CA2019217A1 - Magnetic protein conjugates, a process for the preparation thereof and the use thereof - Google Patents
Magnetic protein conjugates, a process for the preparation thereof and the use thereofInfo
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- CA2019217A1 CA2019217A1 CA002019217A CA2019217A CA2019217A1 CA 2019217 A1 CA2019217 A1 CA 2019217A1 CA 002019217 A CA002019217 A CA 002019217A CA 2019217 A CA2019217 A CA 2019217A CA 2019217 A1 CA2019217 A1 CA 2019217A1
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- Prior art keywords
- magnetic
- formula
- protein conjugate
- cells
- compound
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
- A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/20—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations containing free radicals, e.g. trityl radical for overhauser
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
- G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
- G01N2446/90—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids characterised by small molecule linker used to couple immunoreagents to magnetic particles
Abstract
Abstract of the disclosure 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 I
with n = 1-6, preferably with n = 2 or 3, in which M is a dispersible, magnetically reacting material or particle which carries amino groups, and P is a protein, to a process for the preparation thereof and to the use thereof for the specific removal of cells or soluble antigens, receptors, substrates, cofactors or carbo-hydrate determinants from aqueous salt solutions or body fluids or as part of a diagnostic method or as a diagnos-tic aid, preferably for the removal of cells, preferably for bone marrow depletion or for HLA typing.
M-NH-CO-(CH2)n-S-S-P I
with n = 1-6, preferably with n = 2 or 3, in which M is a dispersible, magnetically reacting material or particle which carries amino groups, and P is a protein, to a process for the preparation thereof and to the use thereof for the specific removal of cells or soluble antigens, receptors, substrates, cofactors or carbo-hydrate determinants from aqueous salt solutions or body fluids or as part of a diagnostic method or as a diagnos-tic aid, preferably for the removal of cells, preferably for bone marrow depletion or for HLA typing.
Description
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, ,', ,,, , . . ' ' , ' ~
.','.' ', 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 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 , . .
, .....
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-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 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
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 , 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.
2~9217 ~ 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 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
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, ,', ,,, , . . ' ' , ' ~
.','.' ', 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 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 , . .
, .....
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-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 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
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 , 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.
2~9217 ~ 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 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
Claims (25)
1. A magnetic protein conjugate of the formula I
M-NH-CO-(CH2)n-S-S-P I
with n = 1-6, preferably with n = 2 or 3, in which M is a dispersible, magnetically reacting material or particle which carries amino groups, and P is a protein.
M-NH-CO-(CH2)n-S-S-P I
with n = 1-6, preferably with n = 2 or 3, in which M is a dispersible, magnetically reacting material or particle which carries amino groups, and P is a protein.
2. A magnetic protein conjugate as claimed in claim 1, wherein the sulfhydryl group or sulfhydryl groups of the protein P are either present in the natural way or generated by reduction of disulfide linkages or introduced by chemical reaction into the pro-tein.
3. A magnetic protein conjugate as claimed in claim 1, wherein P is a polyclonal immunoglobulin.
4. A magnetic protein conjugate as claimed in claim 1, wherein P is a monoclonal antibody or a Fab, Fab' or F(ab')2 fragment.
5. A magnetic protein conjugate as claimed in claim 1, wherein P is an antigen or a residue of an enzyme, hormone, lectin or growth factor.
6. A magnetic protein conjugate as claimed in claim 4, wherein P is a monoclonal antibody of the IgG or IgM class.
7. A magnetic protein conjugate as claimed in claim 4, wherein P is a monoclonal antibody which is direc-ted against an antigen which is present in dis-solved form in aqueous salt solutions or body fluids.
8. A magnetic protein conjugate as claimed in claim 4, wherein P is a monoclonal antibody which is direc-ted against an antigen which is expressed on cells, especially on cells of the myeloid or lymphatic system or of the peripheral blood, especially on B
lymphocytes, T lymphocytes or the precursor cells thereof or on tumor cells, especially on tumor cells of the bone marrow.
lymphocytes, T lymphocytes or the precursor cells thereof or on tumor cells, especially on tumor cells of the bone marrow.
9. A magnetic protein conjugate as claimed in claim 4, wherein P is a monoclonal antibody which is direc-ted against an antigen which is expressed on bacteria, mycoplasmas or protozoa or else on viruses.
10. A magnetic protein conjugate a claimed in claim 1, wherein P is an antigen.
11. A magnetic protein conjugate as claimed in claim 1, wherein M is a dispersible particle with a metal oxide core and an enveloping coat carrying amino groups, it being possible for a group of para-magnetic substances to be embedded in the metal oxide core.
12. A magnetic protein conjugate as claimed in claim 11, wherein the diameter of the particles is between about 0.1 µ and about 100 µ, but preferably between about 0.1 µ and about 1.5 µ.
13. A compound of the formula III
III
in which M has the meaning specified in claim 1.
III
in which M has the meaning specified in claim 1.
14. A compound of the formula IV
M-NH-CO-(CH2)n-SH IV
in which M has the meaning specified in claim 1.
M-NH-CO-(CH2)n-SH IV
in which M has the meaning specified in claim 1.
15. A process for the preparation of a magnetic protein conjugate of the formula I
M-NH-CO-(CH2)n-S-S-P I
which comprises reacting magnetic particles carrying amino groups with a compound of the formula II which reacts with amino groups II
in which n is 1-6, with the formation of an amide linkage to give a compound of the formula III
III
and converting the latter by reductive cleavage of the disulfide linkage into a compound of the formula IV
M-NH-CO-(CH2)n-SH IV
which is finally reacted with a protein P having disulfide linkages to give a compound of the formula I.
M-NH-CO-(CH2)n-S-S-P I
which comprises reacting magnetic particles carrying amino groups with a compound of the formula II which reacts with amino groups II
in which n is 1-6, with the formation of an amide linkage to give a compound of the formula III
III
and converting the latter by reductive cleavage of the disulfide linkage into a compound of the formula IV
M-NH-CO-(CH2)n-SH IV
which is finally reacted with a protein P having disulfide linkages to give a compound of the formula I.
16. A process for the preparation of a magnetic protein conjugate of the formula I, which comprises react-ing magnetic particles M carrying amino groups with iminothiolane to give a compound of the formula IV
M-NH-CO-(CH2)n-SH IV
in which n is 3, and reacting the compound of the formula IV with a protein P having disulfide linkages to give a compound of the formula I.
M-NH-CO-(CH2)n-SH IV
in which n is 3, and reacting the compound of the formula IV with a protein P having disulfide linkages to give a compound of the formula I.
17. A process for the preparation of a magnetic protein conjugate of the formula I, which comprises react-ing magnetic particles M carrying amino groups as in claim 15 to give a compound of the formula III, and reacting the latter with a protein P carrying sulfhydryl groups to give a compound of the formula I.
18. The process for the preparation of a magnetic protein conjugate as claimed in at least one of claims 15, 16 or 17, wherein the linkage between protein P and the magnetic particle with spacer is stabilized by addition of an N-substituted male-imido compound, preferably by addition of N-(C1-C6-alkyl)maleimide, particularly preferably by addi-tion of N-ethylmaleimide, or by addition of a maleimidocarboxylic acid or by addition of iodo-acetamide or bromoacetamide.
19. A method for removing a dissolved antigen, anti-body, receptor, substrate, cofactor or carbohydrate determinant from aqueous salt solutions or body fluids, which comprises the solution being incub-ated with a suitable magnetic protein conjugate of the formula I and, after specific adsorption of the component which is to be removed, the magnetic protein conjugate being separated out by magnetic means, and the specifically adsorbed component being, where appropriate, eluted again from the magnetic protein conjugate or eliminated by reduc-tion of the disulfide linkage of the sparer of the compound of the formula I.
20. A method for removing cells from aqueous salt solutions or body fluids, which comprises the cell suspension being incubated with a suitable magnetic protein conjugate of the formula I and, after specific adsorption of the cells which are to be removed, the magnetic protein conjugate being separated out by magnetic means, and the specifi-cally adsorbed cells or particles being, where appropriate, detached again from the magnetic protein conjugate or eliminated by reduction of the disulfide linkage of the spacer of the compound of the formula I.
21. A method for the positive selection of soluble components as claimed in claim 19 or of cells as claimed in claim 20, which comprises reductive cleavage of the disulfide linkage of the spacer and removal of the magnetic particles using a magnet or by centrifugation.
22. The use of a magnetic protein conjugate as claimed in claim 1 for the specific removal of cells or soluble antigens, receptors, substrates, cofactors or carbohydrate determinants from aqueous salt solutions or body fluids or the use as part of a diagnostic method or as a diagnostic aid.
23. The use of a magnetic protein conjugate as claimed in claim 1 for removing cells as claimed in claim 8 or 9, preferably for bone marrow depletion, or for HLA typing.
24. The use of a magnetic protein conjugate as claimed in claim 1 for the positive selection of cells or soluble antigens, receptors, substrates, cofactors or carbohydrate determinants, but especially for the positive selection of cells, in particular for the positive selection of cells as claimed in claim 8.
25. A magnetic protein conjugate of the formula I as claimed in claim 1 and substantially as described herein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3919923.1 | 1989-06-19 | ||
| DE3919923A DE3919923A1 (en) | 1989-06-19 | 1989-06-19 | MAGNETIC PROTEIN CONJUGATES, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2019217A1 true CA2019217A1 (en) | 1990-12-19 |
Family
ID=6383013
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002019217A Abandoned CA2019217A1 (en) | 1989-06-19 | 1990-06-18 | Magnetic protein conjugates, a process for the preparation thereof and the use thereof |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0403960A2 (en) |
| JP (1) | JPH0341098A (en) |
| AU (1) | AU5717990A (en) |
| CA (1) | CA2019217A1 (en) |
| DE (1) | DE3919923A1 (en) |
| IE (1) | IE902195A1 (en) |
| PT (1) | PT94399A (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4033714C3 (en) * | 1990-10-24 | 1995-09-07 | Brahms Diagnostica Gmbh | New hapten analysis based on the principle of an antigen-antibody reaction |
| GB9105031D0 (en) * | 1991-03-09 | 1991-04-24 | Fisons Plc | Processing analytical reagents |
| AU2593192A (en) * | 1992-09-14 | 1994-04-12 | Oystein Fodstad | Detection of specific target cells in specialized or mixed cell population and solutions containing mixed cell populations |
| NO180658C (en) | 1994-03-10 | 1997-05-21 | Oeystein Fodstad | Method and Device for Detecting Specific Target Cells in Specialized or Mixed Cell Populations and Solutions Containing Mixed Cell Populations |
| NO961031D0 (en) | 1996-03-13 | 1996-03-13 | Det Norske Radiumshospital Tum | Procedure for killing unwanted target cells |
| KR20030047499A (en) * | 2001-12-11 | 2003-06-18 | 김근상 | Multi-terminal plug and jack |
-
1989
- 1989-06-19 DE DE3919923A patent/DE3919923A1/en not_active Withdrawn
-
1990
- 1990-06-14 EP EP19900111263 patent/EP0403960A2/en not_active Withdrawn
- 1990-06-18 JP JP2157800A patent/JPH0341098A/en active Pending
- 1990-06-18 IE IE219590A patent/IE902195A1/en unknown
- 1990-06-18 CA CA002019217A patent/CA2019217A1/en not_active Abandoned
- 1990-06-18 PT PT94399A patent/PT94399A/en not_active Application Discontinuation
- 1990-06-18 AU AU57179/90A patent/AU5717990A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0341098A (en) | 1991-02-21 |
| AU5717990A (en) | 1990-12-20 |
| IE902195A1 (en) | 1991-01-02 |
| EP0403960A2 (en) | 1990-12-27 |
| IE902195L (en) | 1990-12-19 |
| PT94399A (en) | 1991-02-08 |
| DE3919923A1 (en) | 1990-12-20 |
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| Date | Code | Title | Description |
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| FZDE | Dead |