CN1059278A - Antibody conjugates - Google Patents

Abstract

A kind of soluble antibody conjugate, it contains and the antibody that can be connected by the structure of T cell recognition, can cause T cytolysis target cell, and these target cells can be by antibody recognition.This conjugate is characterised in that its structure is a superantigen.

An important mode is the method for dissolving target cell, and wherein target cell contacts with the conjugate of target cell dissolving effective dose.This dissolving method can part be treated cancer, autoimmune, parasitic infection and fungus, virus and bacterial infection effectively.

This description has also been described for example synthetic and the pharmaceutical composition and its preparation method of this conjugate.

Description

Antibody conjugates

The present invention relates to can activating cell toxin T cell (CTLS) antibody conjugates.This conjugate is favourable for destroying unwanted cells, and these cells and cancer form, autoimmune generation, parasitic infection and antibacterial, viral relevant with fungal infection.

Once attempted in the past few years antibody is used in combination with the medicament (cytotoxic agent, cytotoxin) that target cell is directly produced toxic effect, so that the selection to target cell to be provided, and prevent to reduce to minimum to the non-specific influence of other cells and with this influence.The coordination compound that this conjugate of being advised relates to coordination compound (utilizing bonding that molecule is provided) from covalent bonding and non-covalent bonding to simple mixtures (for example, people such as Ghose, J.Natl.Cancer Inst.61(1979) people such as 657-676 and Carlsson, Biotechnology7(1989) 567-73).The cytotoxin of being advised is not subunit A, gelonin and Pseudomonas aeruginosa (Pseudomonas aeruginosa) exotoxin A (the Takeda chemical Ind. of diphtheria toxin, diphtherotoxin, ricin, ricin, people such as EP-A-336405 and Pastan, WO-A-88/00703, these two pieces of patents all by relevant in first to file, SE-9002479 quotes.

Along with the appearance of hybridoma technology and the available of monoclonal antibody, can use the coordination compound notion between antibody and the cytotoxic agent to make cytotoxic agent point to purpose target cell group more single-mindedly.

In view of the destruction of the cytotoxic agent of being familiar with, there is the people once to advise replacing cytotoxic agent, to excite the T-lymphocyte and to activate CTLS with immunostimulant to other cells except that target cell.Suggestion relates to and the conjugated antibody of following substances particularly:

(ⅰ) antibody of direct anti-TXi Baoshouti or can with the bonded chemical compound of TXi Baoshouti (Mass.Inst Techn., EP-A1-180171);

(ⅱ) can activating cell the chemical compound of toxin T cell, for example antigen, mitogen, other foreign proteins and peptide (Neorex Corp., Ep-A1-334300);

(ⅲ) MHC antigen, (Behringwerke AG, Ep-A1-352761);

(ⅳ) make) by the individual antigen (Med.Res.Counc.Wo-A-90/11779(publ.1990-10-18) of treatment with anti-this antigenic immunity; And

(ⅴ) a kind of unnamed bacterial enterotoxin (Ochi and Wake, UCLA-Sym-posium: the cellular immunity of cancer and immunotherapy, 27 days-February 3 January nineteen ninety, summary CE515, the 109th page).

But, the effect of the immunostimulant of being advised up to the present be not too single-minded be to be exactly that its effect is too general.For example traditional antigen can only activate 1/10 ⁵The T cell, and mitogen can activate most of T cells.

Have realized that some medicament mediation T cell moderate ratio activation; Be that they can still not reach people such as 100%(Fleischer far away, J.Exp.Med.167(1988) 1697-1707 with higher frequency activating T cell; And people such as White, cell56(1989) 27-35, two pieces of articles are all by with reference to being incorporated into this paper).Such medicament is than the more effective activator of traditional antigen, so they are named as superantigen (superantigens) (referring to kappler and marrack, Science248:705, (1990)).Confirmed further that known up to now superantigen can be attached to class on the target cell, and can activate the cytotoxin T cell that has distinctive TXi Baoshouti V β chain (people such as Dohlsten, Immunol.71(1990) 96-100; With people such as Hedlund, Cell.Immunol129(1990) 426-34, two pieces of articles are all by with reference to being bonded to herein).Disclosed data show that MHC is in conjunction with the prerequisite that is combination of T cell and generation activation.Can not get rid of the superantigen that to find from now on by TXi Baoshouti V α chain works or other surface textures of only finding are had an effect on the T cell subsets.

People such as Platsoucas have also described immunoregulation effect (Cell Immunol.97(1986) 371-85 of superantigen staphylococcal enterotoxin A (SEA)).

Most of present known superantigens discerned earlier, and all superantigens all derive from microorganism as toxin.Staphyloentero-toxin for example is enterotoxication and can activating T cell, and these two kinds of effects are (people such as Fleischer, the Cell.Immunol.118(1989) 92-101 that can differentiate mutually; People such as Alber, J.Immunol 144(1990) 4501-06; And Infect.Immun.59(1991) 2126-34).

Once the someone advised using superantigen in the past, carried the antigenic cytolysis of II class MHC (Pharmacia AB, Wo-A-91/04053, open day on April 4th, 1991) with guiding CTL mediation.W-A-91/04053 comprised, but clearly do not mention, and is attached to the superantigen in the covalency immunity conjugate.

, lack the cell of II class MHC or express the proteic cell of finite quantity II class MHC can not be in conjunction with the superantigen of q.s to guide its dissolving effectively by CTLS.Therefore carry the antigenic general abundant cell of II class MHC and do not have abundant II class MHC antigen on the most tumors cell, superantigen should be very low for the single-minded value of killing these unwanted cells.

But we have found that,, so just can utilize superantigen to realize the single-minded cell killing effect that mediates by CTLs if superantigen is covalently attached on the antibody (this antibody resists the cell of being killed and has specific antigenic determinant).Immune activation can be induced and be lacked the antigenic target cell expression of II class MHC, and these can strengthen required dissolution.

The present invention relates to new antibody conjugates

(ⅰ) contain (1) antibody and (2) a kind of superantigen, i.e. identification (interacting or combination) and activating T cell, the particularly antibody conjugates of CTL structure at anti-target cell.

(ⅱ) destroy the method for target cell, particularly relate to mammiferous Therapeutic Method, and the activating T cell method of CTLs for example specifically;

(ⅲ) method of synthetic these conjugates; And

(ⅳ) contain the pharmaceutical composition of these conjugates and the preparation method of said composition.

The method of destroying target cell comprises kills some cell by the Therapeutic Method of treatment cancer, autoimmune, viral infection, bacterial infection, fungal infection, parasitic infection and other diseases with its objective is pin-point accuracy.Conjugate of the present invention can be used for pharmaceutical compositions, and these pharmaceutical compositions will be used to destroy the target cell relevant with the disease of just having mentioned.The individuality of receiving treatment is normal animal, mainly is the people.

The superantigen part of conjugate

New antibody conjugates is characterized in that the structure by the T cell recognition is a superantigen.This conjugate is that normality is soluble under physiological pH, dissolves in the serum external.

Preferred superantigen is selected from staphyloentero-toxin (SEs) (for example SEA, SEB, SEC, SED and SEE), toxoid, its active fragment or peptide, and to activating other materials that CTLs has the same function mode.These superantigens can comprise other microbial products (antibacterial and virus), for example obtain from aureus strain, as toxic shock syndrome, TSS toxin (TSST-1), from streptococcus obtain as pyrogenic exotoxin A, bacterial exoprotein and the albumen that produces by mycoplasma arthritidis (mycoplasma arthridis), the interaction and the superantigen of they and T cell have same ability.By cultivating its natural product or genetically engineered cell (recombinant technique) or also can synthesizing, obtain superantigen by synthetic peptide.Be used for superantigen of the present invention when the experimental model that is used for described in this description, should produce and the similar effect of this description experimental section.

Preferred superantigen has the homotype of multiform T cell surface protein that can be relevant with activation CTLs, preferably with the ability of TXi Baoshouti V β link and about 1-40%.

The antibody moiety of conjugate

Though can use polyclonal antibody, as long as they can provide enough narrow specificity, preferred antibody is monoclonal antibody.Expressing antibodies is meant antibody generally, therefore comprises other molecules that antibody activity fragment and other and antibody binding capacity are similar, as long as they have suitable specificity antibody neoantigen and affinity to problematic target cell.This comprises genetic engineering (reorganization produces) antibody and antibody derivatives or other similar binding constructs.An example is, this antibody is that the antigenic determinant to tumor cell for example has narrow spectrum with the Cancer-Related determinant of colon (antigenic determinant, construction).It will also be appreciated that this antibody can not need the antigenic determinant of cell may have specificity to the cell of autoimmune response, virus infected cell, antibacterial, parasite, fungus or other for those.According to the effectiveness of conjugate, this antibody can be had an effect with antigen, and this antigen will make the antibody internalization after combination, be not preferred although be sure of this antibody specificity.

The monoclonal antibody that the present invention studied is that antagonism GA-733 family is (referring to for example EP-A-376,746 and people such as the document wherein quoted and Larsson, Int.J.Canc.42(1988) people such as C242(Larsson 877-82), Int.J.canc.42(1988) 877-82, with Thy-1.2(mab C, people such as Opitz, Immunobiol160(1982) 438-) antigenic C215 antibody.Can expect that it will be useful that other target cell surface textures are had specific monoclonal.The monoclonal preparation that resists the antigenic determinant of selected target cell is that this area is well-known.Referring to for example above-mentioned publication.Express, for example resist the monoclonal antibody of C242 antigenic determinant or C215 antigenic determinant, comprise the antibody that reacts with cross reaction table antigenic determinant.

In three kinds of monoclonals being tested up to now, the C215-conjugate may be least important because they too continually with the reaction of the antigenic determinant of the tumor antigen of on normal cell, expressing.Show that according to specific data the C242-conjugate is better, although our result shows that they may need higher dosage.The Mab C of anti-Thy-1.2 for target hit human cancer cell may be worth not high because Thy-1.2 antigen is to have specific to inhuman mammalian tumor cell.

The hybridoma cell line that produces the C242 monoclonal antibody in January 26 nineteen ninety be preserved in European animal cell culture preservation committee (Porton Down, Salisbury, wilts, U.K.), preserving number is ECACC 90012601.

The banded structure of superantigen and antibody

In the preferred conjugate of the present invention, superantigen is passed through a covalent bond (B-) with the antigen covalent coupling.If when this conjugate is applied to killed cell, does not decompose when for example being applied to animal is important, so this bonding should be a metabolic stability in the sufficiently long time that is effective basically, it is favourable not bringing autoimmune response to raise when furtherly, carrying out this bonding.Keep at this conjugate on the meaning of target cell binding specificity (antitumor, antiviral etc.) efficiently and efficient activating cell toxin T cell ability, this bonding generally should be inert.

In scientific literature and patent documentation, in the bonding structure of immune conjugate, advised several functional groups, therefore key-the B-in our new conjugate can comprise and is selected from following structure:

(ⅰ) amide and hydrazides (amide=-CONR ₁-or-NR ₁CO-, wherein each free valency combines R with saturated carbon atom ₁Can be hydrogen or low alkyl group (C for example _1-6) alkyl substituent or α-N-substituent group, the preferably hydrophilic amino acid of naturally occurring a-amino acid; Hydrazides=-CONHNH-or-NHNHOC-, wherein each free valency combines with saturated carbon atom);

(ⅱ) thioether and disulphide (Sr-, wherein each free valency directly combines with saturated carbon atom respectively, S is a sulphur atom, r is 1 or 2 integer);

(ⅲ) straight chain, side chain or cyclic hydrocarbon chain, they are saturated and can replace with one or more hydroxyls or amino;

(ⅳ) ether (O-, wherein each free valency directly combines with saturated carbon atom);

(ⅴ) primary amine or two replace hydrazines (NH-or-NH-NH-, wherein each free valency directly combines with saturated carbon atom respectively).

The length of bridge should promptly be less than for example＜100 atom of 180 atoms in conventional in the art scope, but more than 3-6 atom, preferably more than 16 atoms.

Preferred bonding is hydrophilic, and should not contain any aromatic ring.The preferred hydrophilic-structure that can form bonding-B-part is the polypeptide chain of (ⅰ) naturally occurring hydrophilic a-amino acid (for example aspartic acid and amide thereof, glutamic acid and amide thereof, lysine, arginine, glycine, threonine, serine and histidine is perhaps arranged), (ⅱ), oxa-alkylidene chain (O(CH for example ₂) _n) _n'-, wherein n is the integer of 2-5, preferred 2-3, and n ' can be the integer of 1-20; (ⅲ)-the S-(thioether) ,-O-(ether) and unsubstituted amide (CONH-), all these groups all are connected the short unsubstituted hydrocarbon chain (C that preferably contains 1 or 2 carbon atom _1-4) on.

Can use (F-(pro _n) _mThe hydrophilic-structure of F type is represented hydrophilic amino acid, wherein F represented amino acid sequence, preferably 4-8 residue, wherein each aminoacid is independently selected from serine, glycine and threonine, m is the integer of 1-4, n be 4-8 integer (cetus Corp., Wo-A-85/03508).

Key-B-can single-minded location or is connected at random on the superantigen part of antibody or conjugate.Possible position is amino terminal, carboxyl terminal and lysine residue (ω amino group).If antibody or superantigen have carried thiol group or disulfide group group (being respectively cystine or cysteine), these groups can be used for covalent coupling, unless their active optional to this conjugate active part.When having carbohydrate structure, it can be oxidized into aldehyde radical, be used to connect other parts (cf.Cetus Corp., EP-A-240,200) of conjugate then.

Conjugate of the present invention should not contain the ester bond and the unsettled amido link of any significant quantity, particularly can not form threonine and histidine residues respectively.If in building-up process, formed this key, can use azanol to be removed (people such as Endo, Cancer Res.48(1988) 3330-3335).

The number of the superantigen part that every antibody active site exists is generally 1-5, preferred 1 or 2.

A kind of preferred model is, the superantigen in each antibody of this conjugate, and/or used binding site in superantigen and the antibody moiety, and/or bonding-B-etc. should be uniformly actual.In other words, in the conjugate all independently these variablees of conjugated molecule should be identical.

This material should be substantially devoid of not conjugated antibody or not conjugated superantigen.

Superantigen and the most correct ratio of antibody, the bond structure of promptly best conjugate are to depend on selected monoclonal (comprise kind, subspecies, the clone of generation, specificity) and the superantigen of selecting.

The experimental model that provides with this description can screen optimal parameter and other superantigens and other antibody.

The embodiment of broad research to the application date according to the present invention, chain-B-contains following structure:

Free valency in the formula I is connected with active part respectively.This connection can directly take place, and perhaps takes place by further bivalence inertia structure, and this bivalence inertia structure is included among bridge-B-.

N is the integer greater than 0,1-20 for example, preferred 2 or greater than 2, under many circumstances less than 10.M is 1 or 2.

S is a sulphur atom, and it is directly connected to each valency (Sr-=thioether or disulfide bond) of saturated carbon atom.R is 1 or 2 integer.

Y is-NH-,-NHNH-or-NHN=CH-, the CO base shown in the right-hand member combines in its left end and the formula I, its right-hand member and saturated carbon atom or carbonyl (having only when Y=-NHNH-) combine.

R is alkylidene (1-4 carbon atom arranged, 1 or 2 carbon atom is arranged usually) preferably, and it can be replaced by one or more (1-3, preferably＜2) hydroxyl (OH).

The preparation of antibody one superantigen conjugate

Antibody conjugates of the present invention may they obtain by enrichment and purification from the cell culture medium that produces this conjugate or from other synthetic their media.

By conjugate synthetic technology known in the art, promptly genetic engineering (recombinant technique) or carry out traditional coupling reaction at suitable functional group place by means of suitable antibody and superantigen can synthesize our new conjugate.Useful functional group that exist in the protein and common is:

(ⅰ) carbohydrate structure.This structure can be oxidized into aldehyde radical, aldehyde radical then with contain H ₂The chemical compound reaction of NNH-group, formation-C=NH-NH-group.

(ⅱ) thiol (HS-).This thiol can be reacted with the chemical compound that contains the thiol reactive group, forms thioether group or disulfide group.Proteinic free thiol is present in the cystine residue, and can be introduced in the albumen by the disulfide bond in thiolysis or the natural cysteine residues of cracking.

(ⅲ) free amine group (H in the amino acid residue ₂N-).Amine groups can with contain for example chemical compound reaction of activatory carboxyl of electrophilic group, form amide groups.Free amine group is the amino terminal or the omega-amino-of lysine residue preferably.

(ⅳ) free carboxy in the amino acid residue.Carboxyl can change into reactivity (activatory) carboxyl, with the chemical compound reaction that contains amino, forms amide groups then.But must take preventive measures, the amino formation amide that main and carboxyl together are present in the same albumen reduces to minimum.Free carboxy is the carboxyl terminal or the carboxyl of binary a-amino acid preferably.

Carried H ₂NNH-base, thiol reactive group, activatory carboxyl or amino chemical compound can be difunctionality coupling agent or antibody or superantigen.Remove H ₂Outside the NNH-group, these groups directly combine with saturated carbon atom, and H ₂NNH-also can combine with carbonyl carbon.These groups can be introduced in antibody or the superantigen by general derivatization.

Recombinant technique provides effective method for the preparation conjugate, and in the method, each partly is attached to the terminal amino group of another part specifically from the terminal carboxyl group of a part.Can be inserted into the corresponding to bond structure of applied this technology.

Select agents useful for same, make them that key-B-as defined above can be provided.Common difunctionality reagent has general formula Z-B '-Z ', and wherein Z is to form identical functional group each other with Z ', and they can locate covalent coupling the functional group who is present on the protein.As mentioned above.B ' is the inertia bridge, and it can contain the structure identical with the key-B-that provides above.Particularly Z and Z ' can be identical or different, they be selected from thiol, thiol reactive group, activatory carboxyl ,-CONHNH ₂Deng.The definition of these groups is as follows under the title of face " new reagent ".

Our method of employing of conjugate used in the experimental section below may further comprise the steps:

(ⅰ) with antibody or superantigen and contain the thiol reactive group and the organic reagent of amino-reactive group reaction, form the antibody or the superantigen that carry the thiol reactive group, and

(ⅱ) with the remainder of superantigen and antibody with contain thiol or protected the organic reagent reaction of thiol and amino-reactive group, formation is loaded with thiol or is protected the superantigen or the antibody of thiol, so

(ⅲ) step (ⅰ) and (ⅱ) products obtained therefrom react to each other respectively, form conjugate, superantigen is connected with antibody by disulfide bond or thioether in this conjugate.

Applied as protein chemistry, the coupling condition of every kind of group itself is known.Coupling can be carried out in segmentation, perhaps finishes in one step by functional group in the middle of producing, and this centre functional group can be connected with initiation material by the inertia spacerarm.In general, generation condition synthetic and purification/this conjugate of recovery is non-degeneration to contained protein always.

This normally refers to hydrated matrix respectively, and pH is 3-10, and temperature is 0 °-50 ℃.Accurate value depends on the conjugate of reactive group and recovery.See following title " new reagent " for details.

New reagent (what the present invention developed).

For chemosynthesis has the conjugate of key-B-, we have developed a kind of new Heterobifunctional reagent, and this reagent has the general formula II:

M is identical with the implication of above-mentioned formula I with n.Z ₁Be the reactive electrophilic group of HS-, thiol (SH) or protected thiol (for example AcS-), and regulation thiol and hydroxyl can not combine with the same carbon atom among the R.The example of the reactive electrophilic group of HS-is:

(ⅰ) with the bonded halogen of saturated carbon atom, the form of preferred alpha-halogen-alkyl carbonyl (Z for example ₁CH ₂CO-);

(ⅱ) activatory thiol preferably is called the group (SSR of reactive disulfide bond ₁), this disulfide bond combines with saturated carbon atom;

(ⅲ) 3,5-dioxy-1-azepine-ring penta-3-alkene-1-base.

The definition of reactive disulfide bond can be referring to for example EP-A-128885, and this patent is combined in herein by reference.

Z ₁' be activatory carboxyl, i.e. an electrophilic group.For example carboxylic acid halides (COCl ,-COBr and-COI), mixed acid anhydride (COOOCR ₁), reactive ester for example N-succinimido oxygen carbonyl ,-C(=NH)-OR ₂, 4-nitrobenzophenone carboxylate (CO-OC ₆H ₄NO ₂) etc.R ₁And R ₂Can be low alkyl group (C ₁-C ₆), R ₂It also can be benzyl.

One of our new reagent advantage is that they can produce structure (OCH ₂CH ₂) _nMiddle n is the conjugate of the homogeneous of integer, and promptly for each given independent conjugate molecules, n is identical.

With Z ₁' and Z ₁The functional group of reaction can be present in natural antibody bioactive molecule or the natural super antigen or can be directed to wherein Z ₁' and Z ₁End can carry out selective reaction with known method own and these type group with suitable antibody or superantigen then.

Known technology comprises the chain elongation that begins from our new reagent or by the effect of conjugated chemical compound chain elongation.

The chain elongation effect that our new reagent place uses can for example produce wherein-and B-is a conjugate as described below

NHN=-; The sp that N=carbohydrate structure (when it is glycoprotein) common and oxidation from antibody or superantigen produces ²The combination of-hydridization carbon atom

NHN=; N=combines with carbon atom described in (2),

R, R ' and R " be with formula I in the alkylidene selected of R the same manner.The implication of r is same as above.

Can begin to prepare this reagent (formula II) from chemical compound with formula III:

M equals integer 1 or 2.N equals integer 1-20, for example 2-20 or 3-9.

Some chemical compound with formula III (m=1 and 2 wherein, synthetic people such as (, Tetrahedron Letters 29(1988) Jullien 3803-06 n=1-10) had been described in the past; Houghton and Southby, Synth.Commun.19(18) (1989) 3199-3209; And EP-A-410,280(1991 is open January 20) and Slama and Rando, Carbohydrate Research 88(1981) 213-221 and Biochemistry 19(1980) 4595-4600).

By formula III chemical compound and known formula Z-B '-Z ' itself Z=Z wherein ₁, B '=R ", R and R ' be as defined above and, Z '=the difunctionality reagent reacting of activatory carboxyl can synthesize the new reagent of formula II as defined above.After the reaction ,-COOH functional group transforms into activatory carboxyl, for example Z ₁'=activatory ester such as N-butanimide oxygen carbonyl, 4-nitrobenzophenone oxygen carbonyl, 2,4-dinitrophenyl oxygen carbonyl etc.

The noval chemical compound of formula III and new derivant thereof contain the polyethers with following general formula:

N is integer 2-20, preferred 3-20 or 3-9.X be comprise its protonated form ( ⁺H ₃N-) H ₂N-or can transform into H ₂N-(is preferably by hydrolysis or reduction reaction) the H of replacement ₂N-.For example unsubstituted amino (H ₂N-); Nitro; Acylamino-(carbon acylamino) for example contains rudimentary acylamino-(formamido group, the acetylamino of acylamino-... hexanamido), they have electron-withdrawing substituent on the alpha-carbon atom of acyl moiety, preferably CF ₃CONH-, CH ₃COCH ₂CONH-etc.; Can be cyclosubstituted phthalimido, carbamoyl (carbamato) (R particularly ₁' OCONH-and (R ₁' OCO) (R ₂' OCO) N-, N-(tertbutyloxycarbonyl for example) amino (Boc), N-(benzyloxycarbonyl group) amino) and two (N-(benzyloxycarbonyl groups)) amino (being respectively Z and diZ), they can be cyclosubstituted, alkylamino, be α carbon in the aroma system wherein with the bonded carbon atom of nitrogen-atoms, for example the N-benzyl is amino and dibenzyl amino, N-trityl amino (triphenyl methylamino) etc., comprise that wherein methine carbon atom (comprising benzylic carbon atoms) is by the similar group of silicon atom (si) replacement, N for example, N-two (tert-butyl group silicyl) amino; And 4-oxygen-1,3,5-triazines-1-base, be included in its 3-and/group that the 5-position is replaced by low alkyl group.

Above-mentioned and later R ₁' and R ₂' represent low alkyl group, the particularly second month in a season and tertiary alkyl, and may be the methyl that cyclosubstituted 1-3 phenyl replaces.Low alkyl group and lower acyl have 1-6 carbon atom.

Y is that (COOH comprises-COO carboxyl ^-) change into the group of carboxyl, preferably be hydrolyzed or the group of oxidation.The most important thing is ester group, wherein the corresponding atom of carbonylic carbon atom or ortho esters combines with the methylene of formula I right-hand member.Alkyl ester group (COOR for example ₁'); Ortho acid ester group (C(OR ₃') ₃) and reactive as defined above ester group.R ₃' implication and aforementioned R ₁' definition identical.

Other groups Y is-CHO ,-CN ,-CONH ₂,-CONR ₁' R ₂', R wherein ₁' and R ₂' identical with aforementioned implication.

By known chemical combination method itself, can synthesize the formula IV chemical compound from known initiation material.Suitable route of synthesis is:

A, form this chain.

B, conversion functional end-group.

C, symmetric polyethers is transformed into asymmetrical ether.

D, disymmetric chain cracking is become two identical segments.

Has recurring unit-OCH ₂CH ₂-initiation material easily can buy.The few ethylene glycol that for example contains 2-6 recurring unit.Other suitable compound with same end group are corresponding dicarboxylic acids and diamidogen.

Initiation material easily with different end groups is ω-monobasic hydroxy-acid, and wherein end group is separated by pure poly(ethylene oxide) bridge.This have nearly existing in the prior art describe (Nakatsuji, Kawamura and Okahara, Synthesis(1981) the 42nd page) of chemical compound of 5 recurring units.

Pharmaceutical composition and preparation thereof

Pharmaceutical composition of the present invention contains prescription known in the art, just contains our new conjugate now.Therefore the form of said composition can be solution, tablet and the ampulla etc. of lyophilizing granular materials, sterilization or sterile preparation.Can have excipient, for example maybe can there be other inert solid or fluent material in water (preferably being buffered to for example PBS of physiology pH value).

In general, by this conjugate and one or more water-insoluble or water-soluble, aqueous or water-free mixed with excipients, be dissolved in this excipient, combine or chemical combination with this excipient, can prepare said composition, if desired, mix with suitable additive and adjuvant.This excipient and condition definitely can not have a negative impact to the activity of this conjugate.Therefore contain water in the described excipient.

Medication and using method.

Usually this conjugate is sold and is taken with deployed in advance dosage,, can be sure of that the dosage range that each dosage contains the conjugate of effective dose is 10 μ g-50mg according to present result.Dosage (B-) etc. changes along with different cases and according to the type of patient's body weight and age, route of administration, disease, antibody, superantigen, key accurately.

Route of administration is about to conjugate of the present invention target cell dissolving effective dose or the treatment effective dose and contacts with target cell as generally known in the art.The approach that specializes above is meant that mainly parenterai administration for example gives mammal (for example people) injection or infusion (subcutaneous, vein, intra-arterial, intramuscular).This conjugate can be to individual part or system's medication of being treated.

" target cell dissolving effective dose " is meant and can activates CTL effectively and it is produced the amount of doing in order to the destruction target cell.

Claims have defined the present invention, and this also is the part of description of the present invention.The present invention will illustrate by some embodiment, but these embodiment are not the restrictions of general concept that we are found.Experimental section is illustrated in the prepared conjugate activating T cell of embodiment in the chemosynthesis of part 1 conjugate and the II part 4 for the effect that dissolves target cell.

The experimental section part 1

The preparation of omega-amino--PEG-carboxylic acid.

8-hydroxyl-3,6-two evil-sad isopropyl esters (1)

(23g 1.0mole) adds in the diethylene glycol (500ml) in batches with the sodium of small pieces under blanket of nitrogen.After sodium reacts completely, mixture is cooled to room temperature, and under agitation add bromoacetic acid (76g, 0.5mole).After 18 hours, under about 4mmHg condition, steam excessive diethylene glycol 100 ℃ of reactions.Add isopropyl alcohol (400ml) then and add in batches chloroacetic chloride (51g, 0.65mole).After 18 hours, mixture is cooled to room temperature 65 ℃ of stirrings, and (3.5g neutralizes 0.15mole) with sodium acetate.Mixture is filtered, and filtrate is evaporated near dry, then with in its water-soluble (200ml).With 1 (3 * 50ml) aqueous phase extracted.The organic facies water (20ml) that merges is washed with the aqueous phase extraction product of dichloromethane (50ml) from merging, and evaporation obtains oil (55g) then.

11-hydroxyl-3,6,9-three evil-hendecanoic acid isopropyl esters (2).

Under blanket of nitrogen, (23g 1.0mol) adds in the triethylene glycol (700ml) in batches with the sodium of small pieces.When sodium reacts completely, mixture is cooled to room temperature, and under agitation add bromoacetic acid (76g, 0.5mole).After 18 hours, under the 4mmHg condition, boil off excessive triethylene glycol 100 ℃ of reactions.Add isopropyl alcohol (400ml) then, and add in batches chloroacetic chloride (51g, 0.65mole).After 18 hours, mixture is cooled to room temperature 65 ℃ of stirrings, and (3.5g neutralizes 0.15mole) with sodium acetate.Mixture is filtered and filtrate is evaporated near dry, then with in its water-soluble (200ml).With 1 (3 * 50ml) aqueous phase extracted.The organic facies that water (20ml) washing merges.With the aqueous phase extraction product of dichloromethane (50ml) from merging, evaporation obtains oil then. ¹H-n.m.r.（CDCl ₃）;δ1.26（d，6H）;3.07（s，2H）;3.6-3.8（m，12H）;4.11（s，2H）;5.09（m，1H）

The 8-(N-phthalimido)-3.6 ,-two evil-capryl alcohol (3).

With 8-chloro-3, (365g, 2.2mole is from triethylene glycol and SOCl for 6-two evil-capryl alcohol ₂Preparation) be dissolved in the dimethyl formamide (400ml), and under agitation add the adjacent two formyl Asias of benzene by (370g, 2.0mole).110 ℃ stir 18 hours after, the pressure reducing and steaming dimethyl formamide.Under 40-50 ℃, residue is suspended in the toluene (1.5 liters), and leaches potassium chloride.Cooling (10 ℃) makes the product crystallization.By the concentrated mother liquor complex crystallization method of laying equal stress on, can from mother solution, obtain second fraction.

¹H-n.m.r.（CDCl ₃）;δ2.9：0（s，1H）;3.51-3.58（m，2H）;3.60-3.68（m，6H）;3.73-3.78（t，2H）;3.89-3.94（t，2H）;7.70-7.89（m，4H）。

The 17-(N-phthalimido)-3,6,9,12,15-five evil-heptadecanoic acid isopropyl esters (4).

-5 ℃ of pacts with under stirring, with pyridine (2.8ml, 35mmole) drips of solution in dichloromethane (30ml) adds to the 8-(N-phthalimido)-3,6-two evil-capryl alcohol (3) (8.5g, 36mmole) and Trifluoromethanesulfonic anhydride (10.2g is 36mmole) in the solution in dichloromethane.After about 30 minutes, with 0.5M hydrochloric acid and water washing organic facies.Drying (Na ₂SO ₄) and filter after, add 8-hydroxyl-3,6-two evil-sad isopropyl esters (1) (12g, 48mmole) and Na ₂PO ₄(6.5g, 46m-mole), in room temperature with this mixture vigorous stirring 20 hours.Reactant mixture is filtered and evaporated filtrate.Residue is distributed between 1 and water.The evaporation organic facies obtains oil (13g).

¹H-n.m.r.（CDCl ₃）;δ1.26（d，6H）;3.58-3.76（m，18H）;3.90（t，2H）;4.11（s，2H）;5.09（m，1H）;7.70-7.89（m，4H）。

The 17-(N-phthalimido)-3,6,9,12,15-five evil-heptadecanoic acids (5).

With the 17-(N-phthalimido)-3,6,9,12,15-five evil-heptadecanoic acid isopropyl esters (4) (13g) are dissolved in oxolane (50ml) and the concentrated hydrochloric acid (50ml).After 16 hours, water (200ml) dilutes this solution at room temperature reaction, and oxolane is removed in decompression.Wash water with toluene (1 *), and extract with dichloromethane (2 *).Dry (Na ₂SO ₄) and evaporate organic facies, obtain oily product (8.5g).

¹H-n.m.r.（CDCl ₃）;δ3.57-3.76（m，18H）;3.91（t，2H）;4.11（s，2H）;4.8（br，2H）;7.65-7.90（m，4H）

17-amino-3,6,9,12,15-five evil-heptadecanoic acid isopropyl esters (6)

With the 17-(N-phthalimido)-3,6,9,12,15-five evil-heptadecanoic acids (5) (8.5g) are dissolved in 150ml ethanol and the 3ml hydrazine hydrate.Solution stirring at room 16 hours, is added HCl(100ml, 3M), then this solution was refluxed 3 hours.After being cooled to room temperature and filtration, regulate pH(pH9, NaOH) filtrate is evaporated near doing.Add entry and be evaporated to closely quick-dryingly once more, regulate the pH(pH4 of this solution, HCl), be evaporated to dried then.Handling this product in room temperature with isopropyl alcohol (100ml) and chloroacetic chloride (2ml) spends the night and evaporates.In water, collect residue, and under alkaline pH (7-11), use dichloromethane extraction.Evaporation obtains product (3.3g).

¹H-n.m.r（CH ₃OD）：δ1.26（d，6H）;3.17（t，2H）;3.65-3.80（m，18H）;4.16（s，2H）;5.07（m，1H）

The molecular formula of synthetic amino-PEG-carboxylic acid.

Chemical compound 1:n=1

Chemical compound 2:n=2

Chemical compound 3, the phtN-=N-phthalimido

Chemical compound 4, the phtN-=N-phthalimido

Chemical compound 5

Chemical compound 6

The preparation of difunctionality reagent and coupling product

Structural formula is being listed in one page separately.

Embodiment 1.17-iodacetyl amino-3,6,9,12,15-five dislikes the preparation of the N-hydroxy-succinamide ester of heptadecanoic acid

A.17-iodacetyl amino-3,6,9,12,15-five dislikes the preparation of heptadecanoic acid (A) with 17-amino-3,6,9,12,15-five dislikes heptadecanoic acid isopropyl ester (referring to the I part of experimental section), and (1.1g 3.2mmole) is dissolved in the 3ml1M sodium hydroxide solution, and places 30 minutes in room temperature.Add 1.5ml6M hydrochloric acid, be evaporated to this mixture dried.Be dissolved in the dichloromethane residue and filtration, obtain 545mg17-amino-3,6,9,12 after the solvent evaporated, 15-five dislikes heptadecanoic acids.With 460mg(1.39mmoles) this chemical compound is dissolved in the borate buffer of 10mlpH8.4.With nitrogen this solution is outgased.Drip 432mg(1.52mmoles with 1 fen clock time) the 2-iodoacetic acid-solution of N-succinimide ester in the 5ml dioxanes is 8.4 by adding 5MNaOH maintenance pH.This reaction solution was stirred 15 minutes, feed nitrogen simultaneously.According to thin layer chromatography (eluant: CH ₂Cl ₂-MeOH 60: 35), be reflected in a lot of minutes fully.After 15 minutes, the pH of this reaction solution is transferred to 3, the freezing and lyophilizing with this solution.Employing contains the 0-13% gradient acetonitrile of 0.1% trifluoroacetic acid, at reversed-phase column PEP-RPC HR 30/26(Pharmacia Biosystems AB) on make the reactant mixture layering, isocratic separates in 13% acetonitrile, 0.1%TFA then.The component at required peak is merged lyophilizing, obtain 351mg17-iodacetyl amino-3,6,9,12,15-five evil-heptadecanoic acids (A).Productive rate: 76%.

By its NMR spectrum, the structure of establishing this product.Represent with δZhi ¹H NMR composes (D ₂O):

-OCH ₂CHO-3.71-3.76，-NHCH ₂CH ₂O-3.65t，

-HNCH ₂CH ₂O-3.41

B.17-iodacetyl amino-3,6,9,12,15-five dislikes the preparation of the N-hydroxy-succinamide ester (B) of heptadecanoic acids

In reaction bulb, take by weighing N-Hydroxysuccinimide (4.5mg, 39 μ mole).With 17-iodacetyl amino-3,6,9,12,15-five evil heptadecanoic acids (A) (18.3mg, 39 μ mole) are dissolved in the exsiccant dioxanes of 0.55ml and are added in the reaction bulb.Make the reaction bulb degassing with nitrogen, in reaction bulb, drip 8.0mg(39 μ mole then) solution of dicyclohexylcarbodiimide in the dry dioxanes of 0.15ml.In reaction bulb, charge into nitrogen, airtight and place dark.Reaction solution was stirred 3.5 hours.Remove by filter formed precipitation.By the percent that product B in the NMR assay determination filtrate forms, be 89%.

Embodiment 2.(17-iodacetyl amino-3,6,9,12,15-five dislikes the heptadecanoyl amino)-preparation of immunoglobulin (C)

A. monoclonal antibody Mab C215

The monoclonal antibody (Mab C215) (34mg, 0.218 μ mole) of IgG2a immunoglobulin like protein is dissolved in the 0.1M borate buffer that 17.7mlpH8.1 contains 0.9% sodium chloride, adds in the reaction bulb.146 μ l are contained 3.6mg(6.4 μ mole) 17-iodacetyl amino-3,6,9,12, the dioxanes injection of solution that 15-five dislikes the N-hydroxy-succinamide ester (B) of heptadecanoic acid is advanced in this buffer, makes in 25 minutes in stirring at room to react completely.Cover reaction bulb with folie and make its lucifuge.The 0.1M phosphate buffer that contains 0.9% sodium chloride with pH7.5 is as eluant, at Sephadex G 25k 26/40 post higher slice.Remove excessive reagent B.The component that will contain required product C merges.In the Amicon chamber, this solution (22ml) is concentrated into 8ml by the YM30 filter.Measure concentrate and replacement degree with the amino acid analysis method, the result is respectively 4.7mg/ml and 18 interval base/each Mab C215.

B. monoclonal antibody Mab C242

According to the described method of embodiment 2.A, with the monoclonal antibody (Mab C242) of IgG1 immunoglobulin like protein respectively with the 17-iodacetyl amino-3 of 15 times, 20 times and 22 times molar excess, 6,9,12, N-hydroxy-succinamide ester (B) reaction of 15-Wu Evil heptadecanoic acid, obtain nine, 12 and 14 (17-iodacetyl amino)-3,6,9,12,15-five dislikes heptadecanoyl amino)-Mab C242(C).

C. monoclonal antibody Mab C

According to the described method of embodiment 2A, with the monoclonal antibody (Mab C) of IgG2a immunoglobulin like protein respectively with the 17-iodacetyl amino-3 of 14 and 18 times of molar excess, 6,9,12, the N-hydroxy-succinamide ester reaction of 15-Wu Evil heptadecanoic acid, obtain four and seven (17-iodacetyl amino-3,6,9,12,15-five dislikes heptadecanoyl amino)-Mab C.（C）

Embodiment 3.2-sulfydryl propionamido-Eu ³⁺The preparation of the staphylococcal enterotoxin A of one labelling (SEA)

A.Eu ³⁺The SEA(D of labelling) preparation

The lyophilized products that SEA(is obtained from Toxin Technology Inc.) (2mg 72nmole) is dissolved in the 722 μ l milli-Q water, and adds in the 15ml polypropylene tube.The 0.1M borate buffer solution that adds 100 μ l pH8.6 is added in 2160 nmoles Eu among the 178 μ l milli-Q then ³⁺-chelating agen (Pharmacia WallacOy).Make in ambient temperature overnight and to react completely.As eluant, on Sephadex G25 PD10 post (Pharmacia Biosystems AB), reaction solution is removed excessive reagent with the phosphate buffer of 0.1M pH8.0 by layering.The component that will contain required product D merges.In the Amicon chamber, this solution (3ml) is concentrated into the 0.8ml volume by the YM5 filter.Measuring its concentration with the amino acid analysis method is 1.7mg/ml.By with EuCl ₃Standard solution compares, and measuring the replacement degree is 0.8Eu ³⁺/ each SEA.

B1.3-(2-pyridine radicals dithio) propionamido Eu ³⁺The SEA(E of labelling) and 3-sulfydryl propionamido Eu ³⁺The SEA(F of labelling) preparation

Will be at the Eu in the 0.1M of the 0.75ml pH8.0 phosphate buffer ³⁺-SEA(1.24mg 44.8nmoles) adds in the 15ml polypropylene tube.With 35 μ l(180nmole) 1.6mg 3-(2-pyridine radicals dithio)-solution of N-succinimide ester in 1ml ethanol of propanoic acid is added in this test tube, with reaction solution stirring at room 30 minutes.Before being reduced into product F, do not separate products therefrom E.In above-mentioned reaction solution, add 20 μ l 0.2M Eu ³⁺-citric acid solution, and add 50 μ l 2M acetic acid pH is transferred to 5.Add the solution of 3.1mg dithiothreitol, DTT (Merck) in 0.1ml0.9% sodium chloride then, and this reaction solution was stirred 20 minutes in room temperature.Add 50 μ l0.9% sodium chloride solutions then cumulative volume is transferred to 1ml.Reaction solution (1ml) is packed on the Sephadex G 25 NAP-10 posts (Pharmacia Biosystems AB), contain the 0.1M phosphate buffer of pH7.5 of 0.9% sodium chloride with required product F eluting by adding 1.5ml.The product F of eluting is collected in the 15ml polypropylene tube, and is used for synthetic product G immediately, to avoid the disulphide reoxidation.

B2.2-sulfydryl propionamido staphylococcal enterotoxin A (SEA) preparation (F2)

According to the described method of embodiment 3B1, the lyophilized products that natural SEA(is obtained from Toxin Technology Inc) or the SEA(rSEA of reorganization preparation) with the 3-(2-pyridine radicals dithio of 2 times of molar excess)-the N-succinimide ester reaction of propanoic acid.

According to people such as Carlsson described (Biochem.J.173(1978) 723-737), be 1.9 mercapto radical propionyl groups/each SEA by the replacement degree of UV-assay.

The preparation of embodiment 4.SEA-monoclonal antibody conjugate (G1 och G2)

A.Eu ³⁺And the conjugate between the Mab C215 (G1)

To the described 4-sulfydryl of embodiment 3B propionamido Eu ³⁺The SEA(F of labelling) add 1.2ml 18 (17-iodacetyl amino-3,6,9,12,15-five dislikes the heptadecanoyl amino) Mab C215(C in the solution) (4mg) in the solution in the 0.1M of the pH7.5 that contains 0.9% sodium chloride phosphate buffer.Place to spend the night to make in room temperature and react completely.Add the 5 μ l(1.2 μ moles of 20 μ l mercaptoethanols in 1ml water then) solution, make unreacted iodine groups sealing.Reaction solution is placed 4 hours after-filtration in room temperature.Then with the 0.002M phosphate buffer of pH7.5 that contains 0.9% sodium chloride as eluant, with filtrate at Superose 12HR16/50 post (Pharmacia Biosystems AB) higher slice.Merging contains the component of required product G and analyzes.The protein content of measuring by the amino acid analysis method is 0.22mg/ml.Pass through Eu ³⁺The replacement degree that algoscopy is measured is each IgG of SEA/.Also this product is carried out the research of immunostimulatory properties and antibody binding capacity.

By improving the amount of chemical compound (F), can obtain higher replacement degree with respect to chemical compound (C).

Conjugate (G2) between B.rSEA and the Mab C215

According to the described method of embodiment 4A, (17-iodacetyl amino-3,6,9,12,15-five dislikes the heptadecanoyl amino) Mab C215(C with 18) with the 2-sulfydryl propionamido-rSEA(F2) reaction of 1.8 times of molar excess.At Phast-Gel ^TMGradient 4-15 goes up and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGI) composition of this conjugate to be analyzed, and with Phast IMAGE(Pharmacia Biosystems AB) coalition is scanned.The gained conjugate by 6% contain three SEA, 15% contain 2 SEA, 28% Mab C215 and the 51% unsubstituted Mab C215 that contains a SEA form.

In another one experiment, use the F2 of 2.7 times of molar excess, the component of the conjugate that obtains is 15% to contain three SEA, 25% and contain Mab C215 and the 26% unsubstituted Mab C215 that two SEA, 34% contain a SEA.

Conjugate between C.rSEA and the Mab C242

C1. according to the described method of embodiment 4A, with 14 (17-iodacetyl amino-3,6,9,12,15-five dislikes the heptadecanoyl amino) Mab C242(C) with the 2-sulfydryl propionamido-rSEA(F2) reaction of 3.2 times of molar excess.Analyze according to the described composition of embodiment 4B, find that it consists of 4% and contains four SEA, 12% and contain three SEA, 28% and contain Mab C242 and the 20% unsubstituted Mab C242 that two SEA, 36% contain a SEA this conjugate.

Carry out same reaction, just in the post layering to remove Mab C242 with at interval between the base and between SEA and the mercapto radical propionyl group before the unsettled coalition, usefulness 0.2M azanol processing reaction product 4 hours.Formed conjugate has following composition: 1% contains four SEA, 12% contains three SEA, 27% and contains Mab C242 and the 24% unsubstituted Mab that two SEA, 36% contain 1 SEA.

C2. according to the described method of embodiment 4A, with 12 (17-iodacetyl amino-3,6,9,12,15-five dislikes the heptadecanoyl amino) Mab C242(C) with the 2-sulfydryl propionamido-rSEA(F2) reaction of 3 times of molar excess.The composition of gained conjugate is 6% to contain two SEA, 26% and contain Mab C242 and the 31% unsubstituted Mab C242 that two SEA, 36% contain a SEA.

C3. according to the described method of embodiment 4A, to nine (17-iodacetyl amino-3,6,9,12,15-five dislikes the heptadecanoyl amino) Mab C242(C) with the 2-sulfydryl propionamido-rSEA(F2) reaction of 3 times of molar excess.The composition of gained conjugate is 13% to contain Mab C242 and the 46% unsubstituted Mab C242 that two SEA, 39% contain a SEA.

Conjugate between D.rSEA and the Mab C

D1. according to the described method of embodiment 4A, with the 2-sulfydryl propionamido-rSEA(F2) reaction of seven (17-iodacetyl amino-3,6,9,12,15-Wu Evil heptadecanoyl amino) Mab C and 5.4 times of molar excess.According to the described method of embodiment 4B the composition of conjugate is analyzed, found that it consists of: 15% contains four SEA, 24% contains three SEA, 29% and contains Mab C, 3% unsubstituted Mab C that two SEA, 19% contain a SEA and 10% dimerization form.

Carry out same reaction, remove between Mab C and the interval base and unsettled coalition between SEA and the mercapto radical propionyl group with the 0.2M azanol.Formed conjugate has following composition: 11% contains three SEA, 24% contains Mab C, 18% unsubstituted Mab C and the 17% dimerization form that two SEA, 30% contain a SEA.

D2. according to the described method of embodiment 4B, with the 2-sulfydryl propionamido-rSEA(F2) reaction of four (17-iodacetyl amino-3,6,9,12,15-five dislikes heptadecanoyl amino) Mab C and 5.7 times of molar excess.This conjugate has following composition: 8% contains four SEA, 18% contains three SEA, 30% and contains Mab C, 5% unsubstituted Mab C and the 12% dimerization form that two SEA, 26% contain a SEA.

Embodiment 5.[17-(3-sulfydryl propionamido)-3,6,9,12,15-five dislikes the heptadecanoyl amino]-rSEA(I) preparation

A.17-[3-(2-propionamido pyridine radicals dithio)]-3,6,9,12,15-five dislikes the heptadecanoyl amino)-rSEA(H) preparation

With 17-[3-(2-pyridine radicals dithio) propionamido]-3,6,9,12, the N-hydroxy-succinamide ester (K) of 15-Wu Evil heptadecanoic acid (0.53mg, 896nmoles) injection of solution in 43 μ l dioxanes is to 3.67mg(128nmoles) rSEA is in 1ml contains solution in the phosphate buffer of pH7.5 of 0.9% sodium chloride.Place to make in 30 minutes in room temperature and react completely.Get 100 μ l and replace degree analyzing.With the remainder refrigerated storage, until it being reduced into product (I).

By going up 100 μ l reaction solution desalinations at Sephadex G50 NICK post (Pharmacia Biosystems AB), measure the replacement degree, and according to people such as Carlsson described (Biochem.J.173(1978) 723-737) use the ultraviolet spectral analysis eluent.2.7 individual interval base and rSEA coupling.

B.[17-(3-sulfydryl propionamido)-3,6,9,12,15-five dislikes the heptadecanoyl amino]-rSEA(I) preparation

The pH that will contain the reaction solution (0.9ml) of product H transfers to pH4.4 with 2HCl, and adds the 2.9mg dithiothreitol, DTT that is dissolved in the 75 μ l0.9% sodium chloride.Made reduction reaction complete with 30 minutes.Reaction solution is added on the Sephadex G25 NAP10 post (Pharmacia Biosystems AB), contain the 0.1M phosphate buffer eluting of the pH7.5 of 0.9%Nacl with 1.5ml, and be used for the product J of synthetic embodiment 11 immediately.

Embodiment 6. contains the preparation of SEA-monoclonal antibody conjugate of double space base under blanket of nitrogen and under the dark condition, with 12 (17-iodacetyl amino-3,6,9,12,15-five dislikes heptadecanoyl amino) Mab C242(C) (4.2mg, 27nmoles, contain in the 0.1M phosphate buffer of pH7.5 of 0.9%Nacl at 1.0ml) with [17-(3-sulfydryl propionamido)-3,6,9,12,15-five dislikes heptadecanoyl amino]-rSEA(I) (1.17mg, 42nmoles), in the above-mentioned buffer of 1ml) reacted 43 hours.Add 1.14 μ mole mercaptoethanols then.After reacting 1 hour again, reaction solution is carried out layering on Superdex 200HR 16/65 post.2mM phosphate buffer eluted product with the pH7.5 that contains 0.9%Nacl.Merge the component that contains required product J, and analyze according to embodiment 4B is described.This conjugate contains Mab C242 and the 66% unsubstituted Mab C242 that two SEA, 25% contain a SEA by 9% and forms.

The experimental section II

The effect of superantigen-antibody conjugates pair cell

The bacteriotoxin that is used for following experiment is from Toxin Technologies(WI; USA) recombiant protein of staphylococcal enterotoxin A of Huo Deing (SEA) or generation from escherichia coli (E.coli).

Antibody is C215, C242 and Thy-1.2mAbs.C215 is the IgG2a mAb that a kind of anti-people of raising clones tumor cell line, the 37kD proteantigen reaction that it and several people's clone cell are fastened.The list of references of these mAbs provides hereinbefore.According to these conjugates of the described preparation of previous section.

Before priority date, only to Eu ³⁺The SEA-C215mAb conjugate of labelling is studied.In a year of priority, checked the result of unlabelled SEA-215, SEA-242 and SEA-Thy-1.2mAb conjugate.The result who introduces is meant unlabelled conjugate now.

Express very low and cytotoxicity clone's tumor cell that can not detection limit in order to measure by the anti-shortage II class MHC of SEA-C215mAb conjugate and not conjugated SEA and C215mAb mediation or II class MHC, the T cell line action effect device cell that we have used various people SEA to enlarge is with one group of clone's tumor cell and II ⁺Class MHC Raji cell is as target cell.Measure according to mAbs dyeing and facs analysis method with anti-HLA-DR, HLA-DP and HLA-DQ, clone tumor cell line Colo205.SW620 and WiDr lack the expression of II class MHC.In the presence of recombinant IL-2(20 unit/ml),, handle the II of using the SEA coating in advance by with repetitive stimulation a little less than the ametycin ⁺Class MHC BSM lymphocyte has been set up the T cell line of SEA amplification from peripheral blood.These T cell lines have strong cytotoxicity to Raji or the BSM cell with the SEA coating, but do not have cytotoxicity to the cell of coating not or with the cell of staphylococcal enterotoxin B (SEB) coating.According to blockade by employing HLA-DR antibody, II ^-The mensuration that the L cell of class MHC Raji mutant cell and HLA-DR transfection carries out (people such as Dohlsten, Immunology71(1990) 96-100), SEA causes that killing action depends on SEA and the interaction of II class MHC on target cell.These T cell lines can be activated by the C215-SEA conjugate, thereby kill C215 ⁺II ^-Class MHC clones tumor cell.Not conjugated on the contrary SEA and C215mAb can only induce limited amount anti-C215 ⁺II ^-The T cell killing action of class MHC clone tumor cell.The cell-mediated cytotoxicity that the staphyloentero-toxin antibody conjugates relies on depends on C215 ⁺Tumor cell conjugation ground combines with SEA-C215mAb's.By the following specificity that fact proved in this combination, promptly not conjugated C215mAb is excessive, and is but uncorrelated with the C242 and the W6/32mAbs that suppress clone's oncolysis.CD + ₄And CD + ₈The T cell is proved and can kills the C215 that SEA-C215 handles ⁺Clone's tumor cell, but can not dissolve the cell that SEA handles.According to the inductive II of killing of SEA that early confirms ⁺The effect of class MHC cell, the T cell be combined in II ^-As if the interaction of the SEA-C215mAb conjugate on the class MHC tumor cell comprised and the interaction of specificity V β TCR sequence with the same manner.This point is proved to be by the SEA specificity rather than from the interaction of the SEB of body specificity T cell line/lineage and C215-SEA conjugate.C242mAb has confirmed to have similar activity to the C215mAb conjugate with the Thy-1.2mAb conjugate.

Chromium labelling and cultivate target cell with SEA

In 37 ℃, 100 μ l volumes, with 0.75 * 10 ⁶Target cell and 150 μ Ci ⁵¹(Amersham Corp., Arlington Hights England) cultivated 45 minutes chromium.With cell be retained in contain RPMI-1640 substrate (Gibco, Paisley, GBR), replenished 2.8%(v/v) 7.5%NaHCO ₃, 1% Sodium Pyruvate, 2%200mML-glutamine, 1%1M Hepes, 1%10mg/ml gentamycin and 10% hyclone (FCS, Gibco, Paisley, GBR) in the complete medium, after the cultivation, with the complete medium that does not contain FCS with cell washing once, and cultivated 60 minutes at 37 ℃, washing and resuspending are in the complete medium that contains 10%FCS.(Costar, Cambridge add 5 * 10 in each hole USA) to 96 hole microdroplet plates at the bottom of the U type ³Target cell.

Cytotoxicity analysis

In each hole, add effector cell with various effectors/target cell ratio.The final volume in every hole is 200 μ l.Each test is done three parts.At 37 ℃ this plate was cultivated 4 hours, collected the chromium that discharges then.γ-register (Cobra Auto-gamma, Packard) middle mensuration ⁶¹The amount of Cr.Calculate cytotoxicity percent by following formula: the * 100 of % cytotoxicity=(X-M)/(T-M), wherein X is the chromium burst size that obtains from test specimen, represent with cpm, M is the spontaneous chromium burst size with the target cell of culture medium culturing, and T cultivates total chromium burst size that target cell obtained with 1% sodium lauryl sulphate.

The result

SEA-C242, SEA-C215 and SEA-be anti--and the Thy-1.2mAb conjugate combines with the cell of the antigenic determinant of expressing corresponding mAbs respectively, and and II ⁺The combination of class MHC cell.On the contrary, not conjugated SEA and II ⁺The combination of class MHC cell.Not conjugated C215, C242 and Thy-1.2mAbs combine with corresponding cell, but do not combine with the Raji cell.(table 1)

Have the SEA-C215mAb conjugate, but when not having not conjugation SEA and C215mAb, the human T-cell is the dissolving II ^-Class MHC SW620, Colo205 and WiDr cell (Fig. 1).In the presence of 10-100ng/ml SEA-C215mAb conjugate, observe clone's oncolysis.Observe at various effectors the high-caliber dissolving of the ratio of target (Fig. 1) with the SEA-215mAb conjugate of anti-SW620.On the contrary, under the ratio of effector to target of all tests, not conjugated SEA or C215mAb can not mediate the cytotoxicity of anti-SW620 cell.This shows the dissolving II ^-The ability of class MHC Colo205 cell only limits to conjugate, and this effect can not be induced by not conjugated SEA and C215mAb.SEA and SEA-C215mAb conjugate have mediated II ⁺The II that class MHCRaji cell and interferon are handled ⁺The T cell killing action of class MHC Colo205 cell, and C215mAb can not.(Fig. 1).

For the dissolving that confirms the mediation of SEA-C215mAb conjugate has comprised that this conjugate combines with the conjugation specificity of C215mAb molecule on target cell, we have carried out the research of blockading with excessive not conjugation C215mAb and mAbC242, and they combine (about the C215mAb combination) with incoherent antigen on clone's tumor cell.Add the mAb C215 cytotoxicity of blockading very doughtily, not influence (Fig. 2) of opposite C242mAb.Similar dissolving by the mediation of SEA-C242mAb conjugate is blockaded by excessive not conjugated C24mAb rather than C215mAb specificity.

At CD + ₄And CD + ₈All having observed the SEA-C215mAb conjugate in the T cell mass induces II class MHC to clone the T cell dissolved ability of dependency (table 2) of tumor cell.SEA can not activate the killing action of any of these T cell subbreed with mediation SW620 cell, but it can induce II ⁺The dissolving (table 2) of class MHC Raji cell.

The SEA-C215mAb conjugate is by the T cell line of SEA amplification, rather than the T cell line that increases by SEB causes SW620 and Raji cytolysis (Fig. 3).When placing the Raji cell, respectively the selective acknowledgment of SEA and SEB has been shown the specificity (Fig. 4) of SEA and SEB system by them.This shows that the SEA-C215mAb conjugate has kept similar V β TCR specificity concerning conjugation SEA not.

Explanation to figure

Fig. 1 .SEA-C215mAb conjugate has caused anti-II ^-The CTL of class MHC clone tumor cell.Above one group on the left side proved do not have (one) or have (concentration of every kind of additive is 1 μ g/ml) under mixture (C215+SEA) condition of SEA-C215mAb conjugate, SEA, C215, C215 and SEA, under the ratio of various effectors to target, the SEA of anti-SW620 cell replys the effect of CTL.Another group has proved anti-II ^-Class C215+MHC clone tumor cell line SW620, Colo2-05 and WiDr, II ⁺Class MHC C215 ⁺Colo205 cell and II that interferon is handled ⁺The SEA-C215 mAb conjugate of class MHC C215-Raji cell and SEA reply the ability of CTLs to target SEA.Effector is 30: 1 to the ratio of target.The not conjugation C215mAb that adds several concentration can not cause the CTL of any anti-these cell lines.For detecting any II class MHC surface expression, use anti-HLA-DR ,-DP ,-mAb of DQ carries out facs analysis to SW620 cell, Colo205 and WiDr cell and fails, but on the Raji cell, but detect rich H LA-DR ,-DP and-expression of DQ, and on the Colo205 cell that interferon is handled, detect rich H LA-DR and-expression of DP.Before being used for the CTL analysis, the Colo205 cell was handled 48 hours with 1000 units/ml recombinant interferon-.

The CTL of Fig. 2 .SEA-C215mAb conjugate and the inductive anti-clone's tumor cell of SEA-C242mAb conjugate depends on the selection of antigen of mAb.By adding not conjugated C215 and C242mAb(30 μ g/ml respectively), the dissolving of the Colo205 cell of the CTL system mediation that SEA replied under blockaded SEA-C215mAb and SEA-C242mAb conjugate (3 μ g/ml) existed.Before adding conjugate 10 minutes, not conjugated mAb or control medium () are added in the target cell.

The CTL that Fig. 3 replys by SEA rather than SEB has mediated the dissolving of clone's tumor cell of SEA-C215mAb conjugate coating.There is not (contrast) or having the SEA-C215mAb conjugate, under the mixture (C215+SEB) of not conjugated C215mAb and SEA and the mixture (C215+SEA) of not conjugated C215mAb and SEB (every kind of additive concentration the is 1 μ g/ml) condition, under effector to the ratio of target is 10: 1 conditions, be used for anti-SW620 and Raji target cell using from body SEA and SEB selectivity T cell line.

Fig. 4 is resisted-Thy-1 by SEA-C242mAb conjugate and SEA-, the inductive cytotoxicity that resists their target cell (being respectively Colo205 tumor cell and EL-4 tumor cell) of 2mAb conjugate.

Table 1

SEA-C215mAb conjugate and C215 ⁺Clone's tumor cell and II ⁺The combination of class MHC Raji cell

Reagent cell Facs analyzes

The SEA-C215mAb Colo205 positive

The Raji positive

The C215mAb Colo205 positive

The Raji feminine gender

The SEA-C242mAb Colo205 positive

The Raji positive

The C242mAb Colo205 positive

The Raji feminine gender

SEA-resists-the Thy-1.2mAb EL-4 positive

Anti--the Thy-1.2mAb EL-4 positive

SEA Colo205 feminine gender

The Raji positive

Contrast Colo205 feminine gender

The Raji feminine gender

The EL-4 feminine gender

Use various contrast additives (PBS-BSA) cultured cell 30 minutes on ice, the washing and by following described the operation.Detect C215mAb and C242mAb and the bonded dyeing of Colo1205 cell and anti--Thy-1.2 and bonded dyeing of EL-4 cell with the anti-Mus Ig of the rabbit of FITC labelling.Resist-SEA serum with rabbit, detect the dyeing of SEA with the anti-rabbit 1g of FTTC-pig then the Raji cell.Use in the face of C215mAb and the described method detection SEA-C215mAb of SEA and Colo205 and the conjugated dyeing of Raji cell.Obtain from Becton and Dickinson at FACS star plus() carry out facs analysis.Determine the truth of a matter with the staining that included only for the second and the 3rd step.

Table 2

CD + ₄And CD + ₈The CTLs dissolving contains clone's tumor cell of C215-SEA conjugate.

The % cytotoxicity

Effector ^A)Target contrast SEA C215-SEA

CD4 ⁺sw620 2 5 50

CD4 ⁺Raji 0 41 43

CD8 ⁺sw620 0 1 23

CD8 ⁺Raji 2 72 68

A) do not exist under (contrast) condition or exist under 1 μ g/mlSEA and the C215-SEA condition at SEA and C215-SEA, under effector to the ratio of target is 30: 1 conditions, use CTL(SEA-3).

Claims (10)

1, a kind of preparation method of conjugate said method comprising the steps of:

(ⅰ) by means of at least a being selected from (a) carbohydrate structure and (b) functional group: thiol, disulfide group, carboxyl and amino structure, according to known method itself, make superantigen and antibody coupling, from antibody and/or synthetic this conjugate of superantigen;

(ⅱ) from the medium of synthetic this conjugate, reclaim conjugate.

2, according to the conjugate preparation method of claim 1, wherein coupling is to carry out on the amino in superantigen and/or antibody.

3, according to the conjugate preparation method of claim 1, wherein coupling may further comprise the steps:

(ⅰ) with antibody or superantigen and contain the thiol reactive group and the organic reagent of amino-reactive group reaction, form the antibody or the superantigen that carry the thiol reactive group;

(ⅱ) with the remainder of superantigen and antibody with contain hydroxyl or protected the organic reagent reaction of thiol, formation is loaded with thiol or is protected the superantigen or the antibody of thiol;

(ⅲ) make from step (ⅰ) and the product that (ⅱ) obtains respectively and react to each other, form the conjugate that superantigen and antibody are connected by disulfide bond or thioether.

4, according to the conjugate preparation method of claim 3, the amino-reactive reagent that wherein contains the thiol reactive group is alpha-halogen acetyl group halides, and the chemical compound that contains thiol or protected thiol is the difunctionality coupling agent with formula II:

(II) wherein m is integer 1 or 2, and n is integer 1-20, Z ₁Be that (same carbon atom combines Z SH) or protected thiol (for example Acs-), and among defence thiol and hydroxyl and the R for the reactive electrophilic group of HS-, thiol ₁' be activatory carboxyl.

5, according to the conjugate preparation method of claim 1-4, wherein said superantigen derives from antibacterial, particularly is selected from staphyloentero-toxin.

6, according to any one conjugate preparation method among the aforementioned claim 1-5, wherein superantigen is SEA.

7, according to any one conjugate preparation method among the aforementioned claim 1-6, wherein each antibody moiety has 1-5, preferred 1-3 superantigen part at least.

8, according to any one conjugate preparation method among the aforementioned claim 1-7, wherein antibody is monoclonal.

9, according to the conjugate preparation method of claim 8, wherein antibody is anti-specifically C242 antigenic determinant.

10, according to the conjugate preparation method of claim 8, wherein antibody is antigenic determinant on the anti-protein specifically, and this antigenic determinant belongs to GA-733 family, particularly the C215 antigenic determinant.

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