Note: Descriptions are shown in the official language in which they were submitted.
PCT/EP91/01223
W092/00763
2086~3~ -
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Immunoreactive compound
The present invention relates to an
immunoreactive compound and a pharmaceutical
preparation comprising the same.
Such an immunoreactive compound can particularly
be used in immunotherapy and in diagnosis.
Immunotherapy is one of the promising
possibilities to fight a number of diseases. The
principle of immunotherapy itself is old. Tt comprises
a targeting moiety which delivers an active substance
to the immediate vicinity of the target.
Thus it can be used to kill, or optionally
stimulate a certain group of cells which share a site
for which a targeting moiety is available. Ligand-
receptor interactions or antibody-antigen in~eractions
are suitable couples of targeting moiety and target,
but others can of course be envisioned ~y the person
skilled in the art.
A probably more elegant way of immunotherapy are
the so called pretargeting strategies. These include,
but are not limited to, prodru~ activation, whereby an
enzyme is coupled to a targeting moiety, which is
administered before or together with a prodrug which
is less toxic than its parent drug and which enzyme
converts the prodrug into the parent drug at the
target site.
- For anti-tumour therapy and tumour localization,
and for cancer diagnosis generally use is made of
antibodies coupled to a label. In anti-tumour therapy
such a label can be e.g. a toxic compound such as
adriamycin, verrucarin, calicheamycin, mitomycinj
ricin a, or any other suitable toxic compound, or an
isotope or, as described above an enzyme.
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PCT/EP91/01223
WO92/00763
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The antibodies generally are targeted against a
particular antigen of the tumour. The antibodies used
for this purpose in most cases are monoclonal
a~tibodies of murine origin. Murine monoclonal
antibodies are easy to obtain according to well
established methods, and against virtually any
antigen.
Nevertheless these antibodies have several
drawbacks. If tumour material is administered to mice
these develop antibodies against almost any antigen of
the tumour material, including the normal antigens of
these cells. In this way it is difficult to obtain
antibodies specific for tumour cells only. Hence,
murine antibodies may be directed to epitopes that are
not tumour-specific according to the human immune
repertoire. Furthermore the use of murine antibodies
is hampered by their inherent immunogenicity in
humans. A solution has been sought in the use of
antibody fragments mainly containing the antigen
binding domain of the murine antibody, which may
overcome the second, but certainly not the first
problem.
A more ideal solution resides in the use of human
anti-tumour antibodies. Human anti-tumour antibodies
can suitably be obtained according to the metho~
described in EP 0151030. A problem is, however, that
this method mainly yields immunoglobulins of the IgM
and/or IgA type. These are in fact pentameric or
dimeric, i.e. they are composed of five or two
monomers interconnected via S-S bridges, whereas each
of these IgM/IgA monomers is composed of two heavy and
two light chains and contains two antigen binding
sites. Each of these monomers roughly e~uals an IgG
molecule in size. Hence a complete IgM molecule is
about five times the size of an intact IgG molecule.
Most IgM's are characterized by antigen-affinities
which are at the low end of the IgG affinity range.
~092/00763 PCT/EP91/01223
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The relatively large dimensions of the IgM
molecules make them less suitable for in vivo use for
immunotherapy and for tumour imaging; it takes a
relatively long time for them to reach the target site
and the clearance of unbound Ig~ taXes at least five
times longer than for Ig~-.The same goes for IgA's,
though not in the order of magnitude as with IgM ' 5 .
A straightforward solution looks to be the
fragmentation of the IgM pentam~r or the IgA dimer
into its monomers. However, it has been reported that
the antigen-affinity of the IgM/IgA monomers is
dramatically lower than the affinity of intact
IgM/IgA; the difference amounts to at least about a
hundred to thousand-fold. This low affinity makes the
IgM/IgA monomer unsuitable for therapeutic and
diagnostic application. A similar lowering of affinity
was found for enzymatically obtained fragments of IgM
and IgA.
The present invention is concerned with the
restoration of the antigen affinity of fragments of
IgM. According to the present invention the af~inity
of antigen binding IgM or IgA fragments can be
restored by coupling them to at least one polypeptide.
Such a polypeptide can advantageously be a human
protein such as human serum albumin, or a (human)
enzyme, or it can be a synthetic polypeptide with a
low immunogenicity such a poly-L-glutamic acid or
poly-L-lysine. The IgM or IgA fragment either can be
an IgM or an IgA monomer, which can be obtained by
reducing the S-S bonds between the monomers, or can be
an antigen binding fragment obtained after enzymatic
cleavage of the IgM or the IgA, e.g. by use of pep~in
or papain. Digestion with pepsin delivers an antibody
fragment generally indicated as F(ab')2 which in turn
is composed of two antigen bindings parts
interconnected by S-S bonds. Reduction of the~e bonds
yields two F(ab') fragments.
WO92/00763 PCT/EP91/01223
~o~6S3~
Bot~ IgM or IgA monomers and F (ab'~ fra~ments
id~ally can be bound to the polypeptide(s) via their
sulphur atom. However, binding of the IgM ~ragment to
the polypeptide can be by any other suitable bond, as
long as the antigen binding characteristics are not
hampered. In this respect it is also convenient to
establish a ~inding via glycosyl groups if present at
the constant region of the IgM or IgA fragment.
The bond between the IgM or IgA f ragment and the
polypeptide can either be a direct link or an indirect
link via a linking group and/or a spacer.
The bond between the IgM sr IgA fragment and the
polypeptide can be established by making available on
both components a group suitable for linking,
optionally reacting either or both linking groups with
a linker and/or spacer, and thereafter reacting the
components to form the desired immunoreactive
compound.
Optionally the polypeptide can be labelled with
one or more therapeutically or diagnostically useful
groups prior to or after the coupling to the IgM or
IgA fragment. Suitable therapeutically useful groups
are e.g. cytotoxic drugs, (optionally chelated)
radioactive atoms, or enzymes for the conversion of
prodrugs into active drugs.
However, if the polypeptide is an enzyme itself,
which is able to convert a prodrug into a drug at the
target site, there is an additional advantage, because
the size o~ the immunoreactive compound plays an
important role in its applicability. Suitable
diagnostically useful groups are e.g. (optionally
chelated) radioactive atoms. The IgM or IgA fragment
advantageously is obtained from human IgM or IgA. This
IgM or IgA is directed against an antigen specific
for, or derived from the tumour, which may be found
either in or on or outside the tumour cells.
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~0 92~00'763 P~/EP91/01223
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Example 1
~. Preparation of immunoreactive Iq~.f- monomers.
Human monoclonal IgM antibodies 16-88 against
tumour associated epitopPs occurring in colorectal
cancer were converted into monomers by reduction with
cysteine.
IgM (3-5 mg/ml) was incubated in lo mmol/l
cysteine in PBS (6.7 mmol/l K/Na phosphate buffer PH
6.5; 0.13 mol/l NaCl) for 3 h at 37 c. suffers were
saturated with nitrogen and the reaction vessel was
closed gas-tight. After incubation, the reaction
mixture was chromatographed on Sephadex G25,
equilibrated with 1 mmol/l cysteine in PBS. Monomers
were precipitated With (NH4)2S04 at 50% sa~uration and
dissolved in a minimal volume of 1 mmol/l cysteine in
PBS PH 7.5. The monomer solution was applied on a
Fractogel TSK HWf 55 (S) column equilibrated in
mmol/l cysteine in PBS PH 7.5. The bed volume of the
column was 45 times that of the volume applied and the
elution rate was 0.06 bed volume/h. Monomers were
usually eluted at a Kav=0.55-0.60, in a predominant
A~80 peak. They were precipitated by (NH4)2S04 at 50%
saturation. After dissolving the precipitate in 0.1
mol/l Sodium phosphate, 0.1 mol/l NaCl, 5 mmol/l EDTA,
1 mmol/l cysteine PH 7.5, residual ammonium sulphate
was removed by gel filtration on Sephadex 525
equilibrated in the EDTA containing PBS buffer
mentioned above.
Solid DTNB (dithionitrobenzoic acid, Ellman's
reagent) was added to the desalted monomer-containing
fraction to a final concentration of 20 mmol/l and
after gently shaking the reaction mixture was
incubated for 3 h at ambient temperature.
PCT/EP91/O~Z23
W092/00763
2 ~ 3 ~
Excess reagent and low molecular ~eight reaction
products were removed by gel filtration on S~phadex
G25 in 0.1 mol/l sodium phosphate; O.lmol/lNaCl;
5mmol/1 EDTA PH 7. 5 (Solution A).
B. Reduction of HSA(-D~PA).
HSA(-DTPA) was dissolved to a concentration of 5
10 mg/ml in 0.1 mol/l sodium phosphate: 0.1 mol/l
NaCl: 5 mmol/l EDTA PH 7.5. To this sol~tion, DTT
(dithio-threitol) was added to a final concentration
of 20 mmol/l and incubation was performed for 30 min.
at ambient temperature. The reaction mixture was then
chromatographed on Sephadex G25 equilibrated in ~he
EDTA/PBS PH 7.5 mentioned above in order to remoYe
excess of reducing agent and low molecular weight
reaction products (Solution B).
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C. Preparation of IqM-HSA(-DTPA) immunoconiuqates.
Immediately a~ter reduction of HSA(-DTPA),
solution A (containing activated monomers) and
solution B (containing reduced HSA) were mixed, the
monomers/HSA(-DTPA) mass ratio being around 0.5.
Incubation was performed overnight at ambient
temperature.
After completion of the conjugation reaction,
non-conjugated HSA(-DTPA) was removed by (NH4)2S04
precipitation at 50% saturation. Conjugate and non-
conjugated monomers were precipitated, whereas HSA(-
DTPA) remains in solution. The precipitate was washed
several times with 50 % saturated (NH4)2S04 and was
dissolved in a minimal volume of 0.1 mol/1 sodium
phosphate, 5 mmol/l EDTA PH 7.5. The solution was
chromatographed on Sephadex G25 equilibrated in the
EDTA phosphate buffer (buffer A) (devoid of NaCl !!).
The protein-containing fraction was then applied on a
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W092/00763 PCT/EP91/01223
5 3 1
Q-Sepharose (Fast Flow) column equllibrated in bu~fer
A in order to separate non-conjugated monomers from
the immunoconjugate by anion-exchange chromat~graphy.
After application of the sample, the Q-Sepharose
column was washed with buffer A until the A280 had
returned to ~aseline level. Proteins retained by the
column were eluted ~y stepwise increasing the NaCl
concentration from 0 - 0.6 mol/l NaCl.
The bulk of the non-conjugated monomers passed
the column in the fall-trough fraction, whereas the
immunoconjugate was retained because of the acidic
character of HSA(-DTPA) and was eluted at 0.3-0.4
mol/l NaCl in buffer A. After desalting the
immunoconjugate on Sephadex G25, the final preparation
was sterilised by filtration through a 0.20 ~m
membrane and stored in small aliquots at 4 C until
use.
Example 2
Preparation of IgM_Fab'-HSA~-DTPA)_conjuqates.
IgM was digested with pepsin according ~o the
method of Putnam. Briefly, whole IgM at a
concentration of 2-5 mg/ml in O.l ~ol/l sodium acetate
buffer PH 4.0 was incubated with pepsin (0.08-0.2
mg/ml) for 8 h at 4 C. The reaction mixture was
chromatographed on Fractogel HW55S equilibrated in
buffer A in order to purify the F~ab')2 fragments
formed from whole, undigestsd I~M and low molecular
weight fragments. In this way, chromatographically
pure F(ab')2 was isolated at a 40-60% yieId.
F(ab')2 was reduced with DTT and coupled to HSA(-
DTPA) in the way described for the monomers.
Purification of F(ab')-HSA(-DTPA) was achieved by
anion exchange chromatography on Q-sepharose and gel
filtration on Fractogel as described above. The final
prep was sterilised by filtration and stored at 4 C.
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Wo 92/OU763
2~8~531
ExamDle 3
Immunoreactivitv_ of IqM, IqM monomers, I~M F~ab~)2
fra~ments. IgM monomer-HSA(-DTPA~ and IqM Fab~ HSA(-
DTPA)-immunocontuaates.
Immunoreactivity was determined by either an
antigen binding assay or competitive EIA.
In the antigen binding assay (Dot-blot ~IA~
dilution series of the samples to ~e tested are
transferred into Immobilon membranes in a Biorad Trans
blot apparatus. Excess protein binding sites are
blocked with 5% Skim milk and the blots are then
incubated with peroxidase-labelled antigen in P~s
buffer. After 2 h of incubati~n at ambient
temperature. The antigen-containing solution is
discarded, the blot is washed three times with PBS-
Tween and the enzyme borinol is detected with a
substrate solution containing 2 mmol/l hydrogen
peroxide and 0.6 mg/ml diaminobenzidine, 0.6 mg/ml
CoC12 as hydrogen donor. Violet coloured spot~ become
visible a~ter 5 min. of incu~ation. The colour
intensity is measured by scanning in a Biorad gel
scanner.
In the competitive EIA, dilution series to be
; tested are incubated with a given amount of
peroxidase-labelled whole IgM for 3 h at ambient
temperature in an microtitre plate coated with 0.1
~g/ml antigen solution. After incubation, the contents
of the wells is discarded and the plates are washed
three times with PBS-Tween buffer. Enzyme activity is
detected with a substrate solution containing
~ tetramethylbenzidine as hydrogen donor. The enzyme
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YVO92/00763 PCT/EP91/01223
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reaction was stopped with 2 mol/l H2S04 and the
absorbance was read at ~50 nm.
Both assays give comparative results with respect
to immunoreactivity of IgM, IgM fragments and
immunoconjugates.
Purified monomers have a specific
immunoreactivity (=IR per mass~ of O.OOl-0.05 times
that of the untreated, whole IgM, whereas F(ab')2
fragments are not immunoreactive in the assays
applied. However purified HSA(-DTPA) conjugates of
monomers exhibit an immunoreactivity identical to that
of whole IgM and F(ab')-HSA(-DTPA) conjugates show
some immunoreactivity comparable to that of purified
monomers.
In conclusion, covalent attachment of a (carrier)
protein, e.g. through disulphide or thioether bridges,
is able to restore the immunoreactivity of IgM
monomers or fragments considerably, even to full
extent in case of monomers.
Example
,
Preparation of Enzyme coniuqate
(Enzyme-)conjugates could be prepared in two
ways:
A: By direct conjugation to SPDP-activated
enzyms
B: By activition of the monomer with DTNB,
followed by reaction with the enzyme, having
~ free -SH groups.
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PCT/EP91/0l223
WO92/~0763
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4.1. Method A
To the enzyme (10 mg/ml in 0,1 M Na2HP04/NaH2PO~
ph 7, 5 ' O ,1 M naCl) 1~10 volume of ~o mM SPDP
(disolved in absolute e~hanol) was added and
incubation was performed for 30 min. at room
temperature in the dar~. The reaction mixture was
chromatographed on a Sephadex G-25 (~) column
equili~rated with the same buffer to remove unreacted
SPDP. The SPDP activated enzyme was added to the IgM-
monomer (which was just before addition also
chromatiographed over Sephadex G-25 (M) equilibanted
in this buffer) in a ratio of 1 : 1 tw/w).
The reaction mixture was incubated for 16 hours
at room temperature in the dark. The conjugate was
-' recovered by addition of an equal volume of 100 %
satured ammonium sulph. The precipitate was washed
three times with 50 % saturated ammonium sulph, before
being dissolved in an appropiate buffer.
(Remark: These final steps could be performed
using HRP as an enzyme, for other enzymes other puri-
fication methods may be necessary).
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l 4.2. Method B
,
Activation of the enzyme:
First the enzyme was activated by SPDP us
described in method A. The activated enzyme was
chromatiografied on a Sephadex G-25 (M) column
equilibrated in 0,1 M NaAc pH 4,5; 0,1 M NaCl. To the
enzyme containing fractions 1/20 volume af lM DTT was
Ldded and incubation was performed for at least 30
minutes at room temperature in the dark.
Just before coupling this mixture was
chromatografied over a Sephades G-25 (M) procedure for
the preparation of IgM-monomer-enzyme conjugates.
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W092/00763 pcT/Epsl/ol223
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4.3. Monomerisation
(Human)IgM was monomerised by incubation at 37C
for 3 h in the following solu~ion (saturated wi~h N2).
5 mM Na2HPO4/NaH2PO4 pH 6,5
65 mM NaCl
O,1 g/L NaN3 10 mM Cysteïne
0,5 U papaïne/g IgM
1-10 g/h IgM
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After this incubation an equal volu~e of 100 %
saturated ammonium sulphate solution was added.
After s~anding at least 2 hours at 4C, the
precipitate was recovered (after centrifugation) and
dissolved in:
50 mM Tris .HCl pH 8
140 mM NaCl
1 mM Cysteïne
To remdve residual ammonium sulphate this
resolution was chromatographed over a Sephades G-25
(M) columns into the same ~uffer.
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4.4. Purification o~ IaM-momomer
;
The above mentioned solution was chromatographed
~ver a Fraktogel HW-55 (S) column (, equilibrated into
the same buffer).
The fractions containing the monomerie IgM,
(,usually the second peak) were pooled and
concentrated by addition of an equal volume of lOO %
saturated ammonium sulphate in a 1 mM Cysteïne-
solution. The precipitate was collected and used for
conjugation.
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PCT/EP91/01223
W092/00763
20 86S 3 1 12
4 . 5 Actlvation of monomerie I~M
The monomerie IgM was chromatographed over
Sephadex G-25 (M) into:
50 mM Tris~HCL pH 8
140 mM NaCl
; 1 mM Cysteïne
To this solution 15 mg DTNB/ml solution were
added (in a solid form) and made to dissolve. The
reaction mixture was incubated for 16 hours at room
temperature in the dark. Unreacted DTNB was removed by
~ chromatography over Sephadex G-25 (M), equilibrated in
; 0,1 ~ Na2HP04/NaH2P04 pH 7,5; 0,1 M NaCl.
The degree of activation of the monomer with TNB
could be determined in the following manner:
conc. of monomer = E 280 (M) (=A)
1,45 x Mw (=180.000)
conc. of TNB = E 330 (M) (=B)
9217
TNB/monomer = B/A
Conjugation: The actived HRP and the activated
monomeric IgM were added to each order in a ratio 1:1
(w/w). Incubation was perfor~ed for 16 hours at room
temperature in the dark. After this period the
~ conjugate was purified as described in method A.
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The immuno reactivity of the enzyme conjugates
was determined by incubation of the conjugate with a
microtiter plate coated with crude antigen mixture of
; a tumour cell line expressing antigens that are
recogni~ed by Monoclonal antibodies 16.88 and 81AV78
(shown in fig. 1). Mab 16.88 recognices specifically a
tumour-associated epitope on cytokeratins, whereas Mab
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W092/00763 13 PCT/~P9~/01223
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81MV78 reacts with a tumour-associated antigen at the
surface of the cells.
However a conjugate of an indifferent antibody a
myecloma IgM with no reactivity towards tumour cells
or cell lines, did not bind to the crude antigen
preparation.
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Mycloma is an antibody (IgM) recognizing an
antigen not present in the mixture of crude antigens.
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