Note: Descriptions are shown in the official language in which they were submitted.
2019217
BEHRINGWERKE AKTIENGESELLSCHAFT HOE 89/B 0 27 - Ma 780
Dr. Ha/Sd
Description
Magnetic protein conjugates, a process for the prepara-
tion thereof and the use thereof
The invention relates to magnetic protein conjugates of
the formula I
M-NH-CO-~CH2)n-S-S-P
with n = 1-6, preferably with n = 2 or 3, to which the
following applies:
M is a dispersible, magnetically reacting material or
particle which carries amino groups, and P is a protein.
P can be a protein in which the sulfhydryl group needed
for bonding in I is either present in the natural way or
generated by reduction of disulfide linkages or intro-
duced by chemical reaction.
P is, in particular, an immunoglobulin or immunoglobulin
residue, preferably a monoclonal antibody or a Fab, Fab'
or F(ab~)2 fragment, an antigen or a residue of an enzyme,
hormone, lectin or growth factor. --
P is preferably a monoclonal antibody of the IgG or IgN
class, in particular a monoclonal antibody which is
directed against an antigen which is present in dissolved
form in aqueous salt solutions or body fluids or a
monoclonal antibody which is directed against an antigen
which is expressed on cells, in which case the cells
expressing tha antigen can be, in particular, cells of
the myeloid or lymphatic system, cells of the peripheral
blood, especially B lymphocytes, T lymphocytes or precur-
sor cells thereof, or tumor cells, especially tumor cells
of the bone marrow. These cells can also be erythrocytes,
,', ,,, , . . ' ' , ' ~
.','.' ',
1 7
- 2 -
bacteria, mycoplasmas or protozoa. However, viruses are
also to be regarded as cells within the scope of the
invention.
M is preferably a dispersible particle with a metal oxide
core and an enveloping coat carrying amino groups, it
being possible for a group of paramagnetic substances to
be embedded in the metal oxide corer preferably a par-
ticle whose diameter is between about 0.1 ~ and about 100
~, but preferably between about 0.1 ~ and about 1.5 ~.
, . -, ~
The invention furthermore relates to a process for the
preparation of~ a magnetic protein con~ugate of the
formula I and to the use of a conjugate of the formula I
for the specific removal of cells or soluble antigens,
receptors, substrates, cofactors or carbohydrate deter-
minants from aqueous salt solutions or body fluids, and
.
to the use as part of a diagnostic aid or as a diagnosticaid, and, in particular, to the use for bone marrow
depletion or for H~A typing.
' ,
A bone marrow transplantation is often the only therapeu-
tic option, inter alia in the treatment of certain types
of leukemia and of panmyelopathy (myelophthisis).
Patients with leukemias and certain lymphoid neoplasms
are occasionally sub~ected to whole-body irradiation with
an extremely high dose and/or aggressive chemotherapy.
Treatment of this type entails complete destruction of
the normal stem cells of the bone marrow, the precursors
; of all blood cells. The patient therefore receives
reinfusion of bone marrow from a suitable donor, from
which cells colonize ~he bone marrow cavities of the
recipient and thus make it possible for the hemopoietic
and immune system to develop anew. This method is called
allogenic bone marrow transplantation.
The T lymphocytes of the donor which are transferred with
the reinfused bone marrow into the patient and which
201 9217
- 3 -
recognize the cells of the recipient as oreign, and
therefore attack and destroy them, are re~ponsible, inter
alia, for the high risk associated with allogenic bone
marrow transplantation. This bone marrow intolerance,
which is often life-threa$ening for the patient, is
called the graft-versus-host reaction or graft-ver~us-
host disease (GVHD). The risks associated with this
graft-versus-host disease can be reduced, on the one
hand, by the patient being reinfused, where possible,
with accurately typed bone marrow from particularly
suitable donors, usually from among relatives. However,
on the other hand, they can al~o be reduced by selective
elimination of undesired cell populations as may be
represented by, for example, T lymphocytes in the donor's
bone marrow before reinfusion into the patient. This
elimination of donor~s T cells can be carried out, for
example, by selective lysis of ths cells which are to be
removed in the presence of complement or by selective
killing of the T cells using so-called immunotoxins or by
another method, for example by magnetic cell depletion of
the bone marrow.
Bone marrow cell depletion of this type can be carried
out in a relatively straightforward manner by incubating
the bone marrow with a murine monoclonal antibody which
is, for example, directed specifically against the T
cells of the bone marrow and, as a consequence, binds
only to the T cells. Such T cells loaded with murine
monoclonal antibodies can now be removed in a ~econd step
by incubating them, for example, with rabbit anti-mouse
immunoglobulin which is bound ta magnetic particles,
which results in the T lymphocytes being loaded in a
specific manner with the magnetic material so that they
can be removed from the bone marrow using a magnet (see
in this connection Vartdal et al., Transplantation
(1987), 43, 366-371 and the literature cited therein).
It is also possible in an analogous manner to remove
other cell populations, such as tumor cells, from the
,..,.,. . . , . . , :
2~92~7
, . . ~
bone marrow, which is of importance for so-called autolo-
gous bone marrow transplantation (see in thi~ connection
Kvalheim et al., Cancer Research (1987), 47, 846-851 and
the literature cited therein). This can also entail, as
described by Rvalheim et al., ibid., the monoclonal
antibody which recognizes the tumor cells being directly
bound to the magnetic particles 80 that the above-
mentioned second antibody (rabbit anti-mouse) i8 no
longer required.~
The method, described above, of bone marrow depletion
using monoclonal or polyclonal antibodies which are bound
to magnetic particles is still very new and requires
further development and testing. Magnetic particles
suitable for this purpose are now commercially available
in a wide variety of forms, and the preparation thereof
~ has been described several times in the patent literature
;~ ~ (see, for example, Chagnon et al., EP 0125995 A2 (prior-
ity US 493991 of May 12, 1983), Advanced Magnetics, or
Ughelstad et al.,~WO 8303920 of Nov. 10, 1983, SINTEF).
It is known of these magnetic particles that they are
composed of a metal; oxide core in which paramagnetic
substances can be embedded, and that the core is sur-
rounded by an enveloping coat which can carry reactive
groups such as, for example, aminophenyl, amino, car-
boxyl, hydroxyl or sulfhydryl groups which can be used
for coupling proteins (Chagnon et al., EP 0125995 A2).
It is known, for example, that particles carrying car-
boxyl groups can be reacted with amino groups of proteins
; in the presence of a condensing agent (Chagnon et al., EP
01~5995 A2).
It is furthermore known to couple proteins to magnetic
particles carrying amino groups by use of glutaraldehyde,
in which case the coupling takes place via the amino
groups in each case (Chagnon et al., EP 0125995 A2).
: , .
It is additionally known that particles carrying hydroxyl
,",''' , ' . ' ' ' ' ' .'''' , ;, . ',, ' ~ ' " ` ' ' ',. , ' ~ ~ ,
201~1~17
, . .
, .....
5 -
groups can be activated by reaction with p-toluenesul-
fonyl chloride and that particles activated in this way
can be reacted with amino groups of proteins (Kvalheim et
al., Cancer Research (1987), 47, 846-851).
It i6 common to all these coupling methods that the
protein is attached to the particles in each case via its
free amino groups. However, ~uch coupling via amino
groups can be a considerable disadvantage with monoclonal
antibodies because this occasionally impairs the specifi-
city and reactivity of the antibodies. This is a conse-
quence of the fact that the amino groups in an antibody
are, as it were, randomly distributed over the entire
molecule and thus also located in the antigen-binding
site of the Fab fragments, which brings about a loss of
specificity on coupllng via these amino groups.
It is additionally known that antibodies can also be
picked up on magnetic particles purely by adsorption,
without any chemical linkage, when the particles are
composed of a styrene/divinylbenzene copolymer which
contains iron oxide, because it iP known that protein
binds non-specifically to polystyrene.
:
However, impairment of the antibo~y specificity and
reactivity must be expected with this method too. Another
serious disadvantage of this method i5 that, however,
antibodies bound by adsorption become detached again on
- bone marrow depletion and thus may al80 be administered
to the patient on reinfusion of the depleted bone marrow,
; which might lead to serious side effects, especially
where there has been previous attempted therapy with
monocIonal antibodies. However, this problem is known and
is to be overcome by covalent attachment of the anti-
bodies to the magnetic particles.
It is also known that polystyrene-based magnetic
particles have the serious disadvantage that they tend to
aggregate and, moreover, attach themselves non-
201921 7
specifically to cells.
Starting from this state of the art, the ob~ect of the
present invention is to develop a method in which mono-
clonal antibodies are coupled to magnetic particles
a) covalently and b) not via their amino groups. Hence,
in other words, the ob~ect of the present invention i8 to
find a coupling method in which the antigen-binding site
of the antibody is not altered or the coupling of the
antibody takes place away from the antigen-binding site.
:
This object according to the invention is achieved by
preparing magnetic protein con~ugates of the formula I.
.
It has already been proposed to convert magnetic par-
ticles carrying amino groups into magnetic particles
which carry as reactive groups maleimido functionalities,
and to conjugate the latter with proteins which have
sulfhydryl groups, it being possible for the sulfhydryl
; groups in the protein to be either already presentnaturally or introduced by chemical means or generated by
reduction of disulfide linkages which are present.
It has now been found that BioMa~ magnetic particles
which carry free amino groups on their surface can be
activated by reaction with 2-iminothiolane (2-It) in such
a way that they can be covalently attached to antibodies
merely by incubation therewith. Magnetic antibody con-
~ugates prepared in this way are new. ~ -
:
It has furthermore been found that BioMa~ magnetic
particles which carry free amino groups on their surface
can also be activated by reaction with N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP) and subsequent
reductive cleavage of the disulfide linkage using dithio-
threitol or mercaptoethanol in such a way that they can
be covalently attached to antibodies merely by incubation
therewith. Magnetic antibody conjugates prepared in this
way are likewise new.
,..~. ,.. - . , . - ,. .. ~ .. : .... , - ..
20~ 9217
- 7 -
It has additionally been found that the said magnetic
particles can, after reaction with SPDP, also be co-
valently attached to antibodies which carry free SH
groups by incubation with the antibodies, without the
necessity previously to activate, using dithiothreitol or
me~captoethanol, the magnetic particles which have been
modified by reaction with SPDP. Magnetic antibody conju-
gates prepared in this way are likewise new.
It has been found, surprisingly, that the loading of the
magnetic particles with antibodies can be increased in
each case when any sulfhydryl groups which are still
present after the coupling step are saturated by reaction
with N-ethylmaleimide or iodoacetamide. Surprisingly,
this also increases the stability of the prepared mag-
netic antibody conjugates.
In particular, it has been found that the disulfide
linkage produced in each case between the magnetic
particle and the antibody can be cleaved again by reduc-
tion with dithiothreitol or mercaptoethanol. Magnetic
particle antibody conjugates which can be cleaved in this
way are likewise new - and the cleavability of the spacer
and the variable spacer length are responsible for two
6pecial advantages of the invention:
1. The cleavable spacer makes possible positive selec-
tion of those antigens or cells which are recognized
by the antibody coupled to the magnetic particles -
specifically by simple magnetic separation. The
magnetic particles can be eliminated from the
depleted antigens or cells again by reduction and
removed using a magnet.
2. The variable spacer length allows the distance
between antibody and magnetic particle to be, within
certain limits, varied and suited to the particular
coupling or separation or depletion problem. This
possibility of choice is a particular advantage
2~217
r~ -- 8 --
especially when particles of different sizes are
used or when antibodies of different classes or
isotype6 are coupled.
It has been found, surprisingly, that the specificity and
reactivity of the antibodies coupled via dii~ulfide
linkages or via the described ~pacers to magnetic par-
ticles is completely retained because the coupling of the
antibody via its hinge region means that there is no
alteration or impairment of its antigen-binding site.
This is responsible for a particular advantage of the
invention compared with hitherto disclosed coupling
methods in which the antibodies are, as described above,
picked up on magnetic particles either purely by adsorp-
tion or via reaction of their amino groups, which may
impair both the specificity and the reactivity of the
conjugated antibodies. Moreover, the present invention
has the advantage compared with coupling by adsorption
that the antibodies are chemically bonded to the magnetic
particles.
It has additionally been found that the magnetic antibody
conjugates according to the invention prove to be parti-
cularly advantageouc, because of their high specificity,
in the depletion of bone marrow, for example.
It has additionally been found that the magnetic antibody
conjugates according to the invention al80 prove to be
; advantageous, because of their hiqh specificity, as part of a diagnostic method or as a diagnostic aid, in parti-
cular, for example, in HLA typing.
In particular, it has been found that the magnetic anti-
body con~ugates according to the invention are suitablefor positive selection of antigens or cells because the
magnetic particles can, after reductive cleavage of the
disulfide linkage of the spacer, be separated again from
the isolated antigens or cells using a magnet or by
centrifugation.
20~ ~217
. g
... .
The preparation of magnetic antibody con~ugates according
to the invention is described by way of example herein-
after for various monoclonal antibodies which are direc-
ted against cells of the bone marrow and for a polyclonal
S rabbit immunoglobulin; however, the said examples do not
:~ restrict the invention. In addition, the use of the
prepared examples of magnetic antibody con~ugates for the
depletion of cells of the bone marrow.is likewise descri-
bed by way of example,~without restricting the use to the
:~ 10: said examples.:
Process for the preparation of magnetic protein con-
15 ` jugates of the formula I
a) Magnetic particles M carrying amino groups are
- reacted in a suitable solvent with a compound of the
,
~ formula II which reacts with amino groups
~ o ~-tCH2 )n-S-S-~ 3 II
0
in which n is 1-6, with the formation of an amide
~; : linkage to give a compound of the formula III
-C0-(CH2)n_S_S_ ~ III
and the latter is converted by reductive cleavage of
1 i ' ' :
the disulfide linkage into a compound of the $ormula
IV
M~NH~C~(CHZ)n~SH IV
which is finally reacted in a suitable aqueous salt-
containing solvent which does not denature proteins,
such as, for example, physiological saline solution
"
2~9217
10 - ~
or a phosphate-buffered saline solution, with a
protein P having disulfide linkage~, such a~ an
antibody, to give a compound of the formula I, or
: '
b) particles M carrying amino groups are reacted in a
suitable aolvent with iminothiolane to give a
compound of the formula IV in which n is 3, after
which this compound of the formula IV is reacted
with a protein P having disulfide linkages in the
manner described above, or
c) particles M carrying amino groups are reacted as
described abo~e to give a compound of the formula
III which is reacted, in a suitable aqueous salt-
containing solven~ which does not denature proteins,
with a protein P carrying sulfhydryl groups, such as
a reduced antibody or a Fab or Fab~ fragment, to
give a compound of the formula I, after which the
linkage between protein P and magnetic particle with
spacer is stabilized, where appropriate, by addition
of a suitable maleimido derivative, for example of
N-ethylmaleLmide, or by addition of iodoacetamide or
bromoacetamide.
Solvents suitable for the coupling of a compound of
the formula II or of iminothiolane to magnetic
particles must be of ~uch a constitution that there
is no impairment of the phy~ical and magnetic
properties of the magnetic particles used in each
case for the coupling, in particular of the size,
dispersibility and surface characteristics thereof~
by the solvent which i~ used. An example of a
solvent suitable or magnetic parti~les a~ are
described, for example~ in EP 0125995 A2 or Wo
8303920 has been found to be a mixture of water and
dimethylformamide.
Determination of the degree o coupling (~g of anti-
body/mg of iron)
2~921 7
..... 1 1
Iron was determined by atomic absorption and nitrogen by
the K~eldahl method. The values for the coupled protein
nitrogen were calculated by the formula
yg P-N ~g Tot-N ~g Tot-N
- = ~sample) - (control)
mg Fe mg Fe mg Fe
where the terms have the following meaning:
P-N: protein nitrogen
Tot-N.: total nitrogen
~; 10 Fe: iron ~ ~
The amount~of protein bound to the particles (~g of
protein/mg of ironj was calculated from the amount of
protein nitrogen per mg of iron by multiplication by the
factor 6.25. The calculated coupling rates are compiled
in Table 1.
Method for depletion of cells
:~ ,
A suspension ~f a cell mixture which is to be depleted in
a salt-containing, preferably physiological a~ueous solu-
tion or in a body ~luid is incubated with a compound of
the formula I at a suitable temperature between, for
example, 0C and 40C, preferably with shaking, likewise
preferably under sterile conditions, for a suitable
period, and then the magnetic particles are removed from
the solution by a ~ultable magnet.
.
Examples of suitable temperatures are 0C, room tempera-
ture or 37C, but room temperature is preferred. The
duration of the incubation depends in each case on the
incubation temperature used and on the binding reactivity
of the antibody and can be, for example, from a few
minutes up to, for example, two hours. Incubation is
preferably at, for example, room temperature for a period
of, for example, 10 to 20 minutes.
2~19217
~`; 12
- ' '
Method for the isolation of soluble bioorganic molecules
, . . .
This method essentially follows the method for depletion
of cells.
Examples
The examples which follow serve to illustrate the inven- -
tion in detail, but do not restrict the invention.
Magnetic particles which have been reacted with mono-
clonal antibodies in the manner described are called
` "magnetobeadsl~ herei~after, with their specificity being
indicated in each case ~by prefixing the particular
antibody name.
,;
Example 1
,
Preparation of a compound of the formula IV in which
n is 3
~ '
-~ 15 3 x 300 ~1 of a commercially available suspension of
magnetic particles (BioMa~, Advanced Magnetics) were each
washed 3x with 10 ml of phosphate-buffered saline solu-
tion, pH 7.2, (PBS) each time and each resuspended in 2
ml of PBS. Then a solution of 2-iminothiolane hydro-
chIoride (2-It HCl) in PBS was added to each:
a) addition of 10 mg of 2-It HCl in 0.5 ml of PBS
b) addition of 2.5 mg of 2-It HCl in 0.5 ml of PBS
c) addition of 0.6 mg of 2-It HCl in 0.5 ml of PBS
,
These suspensions were each shakèn at room temperature
for 1 h. The particles were then removed by centrifuga- -
tion at 3000 x g and in each case washed 3x with 10 ml of
PBS each time.
2~ 92~7
13
,
Example 2
Preparation of a compound of the formula III in which
n is 2
:
3 x 300 ~1 of a commercially available suspension of
magnetic particles (BioMa~, Advanced Magnetics) were each
washed 3x with 10 ml of PBS, pH 7.2, each time and each
was resuspended~in 3 ml of PBS. Then a solution of 5 mg
of N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) in
2 ml of dry dimethylfarmamide (DMF) was added to each,
and the`mixtures~were ~haken at room temperature for 1 h.
The~particles~were then~remo~ed by~centrifugation at 3000
x g, and each waB~ washed~3x with I0 ml of PBS each time.
,
Example 3
Preparation of a compound of the formula IV in which
n i8 2
Three aliquots~of particles~of the formula III prepared
as in Example 2 were each;resuspended in 2.4 ml of PBS,
and 5 mg of dithiothreitol dissolved in 0.1 ml of PBS in
each case were ~added ~to each, and the mixtures were -~
~incubated at room temperature, shaking gently, for -~
~; ~ 30 min. The particles~were then removed by centrifugation
at 3000 x g,~and~ each was washed 3x with 10 ml of PBS
each~time and u6ed~for~the coupling reactions without --
`delay.
Example 4
Coupling of polyclonal rabbit anti-mouse immunoglobulin ~ -
(RAM) to a compound of the formula IV in which n i8 3t -
from Example la, varying the amount of anti~ody
Four aliquots of particles of the formula IV prepared as
in Example la from 300 ~1 aliquots of BioMa~ in each case
were resuspended in
20~9217
: - 14 -
a) 2.5 ml of PBS
b3 2.4 ml of PBS
c) 2.3 ml of PBS
d~ 2.2 ml of PBS
and the suspension was mixed with the following amounts
of RAM:
a) no addition
b) 0.5 mg of RAM in 0.1 ml of PBS
c) 1.O mg of RAM in 0.2 ml of PBS
d) 1.5 mg of RAN in 0.3 ml of PBS
The mixtures (2.5 ml each) were each incubated at room
temperature, while shaking, for 1 h. The particles were
: then removed by centrifugation at 3000 x g, washed 3x
with 10 ml of PBS each time, resuspended in 5 ml of PBS
pH 7.2, sterilized by X-rays and stored at 4C. The
: analytical data are compiled in Table 1.
Example 5
Coupling of the monoclonal antibody BMA 0110 (anti-CD2;
: IgG2b) to a compound of the formula IV in which n i8 3,
from Example la, varying the amount of antibody
'- ,,
Coupling was carried out in analogy to Example 4, adding
the following amounts of BMA 0110 to particle suspensions
a-d of the formula IV:
a) no addition
b) 0.5 mg of BMA 0110 in 0.1 ml of PBS
c) 1.0 mg of BMA 0110 in 0.2 ml of PBS
d) 1.5 mg of BMA 0110 in 0.3 ml of PBS - :
Further processing was carried out in analogy to Bxample
4; the analytical data are listed in Table 1.
Example 6
Coupling of the monoclonal antibody BMA 0110 (anti-CD2;
IgG2b) to a compound of the formula IV in which n is 3,
20~92~7
_ 15 -
from Example lb, varying the amount o~ antibody
Coupling was carried out in analogy to Example 5, but
starting from a compound of the formula IV from Example
lb (instead of Example la).
Example 7
Coupling of the monoclonal antibody BMA 030 (anti-CD3;
IgG2a) to a compound of the formula IV in which n is 3,
from Example la, lb and lc, using the same antibody
concentrations
Aliquots of partlcles~of the formula IV prepared as in
Example la, lb and lc were each resuspended in 2.0 ml of
PBS, and 0.5 ml of a solution of BMA 030 in PBS (corres-
ponding to 1 mg of BMA 030 in 0.5 ml of PBS in each case)
was added to each, and further procescing was carried out
in~analogy to Example 4. The analytical data are compiled
in Table 1.
Example 8
: '~
Coupling of the monoclonal antibody BMA 033 (anti-CD3,
IgG3) to a compound of the formula IV in which n is 3,
from Example la, lb and lc, using the same antibody
concentrations
The reaction was carried out in analogy to Example 7; the
analytical data are listed in Table 1.
,: I , ,
Example 9
. .,:
Couplin~ of the monoclonal antibody BMA 081 (anti-CD8;
IgG2a) to a compound of the formula IV in which n is 3,
~; from Example la, varying the amount of antibo~y
The reaction was carried out in analogy to Example 5; the
analytical data are listed in ~able 1.
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~ 16 -
,
Example 10
Coupling of polyclonal rabbit anti-mouse immunoglobulin
(RAM) to a compound of the formula III in which n is 2,
from Example 2, varying the amount of antibody.
S A solution of 2 mg of RAM in 0.4 ml of PBS was mixed with
2 mg of dithiothreitol and incubated at room temperature
for 30 min. The reduced antibody was isolated by gel
filtration on Sephadex G25 in PBS pH 7.2 in an elution
vo:lume of 4.2 ml.
Aliquots of particles of the formula III prepared as in
Example 2 were resuspended as follows:
a) in 4.3 ml of PBS
b) in 3.6 ml of PBS
c) in 2.9 ml of PBS
These suspensions were mixed with the reduced antibody as
followss
a) addition of 0.7 ml (about 0.3 mg of protein)
b) addition of 1.4 ml ~about 0.6 mg of protein)
c) addition of 2.1 ml ~about 0.9 mg of protein)
: 20 The mixtures (5 ml each) were each incubated at room
temperature, while shaking, for l h. The particles were
then removed by centrifugation at 3000 x g, washed 3x
with 10 ml of PBS each time, resuspended in 5 ml of PBS
pH 7.2, sterilized by X-rays and stored at 4C. The
analytical data are compiled in Table 1.
Example ll
: Coupling of polyclonal rabbit anti-mouse immunoglobulin
~RAM) to a compound of the formula IV in which n is 2,
from Example 3, varying the amount of antibody
Four aliquots of particles of the formula IV prepared as
in Example 3 were resuspended in:
2~217
- 17 -
a) 2.5 ml of PBS
b) 2.4 ml of PBS
c) 2.3 ml of PBS
d) 2.2 ml of PBS
and the suspension was mixed with the following amounts
of RAM:
a) no addition
b~ 0.5 mg in 0.1 ml of PBS
c) 1.0 mg in 0.2 ml of PBS
d) 1.5 ml in 0.3 ml of PBS
The mixtures ~2.5 ml each) were each incubated at room
temperature, whiIe shaking, for 1 h. The particles were
: then removed by centrifugation at 3000 x g, washed 3x
with 10 ml of PBS each time, resuspended in 5 ml of PBS
pH 7.2, sterilized by X-rays, and stored at 4C. The : ~
analytical data are compiled in Table 1. ;:
Example 12
Coupling of the monoclonal antibody BMA 0110 (anti-CD2; ~.
IgG2b) to a compound of the formula IV in which n is 2,
from Example 3, varying the amount of antibody ~ ;
The coupling was carried out in analogy to Example 11;
the analytical data are listed in Table 1.
~ '
,: j , ,
2011 92~L7
~`` -18-
Table 1 Degrees of cou?ling for .nagnetoDeads prepared
in various ways
Ex- Anti- Isotype Speci- Coup- Coupling mixture 3
ample body ficity ling Ab/Fe ) Sp/Fe ) P/Fe )
method (mg/mg) (~mol/mg) (~g/mg)
4b RAM IgG aMIg 2-It/SS 0.1 14.5 110
4c R~M IgG aMIg 2-It/SS 0.2 14.5 173
4d RAM IgG aMIg 2-It/SS 0.3 14.5 229
5b BMA 0110 IgG2b CD2 2-It/SS 0.1 14.5 107
Sc BMA 0110 IgG2b CD2 2-It/SS 0.2 14.5 156
Sd BMA 0110 IgG2b CD2 2-It/SS 0.3 14.5 178
6b BMA 0110 IgG2b CD2 2-It/SS 0.1 3.6 65
6c BMA 0110 IgG2b CD2 2-It/SS 0.2 3.6 78
6d BMA 0110 IgG2b CD2 2-It/SS 0.3 3.6 112
7a BMA 030 IgG2a CD3 2-It/SS 0.2 14.5 71
7b BMA 030 IgG2a CD3 2-It/SS 0.2 3.6 53
7c BMA 030 IgG2a CD3 2-It/SS 0.2 0.9 12
8a BMA 033 IgG3 CD3 2-It/SS 0.2 14.5 69
8b BMA 033 IgG3 CD3 2-It/SS 0.2 3.6 56
8c BMA 033 IgG3 CD3 2-It/SS 0.2 0.9 39
9b Br~A 081 IgG2a CD8 2-It/SS 0.1 14.5 73
9c BMA 081 IgG2a CD8 2-It/SS 0.2 14.5 ~0
9d BMA 081 IgG2a CD8 2-It/SS 0.3 14.5 91
1Oa RAM IgG aMIg SPDP/SH 0.06 3.2 29
1Ob RAM IgG aMIg SPDP/SH 0.12 3.2 36
10c RAM IgG aMIg SPM fSH 0.18 3.2 55
11b RAM IgG aMIg SPDP/red 0.1 3.2 27
11c RA~I IgG aMIg SPDP/red O.2 3.2 51
11d RAM IgG aMIg SPDP/red 0.3 3.2 74
12b BMA 0110 IgG2b CD2 SPM /red 0.1 3.2 27
12c BMA 0110 IgG2b CD2 SPM/red0.2 3.2 47
12d BMA 0110 IgG2b CD2 SPDP/red 0.3 3.2 57
1) Amount of antibody relative to iron (mg/mg) used for the
particular coupling mixture
2) Amount of spacer relative to iron (~mol/mg) used for the
particular coupling mixture
3) Amount of protein coupled relative to iron (~g/mg)
RAM: Rabbit anti-mouse immunoglobulin
CD: Cluster of dif~erentiation
aMIg: Anti-mouse immunoglobulin
