labs, 315-3546, 1:1000 dilution
with PBS containing 0.05% Tween 20 and 0.01%
merthiolate)~
~ ....
i Example 1
1 25 Chymopapain Fragmentation
¦ Ten mg of chymopapain (Sigma) was dissolved ~ ~
¦ in 2.5 mL of a phosphate buffer solution (PBS) ~-
comprising 0.01 M sodium phosphate and 0.15 M NaCl
adjusted to pH 7.4. The chymopapain solution was ~ -
equlibrated with PBS by passing it through a size
exclusion column (PD-10 from BioRad) which had been
! pre-equilibrated with PBS. The enzyme was removed by
elution with 3.5 mL of PBS.
, For these experiments, 10 mg of B72.3 was - -~
¦ 35 equilibrated with PBS using the size exclusion column.
For a typical fragmentation with chymo-
papain, chymopapain was added to the antibody at a
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200821~
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concentration of 4% of the antibody weight (400 ~g
of papain to 10 mg antibody). The proteolysis wa~ -
allowed to proceed for the desired time (usually 3
hours) at 37C after which it was stopped by the
addition of iodoacetamide to a final concentration of
30 mM.
Purification of F(ab')2 Fragments
The purification was achieved by anion
exchange chromatography. A diethylaminoethyl (DEAE)
cellulose anion exchange resin (10 mL of DEAE-
Sephacel obtained from Pharmacia LKB Biotechnology,
Piscataway, NJ) was washed with 5 mM Tris-~Cl, pH
7.5, in a 15 mL scintered glass funnel. Tris is
tris(hydroxymethyl)aminoethane. Two mL of the washed ~ -
suspension were packed into a column (1 cm x 1 cm)
and washed with a further 15 ml of 5 mM Tris-~Cl, pH
7.5.
Chymopapain fragmented B72.3 (0.5 mL,
1.8 mg/mL) was equilibrated with 5 mM Tris-~Cl, p~
7.5 on the size exclusion column (PD-10). The 3.5 mL
volume in which the equilibrated B72.3 fragments were
collected was passed through the DEAE column. The
F(abl)2 fragments were not bound to the column.
Fractions containing the F(ab')2 fragments were
collected until the initial protein peak was eluted.
The fractions containing the F(ab~)2 fragments were
pooled and concentrated.
Analysis of F(ab')2 Fragments
The eluted F(abl)2 fragments were analyzed
30 by electrophoresis described supra and found to be -~
free of the cleaved Fc. After anion exchange
purification, the yield of F(ab')2 fragments was
calculated to be 75-80% using a conventional protein
assay procedure with bovine IgG for calibration. ~ -~
The F(ab')2 fragments retained the ability
to bind antigen using the ELISA described au~ra.
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Western blot analysis indicated that the
fragments had the correct structural characteristics
of F(ab~)2 fragments. More specifically, the
fragments were scored by anti-F(ab~)2 antigens but
not by an anti-Fc antigen. Reduced samples showed
both the light chain and a slightly larger band
(presumably the cleaved heavy chain) when stained for
protein. To analyze the reduced samples, two
anti-F(ab')2 scoring reagents were used. One
seemed to be specific for light chains only, while
the second reagent seemed to cross react with the Fc
region of the heavy chain. Using either probe, the
cleaved heavy chain in the reduced sample was not
detected indicating that it probably originated from
an F(ab') or F(ab')2-like fragment. The anti-Fc
reagent in this test was not as sensitive as the
anti-F(ab~)2 reagents. ~;
Example 2
Fab' Fra~ments from F(ab')2 Fragments
Purified, F(ab')2 fragments from example 1
served as the starting point for the preparation of ~ ;-
Fab' fragments. The approach was to selectively
cleave the inter-heavy chain disulfide bonds while -~
25 leaving the heavy chain-light chain sulfide bonds ~ -
intact. By adding excess DTT to a sample of
F(ab~)2 fragments for an incubation period of 90
minutes at 37C, and subsequently removing the excess
DTT using the size exclusion column, Fab' fragments
30 were produced. ~ -
-~
Example 3
The Reaction of Chymopapain with Other Monoclonal
Antibodies
Other monoclonal antibodies (Table I) were
digested with chymopapain (4%, 4 hours, 37C).
,
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Presence or absence of F(ab~2 fragments was
confirmed using sodium dodecysulfate electrohporesis
which separated the fragments according to size. The
results show that chymopapain is not a general
fragmentation reagent. It only fragments selective
antibodies. -:
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i Comparative Exam~le
Fragmentation of B72.3 with Bromelain
Bromelain is a proteolytic enzyme from
pineapple stem. Initial experiments using the
! 5 preactivated bromelain indicated F(ab')2 fragments
were being produced. Western blot analysis confirmed
that the major polypeptide fragment reacted with the
~ proper detection antigens. For example, the
3 non-reduced sample of F(ab')2 migrating slightly
i~ 10 slower than the 116,000 molecular weight marker
reacted with the anti-F(ab')2 antigen and not with
the anti-Fc antigen. When electrophoresed under
reducing conditions, a light chain and partial heavy
chain were observed. However, overall yield of
F(ab')2 was not good as indicated by the intensity
of the F(ab')2 band in the electrophoretic analysis
which was considerably weaker than the corresponding -~
amount of B72.3. Production of other fragments by
bromelain close in size to the desired F(ab')2
fragments was also observed. It was felt that these
contaminants would be hard to remove during purifica-
tion of F(ab')2 because of their similarity in size
and, presumably, in structure to the desired
fragments.
Fab' Fragments from Bromelain-Produced F(ab~2
Fragments
Attempts were mate to prepare Fab' fragments
from the bromelain-produced F(ab')2. The methods
1 centered on ways to selectively reduce the inter-
1 30 heavy chain disulfide bonds while leaving the light
chain-heavy chain disulfide bonds intact. Conditions
included cleaving F(ab')2 with dithiothreitol
(DTT), ~-mecrcaptoethanol, or cysteine (Cys)
~ followed by treatment with H202 (to reoxidize the
¦ 35 appropriate disulfide bonds), or alkylation with
iodoacetamide (to prevent reformation of the ~ ;
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Z008213
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inter-heavy chain disulfide bonds), or removing
excess reducing reagent. No Fab~ fragment8 were
detected.
The invention has been described in detail
5 with particular reference to preferred embodiments .~ .
thereof, but it will be understood that variations -:~
and modifications can be effected within the spirit
and scope of the invention.
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