LT3909B - New antibody-superantigen conjugates, process for preparing thereof, process for target cell lysis and use of conjugates in pharmaceutical compositions - Google Patents

Abstract

A soluble antibody conjugate having an antibody linked to a structure recognized by T-cells capable of directing T-cells to cell-targeting recognized by antibodies. The conjugate is characterized by the indicated structure is superantigen. An important aspect is the method of cell targeting by which cells are targets contact with effective cell targeting for conjugate content. The lysis method is effective for cancer, autoimmune part of a treatment regimen for diseases, infections with parasites and infections caused by fungi, viruses and bacteria. The description also includes aspects such as conjugate synthesis, pharmaceutical compositions and their preparation.

Description

The present invention is directed to antibody conjugates that can activate. ..cytotoxic ... T-cells. (CTL). Conjugates are used to destroy unwanted cells associated with various cancers, autoimmune processes, parasitic infections, and bacterial, viral and fungal infections.

Attempts have been made to use antibodies in combination with agents that exert direct cellular toxicity to cytotoxic agents (cytotoxic agents, cytotoxins), providing selective cell-target targeting, and preventing and reducing non-specific effects on other cells. The proposed combinations range from covalently linked complexes using molecular moieties and non-covalently linked complexes to simple mixtures (e.g., Ghose et al., J. Nate. Cancer Inst., 61, 1979, 657-676; Karlsson et al., Biotechnology 7). (1989) 567-73). Cytotoxins such as diphtheria toxin, ricin, ricin domain A, gelonin and Pseudomonas aeruginoso exotoxin A are known (Takada Chemical Ind., EP-A-336405 and Pastan et al., WO-A-38/00703; both sources cited in priority application SE9002479).

With the advent of hybridoma technology and the accompanying monoclonal antibodies, it has become possible to use complexes composed of antibodies and cytotoxic compounds to more specifically target cytotoxic agents to the required cell-target population.

Because of the known cytotoxic cell-damaging effects on other cells other than cellular targets, it has been proposed to replace cytotoxic agents with immunostimulants that stimulate T-lymphocytes and activate CTLs. Specific proposals involved antibodies conjugated to:

(i) antibodies directed against T-cell receptors or compounds capable of binding to T-cell receptors (Mass. Inst. Techn., EP-AI180171);

(ii) compounds such as antigens, mitogens, other foreign proteins and peptides that activate cytotoxic T-cells (Noereks Korp., EP-AI334300);

(iii) Basic Tissue Compatibility Antigen Complex (PASKA) (Begringverco AG EP-AI-352761);

(iv) antigens against which the subject is immune (Med. Ros. Coun. WO-A-90 / II779 (published October 18, 1990)); and (v) an unnamed bacterial enterotoxin (Ochi and Wake, UCLA Symposium: Cellular Immunity and Cancer Therapy, January 27-February 30, 1990, Abstracts of Report CE 515, page 109).

However, the immunostimulants proposed so far were either very specific or very general in their function. For example, classical antigens only activate about I of 10 ⁵ T cells, whereas mitogens have the potential to activate most of them.

Some agents have been found to activate T-cells in moderation, i.e. they activate T-cells at relatively high frequencies, but far from 100%. Fleisher et al., J. Exp. Med., 157: 1697-1707 (1988); Wait et al.,

Cell, 56: 27-35 (1989); both articles are included in this description as references. Agents of this type are more potent activators than classical antigens and have therefore been named superantigens / for review see: Keccler and Marrac, Science 248: 705, (1990) /. Further, it was shown in PaLT 3909 B / Doglsten et al., Immunol. 71: 96-100 (1990); Chedlund et al., Cell Immunol. 129, 426-34 (1990), that known superantigens can bind to class II target cell compatibility complex (PASK) molecules on target cells and activate cytotoxic T-cells carrying the beta-chain of the T-cell receptor variable region. Published data indicate that binding to PASCs is a prerequisite for T-cell binding and activation. It cannot be ruled out that in the future, superantigens acting via the alpha chain of the T-cell receptor variable region or other surface structures within T-cell subpopulations will be found.

The immunomodulatory effect of superantigen-Staphylococcus enterotoxin A has also been described by Platsokus et al., (Cell Immunol., 97, 371-85 (1986)).

Most of the now known superantigens have previously been identified as toxins and of microbial origin. Staphylococcal enterotoxins, for example, are enterotoxic and activate T-cells, and these two effects differ from each other / Fleisher et al., Cell Immunol., 118 (1989) 92101; Alber et al., J. Immunol. 144 (1990) _4501-06; and Infact. Immun. 59: 2126-34 (1991).

Previously, the use of superantigens has been proposed to target CTL-mediated murmuration of cells containing Class II Core Tissue Compatibility Complex (PASKA) (Farmacia AB, WO-A-91/04053, published 04.04.1991). WO-A-91/04053 includes superantigens but does not mention covalent immunoconjugates. Cells lacking or expressing threshold levels of PASK class II proteins do not bind sufficient amounts of superantigens to efficiently target their lytic CTL. Thus, due to the high total amount of cells carrying the Class II PASKA and the low amount of the Class II PASKA on most cancer cells, the superantigens should not have a high value in terms of the specific destruction of such unwanted cells.

However, we have discovered that the specific effect of CTL cell killing can be achieved by superantigens if they are covalently linked to an antibody against an epitope that is specific for killing! to the cell. Activation of the immune system can induce expression of PASKA class II in cellular targets devoid of these antigens, which in turn may produce the desired lysis effect.

BRIEF SUMMARY OF THE INVENTION

The present invention encompasses (i) novel antibody conjugates comprising (I) a cell-target antibody and (2) a superantigen. i.e. a structure that recognizes (interacts and / or fuses) and activates T-cells, particularly CTL;

(ii) cell-target destruction techniques, in particular in relation to therapeutic methods for treating mammals, and methods of specific activity of T-cells such as CTL;

(iii) methods for the synthesis of conjugates; and (iv) conjugate-containing pharmaceutical compositions and methods of preparing these compositions.

Techniques for cell-target killing include therapeutic treatments for cancer, viral, bacterial, fungal infections, parasitic infections, and other diseases where the ultimate goal is the killing of certain cells with a high degree of accuracy. In accordance with the present invention, the conjugates may be used in the preparation of pharmaceutical compositions for the disruption of the target targets associated with the aforementioned disorders. The objects of treatment are mammals, first order humans.

Superantigenic Conjugate Part

The novel antibody conjugates exhibit a structure recognized by T-cells as being a superantigen. Conjugates generally dissolve at physiological pH and are soluble in serum in vitro.

Preferably, superantigens are selected from staphylococcal enterotoxins (SEs) such as SEA, SEB, SEC, SEED, and SEE, toxoids, their active fragments or peptides, and other compounds having substantially the same mode of action in CTL activation. Superantigens may include other microbial products (bacterial and viral), such as products from staphylococcal strains, such as Toxic Shock Syndrome Toxins (TSST-I) from Strept. okocci, such as pyrogenic exotoxin A, and bacterial exoproteins and proteins produced by pleuropneumonioid microorganisms with similar ability to interact with T-cells as superantigens interact with them. Superantigens can be produced by growing their natural producers or by cells obtained by genetic engineering (recombinant methods) and potentially also by peptide synthesis. The superantigen used in the frame of the present invention, when tested on an experimental model provided herein, must exhibit activity analogous to that presented in the experimental section of this disclosure.

Optimal superantigens have the potential to bind to T-cell surface 1-30% of the isoform polymorphic protein involved in CTL activation, most preferably to the T-cell receptor variable region beta chain.

Conjugate antibody (AK) part

The antibody is preferably a monoclonal antibody (monAK), although polyclonal antibodies may be used provided they have a sufficiently narrow specificity range. The term antibody is intended to apply to antibodies in their entirety, and thus includes antibody active fragments and other molecules that mimic the binding capacity of antibodies, provided that they exhibit appropriate specificity, avidity, and affinity for the given cell-target. This includes antibodies produced by genetic engineering (recombinant methods), antibody derivatives, and other structures with similar binding ability. In one embodiment of the invention, the antibody is specific for an antigenic determinant of cancer cells, e.g., a determinant associated with colon cancer (epitope, structure). It is also understood that the antibody may be specific for an antigenic determinant on cells responsible for the autoimmune response, cells infected with the virus, bacteria, parasites, fungi, or other unwanted cells. Depending on the efficacy of the conjugate, the antibody may be directed against the antigen internalizing the antibody after fusion, although it is believed that such specific antibodies are not optimal.

In the context of the present invention, the monoclonal antibodies tested are the C215 antibody against the GA-733 family antigen / cf. e.g., EP-A-376746 and references cited therein, and Larsson et al., Int. J. Canc. 42: 87782 (1988), antibody C242 / Larsson et al. Int. J. Canc. 42: 877-82 (1988) and Thy-1.2 / monAK C, Opitz et al., Immunobiol. 160: 438-82 (1982). It is understood that monoclonal PCs specific for other cell-target surface structures may be used. The preparation of monoclonal antibodies to epitopes unique to selected cell-targets is well known. See, for example, the above publications. Expressions such as monoclonal antibodies to the C242 epitope or C215 epitope include antibodies reactive with cross-reactive epitopes.

Of the three monoclonal antibodies tested, C125-conjugates are by far the least interesting because they react with an epitope on a cancer antigen that is very often expressed in normal cells. C242-conjugates appear to be more promising in coming out specificity data, although our results suggest that they may require higher doses. MonAK C against Thy-1.2 may not be of value in the fight against human cancer cells, since Thy-1.2 antigen is specific to non-human cancer cells.

A line of hybridoma cells producing the C242 monoclonal antibody was deposited into the European Collection of Animal Cell Cultures, Porton Down, Selisbury, Wilts, England, 26.01.1990, no. ECACC 90012601.

A structure that binds a superantigen to an antibody

In accordance with the present invention, in a suitable conjugate, the superantigen is covalently linked to the antibody via a covalent bond-bridge (-B-). It is important that the conjugate does not disintegrate when used against cells intended for destruction, such as administration to an animal. Therefore, the bridge must be metabolically resistant for quite a long time to achieve the effect. In addition, the bridge itself should not induce immunological reactions. In general, the bridge should be inert in the sense that the conjugate retains a high specificity for target cell binding (anticancer, antiviral, etc.) and a high ability to activate cytotoxic T-cells.

Several functional groups have been proposed in the scientific and patent literature that may serve as bridges in immunoconjugates. Accordingly, the bridging-in the proposed new conjugates may have structures selected from the group consisting of: (i) amides and hydrazides (amides -CONR _X or -NR _X CO-, where each of the free valences is bound to a saturated carbon atom and R _x may be hydrogen or alkyl, such as a lower alkyl group (C _{x 6} ) or an alpha-N-substituent of a natural alpha amino acid, preferably a hydrophilic amino acid, and hydrazides -CONHNH- or -NHNHOC-, wherein each of the free valencies is combined with a saturated carbon atom;

(ii) thioethers and disulfides (—S _r - wherein each of the free valencies is directly attached to a saturated carbon atom, S is a sulfur atom, and r is an integer equal to 1 or 2);

(iii) straight, branched or cyclic hydrocarbons, which are saturated and may be substituted with one or more hydroxyl or amino groups;

(iv) ethers (-O-, where each free valence is directly bonded to a saturated carbon atom); and (v) primary amines or disubstituted hydrazines (-NH- or -NH-NH-, respectively, wherein each of the free valencies is directly bonded to a saturated carbon atom).

The bridge length is within the range used in the art, that is, less than 180 atoms, e.g., less than 100 atoms, but must be more than 3-6, preferably more than 16 atoms.

A more preferred bridge is hydrophilic and should not have any aromatic cycles. More preferred hydrophilic structures which may form part of the -B- bridge are: (i) polypeptide chains of naturally occurring hydrophilic alpha-amino acids (e.g., aspartic acid and its amide, glutamic acid and its amide, lysine, arginine, glycine, threonine, serine). , and possibly also histidine); (ii) oxyalkylene chains such as / -O (CH ₂ ) _n / _n , wherein n is an integer from 2 to 5, preferably 2 or 3, and n 'may be an integer from I to 20; and (iii) -S- (thioethers), -0- (ethers) and unsubstituted amides (-CONH-), each of which is connected to the short unchanged angliavandenilinėmis circuits (C ₁ _ _4), preferably having I or 2 carbon atoms, .

The hydrophilic amino acids may be present in hydrophilic structures of the type / F- (O ₂ ) n / mF, where F is an amino acid sequence, preferably 4-8 residues, wherein each amino acid is selected from serine, glycine and threonine, m is an integer of 1 to n, and n is an integer from 4 to 8 (Cetus Corp., WO-A-85/03508).

The -B- bridge can be attached either at specific sites on the antigen or superantigen moieties in the conjugate or at random sites. Potential sites include the amino terminus, the carboxyl terminus, and the lysine residue (omega-amino group). If the antibody or superantigen contains a mercapto group or a disulfide group (cystine or cysteine, respectively), these groups can also be used for covalent coupling provided that they do not play a major role in the work of the active conjugate moieties. The carbohydrate structures, if present, can be oxidized to groups of aldehydes, which in turn can be used to fuse with another moiety of the conjugate (Cetus Corp., EP-A-240200).

The conjugate of the present invention need not be rich in ester and labile amide linkages, particularly the tyrosine and histidine moieties formed respectively. If such bonds formed during synthesis, they should be removed using hydroxylamine / Endo et al., Cancer Res. 48: 3330-3335 (1988). The number of superantigenic fragments present in the conjugate per active AK fragment is usually 1-5, usually 1 or 2.

In a preferred embodiment, the conjugate must be substantially homogeneous in terms of the ratio of superantigen / antibody and / or the binding sites used in the superantigen and antibody, respectively, and / or the bridge -B-, and the like. In other words, all individual conjugate molecules forming the conjugate as a substance must be the same with respect to these changing characteristics.

The material should be substantially free of unbound antibodies or superantigens.

The exact ratio of superantigen / antibody, bridge structure, etc. in the optimal conjugate will depend on the monoclonal antibody selected (including class, subclass, clone producing, specificity) and the superantigen selected. The experimental models presented in this description allow the selection of other superantigens and antibodies according to optimal parameters.

According to an embodiment of the invention most closely studied prior to the filing date, the bridge -B- has the structure:

-S _r RCONHCH ₂ CH ₂ (OCH ₂ CH ₂ ) _n O (CH ₂ ) _m COY- (I)

The free valences in formula I are combined with the active moieties, respectively. It is either directly or via kiLT 3909 B on those divalent inert structures on the -B- bridge.

n is an integer greater than O, e.g. 1-20, preferably 2 or more 2, and in most cases less 10. m equals 1 or 2.

S is a sulfur atom, and at each valence is directly attached to a carbon atom (-S _r - is a thioether or disulfide), r is equal to 1 or 2.

Y is -NH-, -NHNH-, or -NHN = CH- groups which are attached at their left end to the CO group shown at the right end of formula I and at their right end with a saturated carbon atom or a carboxyl group (only if Y is -NHNH-).

R is preferably an alkylene group (containing 1 to 4 carbon atoms, most commonly 1 or 2 carbon atoms), which may be substituted by one or more (1-3, most preferably less than 2) hydroxyl groups.

Preparation of antibody-superantigen conjugate

In accordance with the present invention, antibody conjugates may be obtained by saturation and isolation from the culture medium of the cells producing them or from other media in which they have been synthesized.

The synthesis of novel conjugates can be accomplished by methods known in the art, such as by genetic engineering (recombinant methods) or by the appropriate antibody and antigen, by classical condensation reactions with appropriate functional groups. The following functional groups are present and commonly used in proteins:

(i) Carbohydrate structures. This structure can be oxidized to aldehyde groups which, in turn, react with compounds containing the H ₂ NNH- group to form the -C = NH-NH- group.

(ii) Mercapt Group (HS-). The mercapto group may react with a compound having a group capable of interacting with the mercapto group to form a thioether group or a disulfide group. Free mercapto groups are present in proteins in cysteine residues, and can be introduced into proteins by thiolation or cleavage of disulfides in natural cysteine residues.

(iii) Free amino groups on (H ₂ N-) amino acid residues. Amino groups may react with compounds having an electrophilic group, such as an activated carboxyl group, to form an amide group. Preferably, the free amino group is the terminal amino group or omega-amino group of the lysine residue.

(iv) Free carboxyl groups on amino acid residues. The carboxyl group can be converted to an activated carboxyl group, which then reacts with an amine-containing compound to form an amide group. However, care must be taken to minimize the formation of amides with amino groups adjacent to carboxyl groups in the same protein. Often the free carboxyl group is the terminal carboxyl group or the carboxyl group in the dibasic alpha amino acid.

Compounds containing the H ₂ NNH- group capable of reacting with a mercapto group, an activated carboxyl group or an amino group may be a bifunctional condensation reagent, or an antibody or superantigen. The groups are bonded directly to the saturated carbon atom, except for the H ₂ NNH- group, which may alternatively be linked to the carboyl carbon atom of karLT 3909B. The groups may be introduced into the antibody or superantigen by simple substitution.

Recombinant methods are an effective means for obtaining conjugates in which the moieties are specifically linked to one another by a terminal carboxyl group on one portion and a terminal amino group on the other. A connecting structure compatible with the method used may also be introduced.

The reagents used are selected in such a way as to obtain the previously described bridge -B-. conventional bifunctional reagents have the formula Z-B'-Z ', wherein Z and Z' are functional groups compatible with each other to effect covalent attachment to the functional group present in the protein. See also: above, B is an inert bridge, which may have the same structures as the aforementioned bridge -B-. Specifically, Z and Z 'may be the same or different and are selected from the mercapto group, the mercapto group, the activated carboxyl group, -CONHNH ₂ , and 1.1. For a description of these groups, see. hereafter, under New Reagents.

The method of preparing the conjugates used in our experimental part includes:

(i) reacting the antibody and superantigen with an organic reagent having a group capable of reacting with the mercapto group and a group capable of reacting with an amino group to produce an antibody or superantigen having a group capable of reacting with the mercapto group, and (ii) the remaining superantigen and antibody reaction of the partial moiety with an organic reagent having a mercapto group and a group capable of reacting with an amino group to produce superLT 3909 B antigen or an antibody having a mercapto group or a blocked mercapto group, followed by the products of steps (i) and (ii) interact with one another to form a conjugate in which the superantigen is bound to the antibody by a disulfide or thioether linkage.

The condensation conditions for each group in protein chemistry are known. The condensation can take place in stages or in a single step, forming intermediate functional groups which can be attached to the starting materials by inert spacers. In general, the conditions under which synthesis and purification / conjugate isolation are carried out are always such as not to cause denaturation of the proteins used. Typically, it is an aqueous medium with a pH range of 3 to 10 and a temperature of 0 to 50 ° C. The specific conditions depend on which groups are reacting and which conjugate to isolate. See details. section New Reagents.

New Reagents (Developed in Relation to the Invention)

A novel heterobifunctional reagent of general formula II has been developed for the chemical synthesis of conjugates containing the -B- bridge:

Z ₁ RCONHCH ₂ CH ₂ (OCH ₂ CH ₂ ) _n O (CH ₂ ) 'Z'! (II) wherein m and n have the same meanings as in formula (I) above. Z ₁ is an electrophilic group which is capable of reacting with an HS- group, a mercapto (-SH) or a blocked mercapto group (e.g. AcS-), provided that the mercapto and hydroxyl groups cannot be linked to the same carbon atom in the R radical. The electrophilic groups capable of reacting with the HS group are as follows:

(i) halogen bonded to a saturated carbon atom, preferably in the form of an alpha-haloalkylcarbonyl (e.g., Z _L CH ₂ CO-);

(ii) an activated mercapto group, preferably a so-called reactive disulfide (-SSR!) linked to a saturated carbon atom;

(iii) 3,5-dioxo-1-aza-cyclopent-3-en-1-yl.

For the characterization of reactive disulfide, see Ref. e.g., EP-A-125885, which is incorporated herein by reference.

Ζ ′ _γ is an activated carboxyl group, that is, an electrophilic group. Examples include halogen anhydrides of carboxylic acids (-COC1, -COBr and -COJ), mixed anhydrides of carboxylic acids (-COOCRJ, reactive esters such as N-succinimidoyloxycarbonyl, -C (= NH) -O ₂ , 4-nitrophenylcarboxylate (-CO-OC). ₆ H ₄ NO ₂ ) and 1.1 R ₁ and R ₂ may be lower alkyl radicals (C _x -C ₆ ) and in addition R ₂ may also be a benzyl group.

One of the advantages of the new reagents is that they enable the homogeneous conjugate compound to be represented by an integer of n values (OCH ₂ CH ₂ ) relative to n, that is, n being the same for each molecule of the individually conjugated compound.

Functional groups reacting with Z 1 and Z _x may be present in, or introduced into, natural antibody and superantigen molecules. The terminal Z 1 and Z ₄ groups can then be selectively reacted with the corresponding antibody or superantigen by methods known to these types of groups.

Known methodologies include ligation of a chain starting from a new reagent or compounds to be conjugated to CONLT 3909 B. Chain ligation using a new reagent can yield a conjugate in which -B- is:

(1) -COR'-S _r -CONHCH ₂ CH ₂ (OCH ₂ CH ₂ ) _n O (CH ₂ ) _m COY-;

CO is bonded to NH (2) -COR '-S _r -RCONHCH ₂ CH ₂ (OCH ₂ CH ₂ ) _n O (CH ₂ ) _m CONHNH-R -NHN =;

N = usually attached to a sp-inhibited carbon atom derived from an oxidized structure carbohydrate present in the antibody or superantigen (when glycoproteins), (3) -CO (CH ₂ ) _m O (CH ₂ CH ₂ O) _n CH ₂ CH ₂ NHCOR ¹ -S _r -CONHCH ₂ CH ₂ (OCH ₂ CH ₂ ) _n -O (CH ₂ ) _m COY-;

CO - bonded to NH, (4) -CO (CH ₂ ) _m O (CH ₂ CH ₂ O) _n CH ₂ CH ₂ NHCOR '-S _r -CONHCH ₂ CH ₂ (OCH ₂ CH ₂ ) _n -O (CH ₂ ) _m CONHNH-R NHN =;

N = connected in the same way as before (2),

R, R 'and R are alkylene groups selected as in R in formula (I). r has the same meanings as above.

The reagent (Formula II) can be obtained using compounds of Formula III:

NH ₂ CH ₂ CH ₂ (OCH ₂ CH ₂ ) O (CH ₂ ) _m COOH (III) wherein m is 1 or 2, n is an integer from 1 to 20, for example 2 to 20 or 3 to 9.

The synthesis of some compounds of formula III wherein m = 1 and 2 and n = 1-10 has been previously described / Juljen et al., Tetrahedron Letters, 29, 3803-06 (1988); Hougton, Saubi, Synth. Commun. 19 (18) 3199-3209 (1989); and EP-A410280 (Ref. 20.1.91) and Shlama and Rando, Carbohydrate Research 88 (1981) 213-221 and Biochemistry 19 (1980) 45954600).

The novel reagents of formula II can be obtained by reacting a compound of formula III with bifunctional reagents of formula Z-B'-Z ', where Z = Z _X , B' = R, having the same meanings as described above. R and R 'and Z' are an activated carboxyl group as previously described. After the reaction, the -COOH group becomes an activated carboxyl group, e.g., Ζ ' _γ -activated ester such as N-succinimidoyloxycarbonyl, 4-nitrophenyloxycarbonyl, 2,4-dinitrophenyloxycarbonyl, 2,4-dinitrophenyloxycarbonyl, etc.

The novel compounds of formula (III) and their novel derivatives are polyethers of general formula

XCH ₂ CH ₂ (OCH ₂ CH ₂ -) _n OCH ₂ Y (IV) wherein n is an integer from 2 to 20, preferably 3-20 or 3-9; X is an NH ₂ - group, including its protonated form ( ⁺ H ₃ N -), or a substituted -NH ₂ group from which an -NH ₂ group may be obtained, preferably by hydrolysis or reduction. Examples include an unsubstituted amino group (H ₂ N-), a nitro group, an amide (urea) group such as a lower alkylamide group (formylamide) acetylamide ... hexanoylamide), including acylamide groups which have electron-acceptor substituents on the alpha carbon atom of the carbon, especially CF ₃ CONH-, CH ₃ COCH ₂ CONH-, etc .; a phthalimidoyl group which may be substituted on the ring; carbamate group (especially R 1 OCONH- and (R ₁ -OCO) (R _{1 2} -OCO) N- also N- (tert-butyloxycarbonyl) -amino (Bok), N- (benzyloxycarbonyl) amino and di (N benzyloxycarbonyl) -amino (Z and di Z, respectively) substituted in the ring; alkylamino group in which the carbon atom linked to the nitrogen atom is in the alpha position with respect to the aromatic system such as N-monobenzylamino, N, N-dibenzylamino , N-tritylamino (triphenylmethylamino), etc., including analogous groups in which the methyl carbon atom (including the benzyl carbon atom) is replaced by a silicon atom (Si) such as N, N-di (tert-butylsilyl) amino, and 4-oxo -l, 3,5-triazin-1-yl, including lower alkyl substituents at the 3 and / or 5-position.

In the past and further, R ₁ and R ₂ represent a lower alkyl group, especially a secondary and tertiary alkyl group, and a methyl group substituted with 1-3 phenyl groups, which in turn can be substituted. The lower alkyl and lower acyl group have 1-6 carbon atoms.

Y is a carboxyl group (-COOH, including -COO-), or a group which can be converted to a carboxyl, preferably by hydrolysis or oxidation. The most important groups are the ester groups in which the carbonyl carbon atom or the corresponding atom in the orthoesters is linked to the methyl group at the right end of the compound of formula (I). Examples include alkylester groups (-COOR \); orthoether (-C (OR ' ₃ ) ₃ ) r reactive ester groups as previously described. R ' ₃ has the same meanings as previously defined for R'p

Other groups include -CHO, -CN, -CONH ₂ , -CONR ₁ R ₂ , where R _x and R ₂ have the same meanings as before.

The compound of formula IV can be synthesized using known starting materials using known combinations of techniques. Suitable methods of synthesis include:

A) Chain formation.

B) Transformation of terminal functional groups.

C) Transformation of symmetric polyester into asymmetric ether.

D) Splitting the bisymmetric chain into two identical fragments.

Suitable starting compounds having the -OCH ₂ CH ₂ - repeating chain are commercially available. Examples include oligoethylene glycols having 2 to 6 repeating units. Other suitable compounds with the same terminal groups include the corresponding dicarboxylic acids and diamines.

Suitable starting materials having different end groups are omega-hydroxymonocarboxylic acids, which end groups are separated by a pure polyethylene oxide bridge. Such compounds having up to 5 repetitive chains have been previously described / Nakadzui, Kavamura, Okahara, Synthesis (1981), p. 42 /.

Pharmaceutical compositions and their preparation

The pharmaceutical compositions of the present invention are formulated with formulations known in the art, but containing our novel conjugates. The compositions may be in the form of lyophilized particulate, sterile or aseptically prepared solutions, tablets, ampoules and 1.1. They may contain carriers such as water (preferably buffer, physiological pH values such as phosphate salt buffer (FDB)) or other inert solid or liquid.

In general, the composition is prepared by mixing, dissolving, binding or otherwise combining the conjugate with one or more water-insoluble or water-soluble, aqueous or non-aqueous carriers and, if necessary, suitable additives and adjuvants. Carriers and conditions must not adversely affect the activity of the conjugate. The water itself is also among the carriers.

Uses and uses

Typically, the conjugates are solid and used in pre-formulated doses, each containing an effective amount of conjugate, which according to the results is from 10 µg to 50 mg. The exact dosage will vary from case to case and will depend on the patient's weight and age, the route of administration, the nature of the disease, the antibody, the superantigen, the bridge (-B-), and so on.

The route of administration corresponds to those known in the art, i.e. effective for cell-target lysis. The amount of conjugate or therapeutically effective amount of the conjugate of the present invention is in contact with the target cells. In the above cases, this usually refers to parenteral administration, such as injection or injection (subcutaneous, intravenous, arterial, intramuscular) into a mammal such as a human. The conjugate may be administered locally or systemically to the subject.

An effective amount of cell-to-target murmur is understood to be an amount that effectively activates and targets CTL for cell-to-target destruction.

The invention is illustrated by several embodiments which do not limit the invention in any way. Section I of the Experimental Part shows the chemical synthesis of conjugates and Section II shows the activation of T-cells by example of 4 conjugates for cell-target murmur.

THE EXPERIMENTAL PART. SECTION I

Preparation of omega-amino-DEG-carboxylic acid

Isopropyl-8-hydroxy-3,6-dioxaoctanoate (I)

Sodium flakes (23 g, 1.0 mol) were added portionwise to diethylene glycol (500 mL) under nitrogen. After the sodium has completely reacted, the mixture was cooled to room temperature and bromoacetic acid (76 g, 0.5 mol) was added with stirring. The reaction is carried out for 18 hours at 100 [deg.] C. and then excess diethylene glycol is distilled off under a column pressure of about 4 mm Hg. Isopropyl alcohol (400 mL) was added followed by acetyl chloride (51 g, 0.65 mol) in portions. Stir in 16 or. 65 ° C, then cooled to room temperature and neutralized with sodium acetate (3.5 g, 0.15 mol). The mixture is filtered, the filtrate is evaporated to dryness and the residue is dissolved in water (200 ml). The aqueous phase was extracted with 1,1,1-trichloroethane (3 x 50 mL). The resulting organic phases are washed with water (20 mL). The product is extracted from the combined aqueous phases with dichloromethane (50 mL), which is evaporated to an oil (55 g).

Isopropyl-II-hydroxy-3,6,9-trioxa-undecanoate (2)

Sodium (23 g, 1.0 mol) was added portionwise to triethylene glycol (700 mL) under nitrogen atmosphere. After the sodium has completely reacted, the mixture is cooled to room temperature and bromoacetic acid (76 g, 0.5 mol) is added with stirring. The reaction is carried out for 18 hours. At 100 ° C, the excess triethylene glycol is evaporated off by distillation of about 4 mm Hg column slurry at 3909 B. Isopropyl alcohol (400 mL) was added followed by acetyl chloride (51 g, 0.65 mol) in portions. After 18 or. after stirring at 65 ° C, the mixture was cooled to room temperature and neutralized with sodium acetate (3.5 g, 0.15 mol). The mixture is filtered, the filtrate is evaporated to near dryness and the residue is dissolved in water (200 ml). The aqueous phase is extracted with 1,1,1-trichloroethane (3 x 50 ml). The resulting organic phases are washed with water (20 mL). The product is extracted from the combined aqueous phases with dichloromethane (50 ml) to give an oil which is evaporated.

¹ H-NMR (CDCl ₃ ), δ (md): 1.25 (d, 6H); 3.07 (s, 2H);

3.6-3.8 (m, 12H); 4.11 (s, 2H); 5.09 (m, 1H).

8- (N-phthalimidoyl) -3,6-dioxaoctanol (3)

8-Chloro-3,6-dioxa-octanol (365 g, 2.2 mol, obtained from triethylene glycol and SOCl ₂ ) was dissolved in dimethylformamide (400 ml) and potassium phthalimide (370 g, 2.0 mol) was added with stirring. After stirring for 16 hours at 110 ° C, the dimethylformamide was evaporated under reduced pressure. The residue is suspended in toluene (1.5 L) at 40-50 [deg.] C. and the potassium chloride is filtered off. The product is crystallized by freezing (-10 ° C). The second fraction is obtained from the precipitates by concentration and repeating the crystallization procedure.

NMR (CDCl ₃ ), δ (md): 2.90 (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).

Isopropyl-17- (N-phthalimidoyl) -3,6,9,12,15-pentaoxa-heptadecanoate. (4) is added dropwise to a solution of 8- (N-phthalimidoyl) -3,6-dioxaoctanol (3) (8.5 g, 36 mmol) and trifluoromethane sulfuric anhydride (10.2 g, 36 mmol) in dichloromethane with stirring for ca. At -5 ° C, pyridine (2.8 mL, 35 mmol) in dichloromethane (30 mL). After about 30 min, the organic phase is washed with 0.5 M hydrochloric acid and water. After drying over sodium sulfate and filtration, isopropyl-8-hydroxy-3,6-dioxaoctanoate (I) (12 g, mmol) and Na ₂ HPO ₄ (6.5 g, 46 mmol) were added and the mixture was stirred vigorously at room temperature. 20 hours. The reaction mixture is filtered and the filtrate is evaporated. The residue is partitioned between 1,1,1-trichloroethane and water. Evaporation of the organic phase gave an oil (13 g).

¹ H NMR (CDCl ₃ ), δ (md): 1.26 (d, 6H); 3, 58-3, 76 (m, 18H); 3.90 (t, 2H); 4.11 (s, 2H); 5.09 (m, 1H); 7.707.89 (m, 4H).

17- (N-Phthalimidoyl) -3,6,9,12,15-pentaoxa-heptadecanoic acid (5)

Isopropyl-17- (N-phthalimidoyl) -3,6,9,12,15-pentaoxa-heptadecanoate (4) (13 g) was dissolved in tetrahydrofuran (50 mL) and hydrochloric acid (approximately 50 mL). The reaction is carried out for 16 hours at room temperature, after which the solution is diluted with water (200 ml) and the tetrahydrofuran is evaporated under reduced pressure. The aqueous phase is washed with toluene (1x) and extracted with dichloromethane (2x). Dry over sodium sulfate and evaporate the organic phase to give an oil (8.5 g).

^ς Η NMR (CDCl3), δ (md): 3, 57 - ^3.75 (m, 18H); 3.91 (t, 2H); 4.11 (s, 2H); 4.8 (ppm, 2H); 7.65 - 7.90 (m, 4H).

Isopropyl-17-amino-3,6,9,12,15-pentaoxa-heptadecanoate (6)

17- (N-Phthalimidoyl) -3,6,9,12,15-pentaoxa-heptadecanoic acid (5) (8.5 g) is dissolved in 150 ml of ethanol and 3 ml of hydrazine hydrate. The solution is stirred for 16 h at room temperature, then added with HCl (100 mL, 3 M) and heated under reflux for 3 h. After cooling to room temperature and filtration, the mixture was basified to pH 9 with NaOH and the filtrate was evaporated to near dryness, then acidified with HCl to pH 4 and evaporated to dryness. The product is treated with isopropanol (100 mL) and acetyl chloride (2 mL) at room temperature overnight and the solution is evaporated. The residue was dissolved in water and extracted with dichloromethane at an alkaline pH (7-11). Evaporation gave the product (3.3 g).

^{Χ Η} NMR (CDC1 ₃₎ δ (ppm): 1.26 (d, 6H); 3.17 (t, 2H);

3.65-3.80 (m, 18H); 4.16 (s, 2H); 6.07 (m, 1H).

FORMULAS OF SYNTHESIS OF AMINO-DEG-CARBOXYLIC ACIDS

H- (OCH ₂ CH ₂ ) _n CH ₂ CO-OCH (CH ₃ ) ₂

Compound 1: n = I

Compound 2: n = I

PhtN-CH ₂ CH ₂ - (OCH ₂ CH ₂ ) ₂ OH

Compound 3, PhtN- = N-Phthalimidoyl

PhtN-CH ₂ CH ₂ - (OCH ₂ CH ₂ ) ₄ CH ₂ CO-OCH (CH ₃ ) ₂

Compound 4, PhtN - = N - Phthalimidoyl

PhtN-CH ₂ CH ₂ - (OCH ₂ CH ₂ ) ₄ CH ₂ CO-OH

Compound 5

H ₂ N-CH ₂ CH ₂ - (OCH ₂ CH ₂ ) ₄ ch ₂ co-oh

Compound 6

OBTAINING BIFUNCTIONAL REAGENTS AND CONDENSATION PRODUCTS

Example 17 Preparation of N-hydroxysuccinimidic ester of 17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoic acid

A. Preparation of 17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptanoic acid (A)

JCH ₂ CNHCH ₂ CH ₂ (OCH ₂ CH ₂ ) ₄ OCH ₂ COOH (A)

II

Isopropyl-17-amino-3,6,9,12,15-pentaoxa-heptadecanoate (see Experimental Part I) (1.1 g, 3.2 mmol) is dissolved in 3 ml of 1 M sodium hydroxide solution and left at room temperature. minutes. Add 1.6 ml of 8 M hydrochloric acid and evaporate to dryness. The residue was dissolved in dichloromethane and filtered, and evaporated to give 545 mg of 17-amino-3,6,9,12,15-pentaoxa-heptadecanoic acid. Dissolve 460 mg (1.39 mmol) of this compound in 10 mL of borate buffer, pH 8.4. Deaeration of the solution is carried out with gaseous nitrogen. A solution of 432 mg (1.52 mmol) of N-succinimidyl 2-iodoacetate in 5 mL of dioxane is added dropwise over 1 minute, with the addition of 5 M NaOH at pH 8.4. The reaction mixture was stirred for 15 minutes under nitrogen. TLC (eluent: CH ₂ Cl ₂ -MeOH, 50:35) showed complete reaction in a few minutes. After 15 minutes, acidify to pH ₃ , freeze and freeze-dry. The reaction mixture was fractionated on a reversed phase column PHP-RPC HP 30/26 (Farmacia, Biosystems AB) using a gradient of 0 to 13% acetonitrile in 0.1% trifluoroacetic acid followed by partitioning in 13% acetonitrile, 0.1% TFAR (trifluoroacetic acid). Combine the required peak fractions and lyophilize to give 351 mg of 17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoic acid (A). Yield: 76%.

The structure of the product was determined by NMR spectroscopy. ^The following δ (md) values were obtained in the ^X H NMR spectrum (D ₂ O):

JCH ₂ C 4.23 s; OCH ₂ COH 3.76 s; -NHCH ₂ CH ₂ O- 3.41; -OCH ₂ CH ₂ OII

II

3.71 - 3.7 6; -NHCH ₂ CH ₂ O- 3.65 t.

B. Preparation of 17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoic acid N-hydroxysuccinimide ester (B)

II

JCH ₂ CNHCH ₂ CH ₂ (OCH ₂ CH ₂ ) ₄ and ₂ con

II

II

(B)

II

The reaction ampoule weighed hydroxysuccinimide (4.5 mg, 39 pmol). 17-Iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoic acid (A) (18.3 mg, 39 µmol) is dissolved in 0.55 ml of dry dioxane and filled into an ampoule. The ampoule is deaerated with gaseous nitrogen and then treated with 0.8 mg (39 µmol) dicyclohexylcarbodiimide in 0.15 ml of dry dioxane. The ampoule is filled with gaseous nitrogen, sealed and placed in the dark. The reaction mixture is stirred for 3.5 hours and the precipitate formed is removed by filtration. NMR analysis showed that the B product obtained in the filtrate was 89%.

Example 17 Preparation of (17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecamoylamino) -immunoglobulin (C)

A. Monoclonal antibody monAK C215

IgG- [NHCCH ₂ O (CH ₂ CH ₂ O) ₄ CH ₂ CH ₂ NHCCH ₂ J] _n (C),

II II o o n = 4, 7, 9, 12, 14, 18

Immunoglobulin class Ig62a monoclonal antibody (monAK C215) (34 mg, 0.218 µmol) dissolved in 17.7 ml of 0.1 M borate buffer, pH 8.1 containing 0.9% sodium chloride was added to the ampoule. To the buffer solution add 146 μΐ of dioxane solution containing 3.6 mg (6.4 pmol) of 17-iodoacetylamino-3,6,6,12,15-pentaoxa-heptadecanoic acid N-hydroxysuccinimidate ester (B) and stir until completion. at room temperature for 25 minutes. The reaction ampoule is covered with folga to prevent light transmission. Excess reagent B is removed by fractionation on a column of Sephadex G 25 K 26/40 using 0.1 phosphate buffer pH 7.5 containing 0.9% NaCl as eluent. Collect and combine the fractions containing the desired product C. The solution (22 ml) is concentrated in an Amicon lattice through a YM 30 filter to a volume of 8 ml. Concentration and degree of substitution are determined by amino acid analysis. They form 4.7 mg / ml and 18 spacer / monAK C215, respectively.

B. Monoclonal antibody monAK C242

The reaction is carried out according to the procedure described in Example 2A using an N-hydroxysuccinimide ester of immunoglobulin Ig GI class monoclonal antibody (monAK C242) with 15-, 20- and 22-fold 17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoic acid. ) in excess of moles to give nona-, dodeca-, and tetradeca (17-iodacetylaminoLT 3909 B

3, 6,9,12,15-pentaoxa-heptadecanoylamino) -monAK C242, respectively (C).

C. Monoclonal antibody monAK C

The immunoglobulin class Ig G 2a monoclonal antibody (monAK C) was treated with 17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoic acid N-hydroxysuccinimide ester 14- and the procedure described in Example 2A.

18-fold excess of moles to give tetra- and hepta (17-iodoacetylamino-3,6, 9,12,15-pentaoxa-heptadecanoylamino) -monAK C, respectively (C).

Example Preparation of Staphylococcal Enterotoxin A (SEA) Labeled with 2-Mercaptopropionylamino-Eu ³⁺

A. Preparation of Eu ³⁺ -labeled SEA (D)

SEA (lyophilized product from Toxin technology inc.) (2 mg, 72 nmol) was dissolved in 722 μΐ of water and placed in a 15 ml isopropylene tube. 100 μΐ of 0.1 M borate buffer, pH 5.6 was added followed by 2160 nmol of Eu ³⁺ complex with chelates (Farmacia Vallac Oi) 178 μΐ milli-Q. The reaction is complete overnight at room temperature. Excess reagents are removed by fractional column reaction with Sephadex G 25 PD 10 (Farmacia Biosystems AB) using 0.1 M phosphate buffer, pH 8.0 as eluent. The fractions containing the desired product D are combined. Concentrate the solution (3 ml) of konLT 3909 B in an Amicon lattice through a YM5 filter to a volume of 0.8 ml. The concentration determined by amino acid analysis was 1.7 mg / ml. The degree of substitution compared to standard EuC1 ₃ solution was 0.8 Eu ³⁺ / SEA.

Obtaining SEA (E)

BI. SEA (FU) of 3- (2-pyridylthio) propionylamino-Eu-labeled and 3-mercaptopropionylamino-Eu- ³⁺ -labelled

3+ (Eu -SEA- (NHCCH ₂ CH ₂ SS

II

O

) 3-4 (E)

Place 15 ml of isopropylene tube (1.24 mg, 44.5 nmol) in 0.75 ml of 0.1 M phosphate pH 8.0. Add 35 μΐ (180 nmol

Of Eu ³⁺ -SEA buffer,) 1.6 mg

Of N-succinimidyl-3- (2-pyridylthio) -propionate per ml of ethanol solution and the reaction mixture was stirred for 30 min at room temperature. The resulting product E is not isolated prior to reduction to product F _x .

Eu ³⁺ -SEA- (NHCCH ₂ CH ₂ SH) ₃ - ₄ (FJ

II

To the above reaction mixture was added 20 μΐ of 0.2 M Eu ³⁺ citrate solution and 50 μΐ of 2 M acetic acid to adjust the pH to 5.0. Then 3.1 mg of dithiotrentol (Merk) in 0.1 ml-tre 0.9% sodium chloride solution is added and the reaction mixture is stirred for 20 min at room temperature. Then make up to 1 ml with 50 μΐ of 0,9% sodium chloride solution. The reaction solution was applied to a column (1 mL) filled with Sephadex G25 NAP 10 (Farmacia Biosystems AB) and the desired product F _x washed with 1.5 mL of 0.1 M phosphate buffer pH 7.5 containing 0.9% sodium chloride. The isolated product F _{4 is} collected in a 15 ml isopropylene tube and used immediately to synthesize product G to prevent oxidation of the disulfide product.

B2. Preparation of 2-mercaptopropionylamino-taaphylococcal enterotoxin A (SEA) (F ₂ )

SEA (NHCCH ₂ CH ₂ SH) _lr g (F ₂ )

II

Natural SEA (lyophilized product, Toxin Technology Ine.) Or recombinantly produced SEA (rSEA) is exposed to double mole excess of N-succinimidyl-3- (2-pyridylthio) propionate according to the procedure described in Example 3BI.

The degree of substitution detected by UV analysis according to Karlsson et al. / Biochem. J. 173, 723-737 (1978), contains 1.9 mercaptopropionyl groups per molecule of SEA.

Example Preparation of SEA monoclonal antibody conjugate (G _a or G ₂ )

A. Eu ³⁺ -SEA Conjugates with MonAK C215 (G _x ) [NHCCH ₂ O (CH ₂ CH ₂ O) ₄ CH ₂ CH ₂ NHCCH ₂ SCH ₂ CH ₂ CNH-EU ³⁺ -SEA] _m / II II II / 0 0 0

IgG (GJ \

[NHCCH ₂ O (CH ₂ CH ₂ O) ₄ CH ₂ CH ₂ NHCCH ₂ SCH ₂ CH ₂ OH] ₁₈ . _m

II II o o

To a solution of 4-mercaptopropionylamino-Eu ³⁺ -labelled SEA (F) described in Example 3B is added octadeca (17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoylamino) monAK C215 (C) (4 mg) 1, 2 ml of 0.1 M phosphate buffer, tuLT 3909 B, 0.9% sodium chloride, pH 7.5. The reaction is complete overnight at room temperature. Unreacted iodoalkyl groups are blocked by addition of 5 μΐ (1,2 μιηοΐ) mercaptoethanol solution obtained by dissolving 20 μΐ in one ml of water. The reaction solution is left for 4 hours at room temperature and then filtered. The filtrate was fractionated on a column packed with Superos 12 HR 16/50 (Farmacia Biosystems AB) using 0.002 M phosphate buffer pH 7.5 containing 0.9% sodium chloride as eluent. The fractions containing the final product G are combined and analyzed. According to amino acid analysis, the protein content is 0.22 mg / ml. The degree of substitution determined by the amount of Eu ³⁺ is one SEA molecule per IgG molecule. The product was tested for immunostimulatory properties and antibody binding capacity.

Increasing the amount of compound (F) relative to the amount of compound (C) results in a higher degree of substitution.

B. Conjugates of rSEA with MonAK C215 (G ₂ ) [NHCCH ₂ O (CH ₂ CH ₂ O) ₄ CH ₂ CH ₂ NHCCH ₂ SCH ₂ CH ₂ CNH-SEA] / II II II / O 0 0

IgG (G ₂ ) [NHCCH ₂ O (CH ₂ CH ₂ O) ₄ CH ₂ CH ₂ NHC-CH ₂ SCH ₂ CH ₂ OH] _n .

II II o o

Octadeca (17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoylamino) monAK C215 (C) is treated with an excess of 1.8 moles of 2-mercaptopropionylamino-rSEA (F ₂ ) according to the procedure described in Example 4A. Conjugate composition was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (DSN-PAGE) on a PhastGel gradient of 4 to 15, and bands scanned on Phast

With the help of IMAGE (Pharmacia Biosystems AB). The resulting conjugate is composed of 6% monAK C215 with 3 SEA, 15% with two SEA, 28% with one SEA and 51% unchanged monAK C215.

Another assay uses a 2.7 molar excess of F ₂ to give a conjugate of the following composition: 15% monAK C215 with three SEAs, 25% with two SEAs, 34% with one SEA, and 26% unchanged monAK C215.

C. rSEA conjugates with monAK C242

According to the procedure described in Example 4A, tetradeca (17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoylamino) monAK C242 (C) is exposed to a 3.2-fold excess of moles of 2-mercaptopropionylamino-rSEA (F ₂ ). Conjugate composition was analyzed as described in Example 4B, yielding the following results: 4% monAK C242 with four SEAs, 12% with three SEAs, 28% with two SEAs, 36% with one SEA, and 20% unchanged monAK C242.

Following the same reaction, just prior to column fractionation, the product is treated for 4 hours to remove the 0.2 M hydroxylamine unstable bond between the monAK C242 and the spacer and between the SEA and the mercaptopropionyl group. The resulting conjugate is of the following composition: 1% monAK C242 with four SEAs, 12% with three SEAs, 27% with two SEAs, 36% with one SEA and 24% unchanged monAK.

C2. According to the procedure described in Example 4A, dodeca (17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoylamino) monAK C242 is treated with a triple excess of moles of 2-mercaptopropionylamino-rSEA (F ₂ ). The resulting conjugate has the following composition: 6% monAK C242 with three SEAs, 26% with two SEAs, 36% with one SEA, and 31% unchanged monAK C242.

C3. According to the procedure described in Example 4A, nona (17-iodacetylamino-3,6,9,12,15-pentaoxa-heptadecanoylamino) monAK C242 (C) is treated with a triple excess of moles of 2-mercaptopropionylamino-rSEA (F ₂ ). The resulting conjugate has the following composition: 15% monAK C242 with two SEAs, 39% with one SEA and 46% unchanged monAK C242.

D. rSEA conjugates with monAK C

DI. According to the procedure described in Example 4A, hepta (17-iodo-acetylamino-3,6,9,12,15-pentaoxa-heptadecanoylamino) monAK is treated with a 5,4-fold excess of 2-mercaptopropionylamino-rSEA (F ₂ ). The conjugate composition was analyzed as described in Example 4B, yielding the following results: 15% with four SEAs, 24% with three SEAs, 29% with two SEAs, 19% with one SEA, 3% unchanged monAK C and 10% dimer.

The same reaction is carried out in the presence of 0.2 M hydroxylamine unstable bonds between the monAK C and the spacer and between the rSEA and the mercaptopropionyl group. The resulting conjugate has the following composition: 11% monAK C with three SEAs, 24% with two SEAs, 30% with one SEA, 18% unchanged monAK C and 17% dimer.

D2. According to the procedure described in Example 4B, tetra (17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoylamino) monAK C is exposed to a 5,7-fold excess of 2-mercaptopropionylamino-rSEA (F ₂ ). The conjugate is of the following composition: 8% monAK C with four SEAs, 18% with three

SEA, 30% with two SEAs, 26% with one SEA, 5% unchanged monAK C, and 12% dimer.

An example. Preparation of [17- (3-mercaptopropionylamino) -3,6,9,12,15pentaoxa-heptadecanoylamino] -rSEA (H)

A. Preparation of 17- (3- (2-pyridylthio) propionylamino) -3,6,9,12,15-pentaoxaheptadecanoylamino-rSEA (H) rSEA (NHCCH ₂ O (CH ₂ CH ₂ O) ₄ CH ₂ CH ₂ NHCCH ₂ CH ₂ SS

(H)

A solution of 17- (3- (2-pyridylthio) propionylamino) -3,6,9,12,15-pentaoxa-heptadecanoic acid N-hydroxysuccinimide ester (0.53 mg, 896 nmol) in 43 μΐ dioxane was added to 3.65 mg (128 nmol) of rSEA solution per ml of 0.1 M phosphate buffer pH 7.5 containing 0.9% sodium chloride. The reaction is complete within 30 min at room temperature. Take 100 μΐ for the degree of substitution analysis. The remainder is stored frozen until reduced to the product.

The degree of substitution is determined by removing salts from the 100 μΐ reaction mixture on a column containing Sephadex G50 NICK (Farmacia Biosystems AB) and analyzing the eluate by UV spectroscopy according to Karlsson et al. / Biochem. J., 173, 723-737 (1978). The 2.7 spacer is connected to rSEA.

B. Preparation of 17- (3-mercaptopropionylamino) -3,6,9,12,15-pentaoxa-heptadecanoylamino-rSEA (I) rSEA (NHCCH ₂ O (CH ₂ CH ₂ O) ₄ CH ₂ CH ₂ NHCCH ₂ CH ₂ SH) _n (I)

II II o o

The reaction solution containing product H (0.9 ml) was acidified to pH 4.4 with 2 M HCl and 2.9 mg of dithiotrentol dissolved in 75 μΐ of 0.9% sodium chloride solution was added. The reduction occurs within 30 minutes. The reaction mixture is applied to a column of Sephadex G 25 NAP 10 (Farmacia, Biosystems AB) and washed with 1.5 ml of 0.1 M phosphate buffer, pH 7.5 containing 0.9% sodium chloride, and the product is then used immediately. For the synthesis of j in Example II.

An example. Preparation of SEA-monoclonal antibody conjugate J with double spacer

CH ₂ NHCCH ₂ SCH ₂ CH ₂ (OCH ₂ CH ₂ ) ₄ O

II [NHCCH ₂ O (CH ₂ CH ₂ O) ₄ CH ₂ 0 ch ₂ cnhch ₂

CH ₂ CNH-rSEA] _m

II

IgG (J) [NHCCH ₂ O (CH ₂ CH ₂ O) ₄ CH ₂ CH ₂ NHCCH ₂ SCH ₂ CH ₂ OH] _n _ _m

II

II

Dodeca (17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoylamino) monAK C242 (B) (4.2 mg, 27 mmol, 0.1 mL 0.1 M phosphate buffer, pH 7.5) , 9% sodium chloride) reacts with / 17- (3-mercaptopropionylamino) -3,6,9,12,15pentaoxoheptadecanoylamino / -rSEA (I) (1.17 mg, 42 nmol) in a nitrogen atmosphere for 43 hours in the dark

I ml of the above buffer). After that is added

1.14 μτηοΐ mercaptoethanol. After a further 1 h, the reaction mixture is partitioned on a Superdex 200 HR 16/55 column. The product was washed with 2 mM phosphate buffer pH 7.5 containing 0.9% sodium chloride. The fractions containing the final product J are pooled and analyzed as described in Example 4B. The conjugate consists of 9% monAK C242 with two SEAs, 25% with one SEA and 60% unchanged monAK C242.

EXPERIMENTAL CHAPTER II

Effects of superantigen-antibody conjugates on cells

In subsequent experiments, Staphylococcus A enterotoxin (SEA), obtained from Toksin Technology (Wisconsin, USA), or produced as a recombinant protein in E. coli cells was used as a bacterial toxin.

The antibodies were C215, C242 and Thy-1.2 monAK. C215 is an Ig G2a monAK against the human colon cancer cell line and reacts on some human colon cancer cell lines with a protein antigen of 37 kD. References to these monAKs have been made previously. The preparation of conjugates is described in the first section.

Prior to the application date (priority date), only studies with Eu ³⁺ - labeled conjugates SEAC215 monAK were performed. The following year, results for unlabeled conjugates SEA-215, SEA-242 and SEAThy-1.2 monAK were confirmed. The results reported here were obtained with unlabeled conjugates.

To determine the cytotoxicity induced by the conjugate SEAC215 monAK and unconjugated SEA and C215 monAK, we used various human T-cell lines exposed to SEA to express small and undetectable amounts of Class II PASKA in colon cancer cells. , and the colon cell layer and Raji cells expressing Class II PASKA (Class II ⁺ PASKA) as target cells. The colon cancer cell lines Colo 205, SW 620 and WiDr Class II PASKA do not express what was detected by monAK staining against HLA-DR, HLA-DP and HLA-DQ and activated by fluorescence by cellular analysis. T-cell lines treated with SEA were obtained from peripheral blood by weekly restimulation with mitomycin C II ⁺ PASKA lymphoma BSM cells pre-coated with SEA in the presence of recombinant IL-2 (20 units / ml). These T-cell lines exhibited high cytotokLT 3909 B sensitivity to Raji or BSM cells coated with but not coated with SEA or cells coated with staphylococcal enterotoxin B (SEB). This inducible destruction of SEA is dependent on SEA cell-target interaction with Class II PASKA as determined by blocking antibodies HLA-DR, mutant Raji cells not expressing Class II PASKA (L-class II PASKA) and L-cells transfected with HLA-DR / Doglsten et al., Immunology 71: 96-100 (1990). These T-cell lines appeared to be activated by the C215-SEA conjugate, for the destruction of C215 ⁺ Class II PASKA cells in colon cancer. In contrast, unconjugated SEA and C215 monAK appeared to be incapable of inducing more than marginal T-cell destruction in C215 ⁺ Class II PASKA cells of colon cancer. Cell-induced cytotoxicity dependent on staphylococcal enterotoxin-antibody conjugate is dependent on the binding of SEA-C215 monAK conjugate to C215 ⁺ cancer cells. The specificity of this binding was demonstrated by the fact that excess of unconjugated monAK C215, but not C242 and W8 / 32 monAK, inhibits lysis of colon cancer cells. CD4 ⁺ and CD8 ⁺ T cells kill C215 ⁺ cells affected by SEA-C215 in colon cancer but do not lyse cells affected by SEA. Apparently, T-cell interactions with SEA-C215 conjugate Monaco, bound to tumor cells Il'Klasės below, comprises the interaction with T-cells of variable receptor beta-chain sequence specific manner analogous to that previously demonstruotam SEA iššaukiamam Class II ⁺ cells PASKA destruction. This was demonstrated by the interaction of SEA-specific but not autologous SEB-specific T-cell line with the C215-SEA conjugate. C242 monAK conjugates and Thy-1.2 monAK conjugates show activity analogous to that of C215 monAK conjugate.

Chromium labeling and cell-target incubation with SEA

0, 75 x 10 ⁶ cellular targets and 150 µCi ⁵¹ Cr (American Corp., Arlington Heits, England) were incubated in a 100 μΐ volume for 45 minutes. Cells were maintained in complete medium containing RPMI-1640 medium (Gibko, Peizli, UK) supplemented with 2.8% (v / v) 7.5 NaHCO ₃ , 1% sodium pyruvate, 2% 200 mM 1-glutamine, 1% Hepes, 1% mg / ml gentamicin and 10% calf embryo serum (VES, Gibko, Peizli, UK). After incubation, cells were washed once in complete medium without VES, incubated at 37 ° C for 60 min, washed and resuspended in complete medium containing 10% VES. To each of the wells, 96-well microliter plates were supplemented with 5x10 ³ cell-targets per well (U, Costar, Cambridg, USA).

Test for cytotoxicity

Cell effectors / cell-target effector cells were added to the sockets in various ratios. The terminal volume in each socket was 200 μΐ. Each challenge was repeated three times. The plates were incubated at 37 ° C for 4 hours, after which time the chromium released was collected. ^{The 51} Cr content was determined with a gamma counter (Cobra, Auto-gamma, Paccard). The percentage of cytotoxicity was determined using the formula:

% cytotoxicity = (Χ-Μ) / (T-M) x 100 where

X represents the chromium release (in units of imp / min) obtained in the test sample, M represents the spontaneous chromium release by cell-target incubated with medium, and T represents the complete chromium release obtained by incubation of the cell-target with 1% sodium dodecyl sulfate.

Results

The conjugates SEA-C242, SEA-C215 and SEA-anti-Thy-1.2 bind to cells expressing the respective monoclonal antibody epitopes, respectively, and to Class II ⁺ PASKA cells. On the other hand, unconjugated SEA binds only to PASKA class II ⁺ cells. Non-conjugated monoclonal antibodies C215, C242 and Thy-1.2 bind to cells expressing the respective epitopes but not to Raj i cells (Table 1).

Human T-cell lines lyse cells of lines SW620, Colo 205, and WiDr II ^- Class PASKA in the presence of SEA-C215 monAK conjugate, but do not lyse in the presence of unconjugated SEA and C215 monAK (Fig. I). Colon cancer cell lysis occurred at concentrations of 10-100 ng / ml SEA-C215 monAK conjugate. High lithium rates in various effector / target ratios were observed for SEA-215 monAK relative to SW620 (Fig. I). Conversely, unconjugated SEA or C215 monAK does not induce eitotoxicity against SW620 cells in all of the effector-to-target ratios tested. This indicates that the ability to lyse Colo 205 class II 'PASKA cells is conjugate-bound and does not induce unconjugated SEA and C215 monAK. SEA and SEA-C215 conjugate Monaco, but not C215 Monaco induces Class II ⁺ Raji cells and PASKA affect interferon line Colo 205 Class II ⁺ cells PASKA destruction of T-cells.

To demonstrate that specific conjugate binding to the C215 monAK molecule on the cell-target is induced by the inducible SEA-C215 monAK conjugate, an inhibition assay was performed with an excess of unconjugated C215 monAK and C242 monAK (which binds to the foreign antigen on colon cancer cells). C215 for monAK binding). Addition of monAK C215 potently inhibits cytotoxicity whereas C242 monAK shows no effect (Fig. (Fig.2). 2). Similarly, lecithin SEA-C242 monAK conjugate is specifically inhibited by excess unconjugated C242 monAK but not C215 monAK.

The ability of SEA-C215 monAK conjugate to induce T-cell-dependent colon cancer in the SW620 lineage of class II PASKA cells was also observed in CD4 ⁺ and CD8 ⁺ T-cell populations. SEA does not activate any of these T-cell subclasses, nor does it induce cell death of the SW620 lineage, but lyses PASKA Raji class II ⁺ cells (Table 2).

The conjugate SEA-C215 monAK induces SW620 and Raji cell lysis in a line of T-cells affected by SEA but not in a line of T-cells affected by SEB (Fig. (Fig.3). 3). The selectivity of SEA- and SEBlines is shown by their selective response to SEA and SEB, respectively, upon contact with Raji cells (Fig. (Fig.4). 4). This indicates that the monA conjugate of SEA-C215 retains specificity for the T-cell receptor variable region beta chain analogous to that of unconjugated SEA.

DESCRIPTION OF THE FIGURES FIG. SEA-C215 monAK conjugate targets CTL against colon cancer cells of Il'Class PASKA. The top left graph shows the effect of CTL-sensitive SEAs on SW620 cells at various effector / target ratios, in the absence (-) or in the presence of SEA-C215 monAK conjugate, SEA, C215, and C215 and SEA (C215 + SEA). 1 µg / ml. The following graphs show the ability of SEA-C215 monAK conjugate and SEA to target SEA-sensitive CTL against colon cancer SW620, Colo205 and WiDr lines of Il'Class C215 ⁺ PASKA cells exposed to interferon Colo 205 Class II ⁺ PASKA C215 ⁺ cells and Raji II ⁺ class PASKA C215 'cells. EfekLT 3909 has a toroid / target ratio of 30: 1. Addition of unconjugated C215 monAK at some concentrations does not target CTL against the cells of these lines. Activated fluorescence cell analysis used for SW620, Colo 205, and WiDr cells using monAK against HLA-DR, -DP, -DQ did not detect any superficial expression of Class II PASKA, while abundant HLA-DR -, - DP was observed. and -DQ expression on Raji cells, and HLA-DR and -DP on interferon-treated Colo 205 cells. Colo 205 cells were exposed to 1000 units / ml recombinant interferon-gamma for 48 hours prior to use in the CTL assay.

FIG. Dependence of SEA-C215 monAK and SEA-C242 monAK Conjugated Inducible CTL on Colon Cancer Cells on Antigenic MonAK Selectivity. The lysis of Colo 205 cells sensitive to SEA CTL in the presence of SEA-C215 monAK and SEA-C242 monAK (3 µg / ml) conjugates was inhibited by the addition of unconjugated C215 and C242 monAK (30 µg / ml), respectively. Unconjugated monAK or control medium (-) was added to the target cells for 10 min before conjugates.

FIG. Liquid colon cancer cells coated with the conjugate SEA-C215 monAK are induced by CTL responding to SEA but not to SEB. Autologous T-cell SEA- and SEB- selective lines, effector-to-target ratio of 10: 1, pre-cellular SW620 and Raji, in the absence (control) and in the presence of SEA-C215 monAK conjugate, were used. a mixture of unconjugated C215 monAK and SEA (C215 + SEA) and unconjugated C215 monAK and SEA (C215 + SEB) at a concentration of 1 µg / ml for each additive.

FIG. Cytotoxicity induced by SEA-C242 monAK and SEA-anti-Thy-1.2 monAK conjugates against cell LT 3909 B targets (Colo 205 tumor cells and EL-4 tumor cells, respectively).

Table I

Binding of SEA-C215 monAK Conjugate to C215 ⁺ Cells of Colon Cancer and PASKA Cells of Raji Class II ⁺

Reagent The cell Fluorescent analysis SEA-C215 monAK Colo205 Positive Ra j i Positive C215 monAK Colo205 Positive Ra j i Negative SEA-C242 monAK Colo205 Positive Ra j i Positive C242 monAK Colo205 Positive Ra j i Negative SEA-anti-Thy-1.2 monAK EL-4 Positive anti-Thy-1.2 monAK EL-4 Positive SEA Colo205 Negative Ra j i Positive Control Colo205 Negative Ra j i Negative EL-4 Negative Cells were incubated with various additives for control (bovine serum albumin) for 30 min on ice, rinsed and treated as previously described. C215 monAK and C242 monAK bound to Colo205 was determined using FITC-labeled rabbit I g

against mouse antigens. The staining of SEA bound to Raji cells was determined using rabbit antisera against SEA followed by FITC-labeled porcine Ig g against rabbit antigens. The staining of SEA-C215 monAK conjugate bound to Colo205 and Raji cells was determined using procedures previously described for C215 monAK and SEA. Activated fluorescence analysis was performed on a Becton and Diccinson instrument. For the background setting, staining for the second and third stages was used.

Table

Characterizing C215-SEA Conjugate in Colon Cancer Cell Lysis by CD4 ⁺ CTL and CD8 ⁺ CTL

Effector * Target Cytotoxicity%

control SEA C215-SEA CD4 ⁺ SW620 2 5 50 CD4 ⁺ Raji 0 41 43 CD8 ⁺ SW620 0 1 23rd CD8 ⁺ Raji 2 72 68 * CTL (SEA-3) kin, level was 30: 1, used in the ratio effector / it- in the absence of (control) or

in the presence of SEA and C215-SEA at a concentration of 1 µg / ml.

Claims (21)

DEFINITION OF INVENTION A soluble antibody conjugate comprising an antibody and a superantigen, characterized in that (i) the specified antibody is specific for disruption of the cell-target surface structure and (ii) the said superantigen is recognized by T-cells and is capable of activating cytotoxic T-cells (CTL). ), and most often is able to interact with the beta-chain of the T-cell receptor variable region. 2. The conjugate of claim 1, wherein the cellular target is associated with a disease selected from the group consisting of cancer, autoimmune disease, parasitic infections and microbial infections such as viral, fungal and bacterial infection, and an antibody specifically targeted. against the antigenic determinant on the designated cell-target. 3. The conjugate of claim 1 or 2, wherein the disease is cancer and the antibody is directed against an antigenic determinant (epitope) on a tumor cell such as a cell associated with colon cancer. 4. The conjugate of claim 3, wherein the antibody is specifically directed against the C242 epitope. 5. The conjugate of claim 3, wherein the antibody is specifically directed against an epitope in a protein belonging to GA-733, namely the C215 epitope. 6. The conjugate of any one of claims 1-5, wherein at least 1-5, preferably 1-3 superantigens are present per antibody. 7. The conjugate of any one of claims 1-6, wherein the monoclonal antibody is used as an antibody. 8. The conjugate of any one of claims 1-7, wherein the superantigen is bacterial and is selected from the group consisting of staphylococcal enterotoxins. 9. The conjugate of any one of claims 1-8, wherein the staphylococcal enterotoxins SEA are used as a superantigen. 10. The conjugate of any one of claims 1-9, wherein the superantigen and antibody are linked to each other by a covalent organic bridge -B- which preferably has at least one amide structure and is hydrophilic having at least one chain 6 atoms. 11. The conjugate of claim 10, wherein the bridge -B- has the following structure: -S _r RCONHCH ₂ CH ₂ (OCH ₂ CH ₂ ) _n O (CH ₂ ) _m COY- (I) wherein: r is 1 or 2; R is an alkylene group having 1 to 4 carbon atoms; n is an integer from 1 to 20; m is 1 or 2; and Y is -NH-, -NHNH- or -NHN = CH-. 12. The conjugate of any one of claims 1 to 10, wherein it is used to target cell surface targets having a surface structure to which the conjugate antibody is directed. 13. A method of cell-target mutagenesis, wherein the target cell is contacted with an effective amount of an antibody-soluble conjugate containing an antibody bound to a superantigen for the purpose of cell-target mutation, said antibody is cell-specific, and said superantigen is recognizable. T-cells and is able to activate cytotoxic T-cells (CTL). 14. The lysing method of claim 13, wherein the superantigen, antibody, and antibody binding to the superantigen bridge are as defined in any one of claims 1-12. 15. A method of preparing a conjugate comprising binding the antibody and / or superantigen to at least one structure selected from the group consisting of (a) carbohydrates and (b) functional groups: mercapto, disulfide, carboxyl, and amino; further isolates the conjugate from the medium. 16. A process for the preparation of a conjugate according to claim 15, wherein the condensation reaction involves an amino group present in the superantigen and / or antibody. 17. The process for preparing the conjugate of claim 16, wherein the condensation reaction comprises the steps of: (i) reacting the antibody or superantigen with an organic reagent having a mercapto-reactive group and an amino-reacting group to produce an antibody or superantigen having a mercapto-reactive moiety, and (ii) reacting the remainder of the superantigen or antibody. with organic reagents having a mercaptogroup or a blocked mercaptogroup, or a group capable of reacting with an amino group to produce a superantigen or antibody containing a mercaptogroup or a blocked mercaptogroup; followed by the products of steps (i) and (ii) respectively reacting with each other to form a conjugate in which the superantigen is bound to the antibody via a disulfide or thioether group. 18. A process for the preparation of a conjugate according to claim 17, wherein the compound capable of interacting with the amino group and having the group capable of interacting with the mercaptogroup is an alpha-haloacetyl halide, and the compound having the mercaptogroup or blocked mercaptogroup is a bifunctional coupling reagent of formula II: Z ₁ RCONHCH ₂ CH ₂ (OCH ₂ CH ₂ ) _n O (CH ₂ ) _n , z '(II) wherein: m is 1 or 2, n is an integer from 1 to 20, Z _x is a HS-reactive electrophilic a group, a mercaptogroup (-SH) or a blocked mercaptogroup (e.g., AcS-), provided that the mercaptogroup and the hydroxyl group are not attached to the same carbon atom in the R radical; and z \ is an activated carboxyl group. 19. The method of producing the conjugate of any one of claims 1518, wherein the superantigen and antibody indicated are as defined in any one of claims 1-9. The conjugate of any one of claims 1 to 11 for the manufacture of a medicament for treating a mammal, including a human being, selected from the group consisting of cancer, autoimmune diseases, infections 10 viruses, bacteria or fungi, and parasitic infections. Use of a conjugate according to any one of claims 1 to 11 for the preparation of a pharmaceutical composition for 15 for the treatment of cancer, autoimmune diseases, infections caused by viruses, bacteria and fungi, or infestation by parasites.