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 comprising an antibody linked to a structure recognized by T cells capable of directing T cells to lyse target cells recognized by the antibody. The conjugate is characterized in that said structure is a superantigen. An important aspect is the method of lysing the target cells, whereby the target cells are contacted with an amount of conjugate effective for lysing the target cells. The lysing method is part of an effective treatment regimen for cancer, autoimmune diseases, parasitic infections, and infections caused by fungi, viruses, and bacteria. The description also provides aspects such as the synthesis of the conjugate, pharmaceutical compositions, and their preparation.

Description

The present invention provides antibody conjugates that can activate cytotoxic T-cells (CTLs). The conjugates are used to destroy unwanted cells associated with various forms of cancer, autoimmune processes, parasitic infestations, and bacterial, viral, and fungal infections.

Attempts are known to use antibodies in combination with agents that have a direct toxic effect on target cells (cytotoxic agents, cytotoxins), ensuring selective effect on target cells and preventing and reducing nonspecific effects on other cells. The proposed combinations range from covalently linked complexes using molecular bridges and noncovalently 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). Known cytotoxins include diphtheria toxin, ricin, ricin domain A, gelonin and Pseudomonas aeruginosa exotoxin A (Takada Chemical Ind., EP-A-336405 and Pastan et al., WO-A-38/00703; both cited in connection with priority application SE9002479).

With the advent of hybridoma technology and its accompanying monoclonal antibodies, it became possible to use complexes composed of antibodies and cytotoxic compounds to more specifically direct cytotoxic agents to the desired target cell population.

Since cytotoxic cells are known to have damaging effects on other cells that are different from their target cells, it has been proposed to replace cytotoxic agents with immunostimulators that stimulate T-lymphocytes and activate CTL. Specific proposals have concerned 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 Corp., EP-AI334300);

(iii) Major histocompatibility complex antigens (MHCA) (Begringverco AG EP-AI-352761);

(iv) antigens to which the subject is immune (Med. Ros. Counc. WO-A-90/II779 (published 18.10.1990)); and (v) an unnamed bacterial enterotoxin (Ochi and Wake, Symposium UCLA: Cellular Immunity and Cancer Therapy, 27 January-30 February 1990, abstracts of paper CE 515, p. 109).

However, the immunostimulants proposed so far have been either very specific or very general in their action. For example, classical antigens activate only about 1 in 10 ⁵ T-cells, while mitogens can potentially activate most of them.

Some agents have been found to activate T cells moderately, i.e. they activate T cells at a relatively high frequency, but far from 100%. /Fleisher et ai., J. Exp. Med., 157 (1988)1697-1707; Wait et ai.,

Cell, 56(1989)27-35; both articles are incorporated herein by reference. Agents of this type are more effective activators than classical antigens, and have therefore been called superantigens (for review see Keccler and Marrac, Science 248:705, (1990)). It has further been shown (Doglsten et al., Immunol. 71(1990)96-100; Chedlund et al., Cell Immunol. 129(1990)426-34) that known superantigens can bind to major histocompatibility complex (MHC) Class II molecules on target cells and activate cytotoxic T cells containing the beta chain of the variable region of the T-cell receptor. Published data indicate that PASK binding is a prerequisite for T-cell binding and activation. It cannot be ruled out that in the future, superantigens will be discovered that act through the alpha chain of the T-cell receptor variable region or through other surface structures present on T-cell subpopulations.

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

Most of the superantigens now known were previously shown to be toxins and to be of microbial origin. Staphylococcal enterotoxins, for example, are enterotoxic and activate T cells, and these two effects are distinct from each other/Fleisher et al., Cell Immunol., 118(1989)92101; Alber et al., J. Immunol., 144 (1990) ₄ 501-06; and Infact. Immun. 59(1991)2126-34/.

It has previously been proposed to use superantigens to direct CTL lysis of cells bearing Class II major histocompatibility complex antigens (PASKA) (Farmacia AB, WO-A-91/04053, published 1991.04.04). WO-A-91/04053 covers superantigens but does not mention incorporation into covalent immunoconjugates. Cells lacking Class II PASKA proteins, or expressing subthreshold amounts of these proteins, do not bind sufficient amounts of superantigens to effectively direct their lysis by CTLs. Thus, due to the large total number of cells bearing Class II PASKA and the low levels of Class II PASKA on most cancer cells, superantigens should not be of great value in terms of specific killing of such unwanted cells.

However, we have found that the specific effect of cell killing by CTLs can be achieved by superantigens if they are covalently linked to an antibody against an epitope that is specific for the killing cell. Activation of the immune system can induce the expression of Class II PASKA by target cells that do not have these antigens, which, in turn, can make the desired lytic effect possible.

Brief summary of the invention

The present invention includes (i) novel antibody conjugates comprising (I) an antibody against target cells and (2) a superantigen, i.e. a structure that recognizes (interacts with and/or binds to) and activates T-cells, particularly CTLs;

(ii) methods of disrupting target cells, particularly in relation to therapeutic methods of treating mammals, and methods of specific activity of T-cells such as CTLs;

(iii) methods for synthesizing conjugates; and (iv) pharmaceutical compositions containing conjugates and methods for preparing these compositions.

Targeted cell destruction methods include therapeutic methods for cancer, viral, bacterial, fungal infections, parasitic infestations and other diseases, in which the main goal is the destruction of certain cells, achieving a high degree of precision. In accordance with the present invention, the conjugates can be used to obtain pharmaceutical compositions for the destruction of target cells associated with the above-mentioned diseases. The subjects of treatment are mammals, primarily humans.

Superantigenic portion of the conjugate

The novel antibody conjugates have a structure that is recognized by T-cells as a superantigen. The conjugates are generally soluble at physiological pH values, and in vitro they are soluble in serum.

Preferably, superantigens are selected from the group of staphylococcal enterotoxins (SE), such as SEA, SEB, SEC, SĖD, and SEE, toxoids, active fragments or peptides thereof, and other compounds that have substantially the same mode of action in CTL activation. Superantigens may include other microbial products (bacterial and viral), such as products from staphylococcal strains, e.g., toxic shock syndrome toxins (TSST-I) from Streptococcus pneumoniae, e.g., pyrogenic exotoxin A, and bacterial exoproteins and proteins produced by pleuropneumoniae microorganisms that have a similar ability to interact with T cells as superantigens. Superantigens may be obtained by culturing their natural producers or cells obtained by genetic engineering methods (recombinant methods), and potentially also by peptide synthesis. The superantigen used in the framework of this invention, when tested on the experimental model presented in this description, should show a performance analogous to that presented in the experimental part of this description.

Optimal superantigens have the potential ability to bind to 1-40% of the isoforms of a polymorphic protein on the T-cell surface associated with CTL activation, most optimally to the beta-chain of the variable region of the T-cell receptor.

Antibody (AB) portion of the conjugate

The antibody is preferably a monoclonal antibody (mAb), although polyclonal antibodies may also be used, provided that they have a sufficiently narrow range of specificity. The term antibody is applied to antibodies in general, and thus includes active fragments of antibodies and other molecules that mimic the binding ability of antibodies, provided that they have the appropriate specificity, avidity and affinity for a given target cell. This includes antibodies obtained by genetic engineering methods (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 an autoimmune reaction, cells infected with a virus, bacteria, parasites, fungi or other unwanted cells. Depending on the efficiency of the conjugate, the antibody may be directed against an antigen that internalizes the antibody after conjugation, although there is a view that such specific antibodies are not optimal.

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

Of the three monoclonal antibodies tested, the C125-conjugates are by far the least interesting, as they react with an epitope on a cancer antigen that is very commonly expressed on normal cells. The C242-conjugates, based on specificity data, seem more promising, although our results suggest that they may require higher doses. MonAK C against Thy-1.2 may not be of value against human cancer cells, as the Thy-1.2 antigen is specific for non-human, but non-mammal cancer cells.

A line of hybridoma cells producing the monoclonal antibody C242 was deposited in the European Collection of Animal Cell Cultures, Porton Down, Salisbury, Wilts, England, 1990.01.26, No. ECACC 90012601.

Structure that connects superantigen to antibody

According to the present invention, in a suitable conjugate, the superantigen is covalently linked to the antibody via a covalent bridge (-B-). It is important that the conjugate does not degrade when used against cells for destruction, e.g., when administered to an animal. Therefore, the bridge must be metabolically stable for a relatively long time to achieve an effect. In addition, it is necessary that the bridge itself does not cause immunological reactions. In general, the bridge must be inert in the sense that the conjugate retains high specificity for binding to target cells (anticancer, antiviral, etc.) and a high ability to activate cytotoxic T cells.

Several functional groups have been proposed in the scientific and patent literature to serve as bridges in immunoconjugates. Accordingly, the bridge -B in the proposed novel 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 bonded 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-, where each of the free valences is bonded to a saturated carbon atom;

(ii) thioethers and disulfides (—S _r —, where each of the free valencies is directly bonded 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 have one or more hydroxyl or amino group substituents;

(iv) ethers (-0-, where each free valence is directly bonded to a saturated carbon atom); and (v) primary amines or disubstituted hydrazines (-NH- or -NH-NH-, respectively, where each of the free valences is directly bonded to a saturated carbon atom).

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

The preferred bridge is hydrophilic and should not contain any aromatic rings. Preferred hydrophilic structures that can form part of the bridge -B- 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 ,, where n is an integer from 2 to 5, preferably 2 or 3, and n' may be an integer from 1 to 20; and (iii) -S- (thioethers), -O- (ethers) and unsubstituted amides (-CONH-), each of which is linked to short unsubstituted hydrocarbon chains (C ₁ _ ₄ ), preferably containing 1 or 2 carbon atoms.

Hydrophilic amino acids can be in hydrophilic structures of the type /F-(0 ₂ o)n/mF, where F is an amino acid sequence, preferably, of 4-8 residues, where each amino acid is selected from serine, glycine and threonine, m is an integer from 1 to 4, 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 locations. Potential sites include the amino terminus, the carboxyl terminus, and a 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 attachment, as long as they do not play an essential role in the functioning of the active moieties of the conjugate. Carbohydrate structures, if present, can be oxidized to aldehyde groups, which in turn can be used for attachment to another moiety of the conjugate (Cetus Corp., EP-A-240200).

The conjugate proposed in this invention should not contain many ester and labile amide bonds, especially formed at tyrosine and histidine residues, respectively. If such bonds are formed during synthesis, they should be removed using hydroxylamine /Endo et al., Cancer Res. 48(1988)3330-3335/. The number of superantigenic fragments per active AK fragment in the conjugate is usually 1-5, most often 1 or 2.

In a preferred embodiment, the conjugate should be substantially uniform in superantigen/antibody ratio, and/or in the binding sites used in the superantigen and antibody, respectively, and/or in the bridge -B-, etc. In other words, all individual conjugate molecules that make up the conjugate as a substance should be uniform in these variable characteristics.

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

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

According to the embodiment of the invention, the most studied until the date of filing the application, 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 valencies in formula I are connected to the active moieties, respectively. That is, directly, or through kiLT 3909 B those divalent inert structures located in the bridge -B-.

n is an integer greater than 0, e.g., 1-20, preferably 2 or more than 2, and in most cases less than 10. m is equal to 1 or 2.

S is a sulfur atom, and each of its valences is directly bonded to a carbon atom (-S _r - is a thioether or disulfide), r equal to 1 or 2.

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

R is preferably an alkylene group (containing from 1 to 4 carbon atoms, most often 1 or 2 carbon atoms) which may be substituted with 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 can be obtained by saturation and isolation from the culture medium of the cells producing them, or from other media in which they were synthesized.

The synthesis of new conjugates can be carried out by methods known in the art, e.g. by genetic engineering methods (recombinant methods) or by classical condensation reactions from the corresponding antibody and antigen 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) Mercapto group (HS-). A mercapto group can react with a compound containing a group that can interact with the mercapto group to form a thioether group or a disulfide group. Free mercapto groups are found in proteins on cystine residues, and can be introduced into proteins by thiolation or by cleavage of disulfides in natural cysteine residues.

(iii) Free amino groups (H ₂ N-) in amino acid residues. Amino groups can react with compounds containing 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 a lysine residue.

(iv) Free carboxyl groups in amino acid residues. A carboxyl group can be converted to an activated carboxyl group, which then reacts with a compound containing an amino group 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 a dibasic alpha-amino acid.

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

Recombinant methods are effective means of producing conjugates in which moieties are specifically linked to each other by a bond between the terminal carboxyl group of one moiety and the terminal amino group of the other moiety. A linking structure compatible with the method used can also be introduced.

The reagents used are selected in such a way as to obtain the bridge -B- described above. Common bifunctional reagents have the formula Z -B'-Z', where Z and Z' are functional groups compatible with each other, enabling the realization of a covalent bond with a functional group present in the protein. See above, B is an inert bridge, which can have the same structures as those mentioned above for the bridge -B-. In particular, Z and Z' can be the same or different and are selected from a mercapto group, a group capable of reacting with a mercapto group, an activated carboxyl group, -CONHNH ₂ , and 1.1. For a description of these groups, see below, in the subsection New Reagents .

The method of obtaining the conjugates used in the experimental part we use includes:

(i) reacting the antibody and the superantigen with an organic reagent having a group capable of reacting with a 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 a mercapto group, and (ii) reacting the remainder of the superantigen and the antibody with an organic reagent having a mercapto group and a group capable of reacting with an amino group to produce a superLT 3909 B antigen or antibody having a mercapto group or a blocked mercapto group, followed by (iii) reacting the products obtained in steps (i) and (ii) with each other to form a conjugate in which the superantigen is linked to the antibody by a disulfide or thioether bond.

The conditions for condensation are known for each group in protein chemistry. Condensation can occur in stages or in a single step, forming intermediate functional groups that can be attached to the starting materials by inert spacers. In general, the conditions under which the synthesis and purification/isolation of the conjugate are carried out are always such that they do not cause denaturation of the proteins used. Typically, these are aqueous media, pH values in the range of 3 to 10, and temperatures from 0 to 50°C. The specific conditions depend on which groups react and which conjugate needs to be isolated. For details, see the subsection New Reagents.

New reagents (developed in connection with this invention)

A new heterobifunctional reagent with the general formula II was developed for the chemical synthesis of conjugates containing a -B- bridge:

Z ₁ RCONHCH ₂ CH ₂ (OCH ₂ CH ₂ ) _n O (CH ₂ ) “Z'! (II) where m and n have the same meanings as in the above-mentioned formula (I) . Z ₁ is an electrophilic group capable of reacting with an HS- group, a mercapto (-SH) or a blocked mercapto group (for example, AcS-), provided that the mercapto and hydroxyl groups cannot be bonded to the same carbon atom in the R radical. Electrophilic groups capable of reacting with an HS- group are the following:

(i) halogen bonded to a saturated carbon atom, preferably in the form of alpha-halo-alkylcarbonyl (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-I-aza-cyclopent-3-en-I-yl.

The description of the reactive disulfide, see e.g. EP-A-125885, is given in the reference herein.

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

One of the advantages of the new reagents is that they enable the production of a homogeneous conjugated compound in the structure (OCH ₂ CH ₂ ) with respect to n for integer values of n, i.e. n is the same for each molecule of a given conjugated compound.

The functional groups reactive with Z1 and _Z4 may be present in the natural antibody and superantigen molecules or may be introduced into them. The terminal groups Z1 and _Z4 may then react selectively with the corresponding antibody or superantigen by methods known for these types of groups.

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

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

CO 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 bonded to an sp- _hybridized carbon atom derived from an oxidized carbohydrate present in the antibody or superantigen (when they are glycoproteins), (3) -CO( _CH2 ) _{mO ( CH2CH2O} ⁾ _{nCH2CH2NHCOR1} - _{Sr- RCONHCH2CH2 ( OCH2CH2 ) n-} 0( _CH2 ) _{mCOY- ;}

CO - bonded to NH, (4) -CO (CH ₂ ) _m O (CH ₂ CH ₂ O) _n CH ₂ CH ₂ NHCOR' -S _r -RCONHCH ₂ CH ₂ (OCH ₂ CH ₂ ) _n -0 (CH ₂ ) _m CONHNH-R NHN= ;

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

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

The reagent (formula II) can be obtained using compounds having formula III:

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

The synthesis of some compounds of formula III, where m = 1 and 2, and n = 1-10, has been described previously./Juljen et al., Tetrahedron Letters, 29(1988)3803-06; Hougton, Saubi, Synth. Commun. 19(18)(1989)3199-3209; and EP-A410280 (ref. 20.1.91) and Šlama 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 R and R' described above, and Z' is an activated carboxyl group as defined above. After the reaction, the -COOH group becomes an activated carboxyl group, e.g., Ζ' _γ - activated ester, such as N-succinimidyloxycarbonyl, 4-nitrophenyloxycarbonyl, 2,4-dinitrophenyloxycarbonyl, 2,4-dinitrophenyloxycarbonyl, etc.

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

XCH ₂ CH ₂ (OCH ₂ CH ₂ -) _n OCH ₂ Y (IV) where 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 can be obtained, preferably by hydrolysis or reduction. Examples are an unsubstituted amino group (H ₂ N-), a nitro group, an amide (urea) group, such as a lower alkyl amide group (formyl amide) acetyl amide... hexanoyl amide), including acyl amide groups having electron-withdrawing substituents on the carbon alpha-atom in the acyl moiety, especially CF ₃ CONH-, CH ₃ COCH ₂ CONH-, etc.; a phthalimidoyl group, which may have a ring substituent; carbamate group (especially R^OCONH- and (R'11 - OCO) (R' ₂ -OCO) N- as well as N-(tert-butyloxycarbonyl)-amino (Bok), N-(benzyloxycarbonyl)amino and di(N-benzyloxycarbonyl)-amino (Z and di Z, respectively) which have ring substituents; alkylamino group in which the carbon atom connected 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-1,3,5-triazin-1-yl, including those having lower alkyl substituents in the 3- and/or 5-positions.

Hereinafter, R1 and _R2 are a lower alkyl group, especially a secondary and tertiary alkyl group, and a methyl group substituted by 1-3 phenyl groups, which in turn may be substituted. The lower alkyl and lower acyl groups have 1-6 carbon atoms.

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

Other groups are -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, by combinations of known methods. Suitable synthetic routes include:

A) Chain formation.

B) Transformation of terminal functional groups.

C) Transformation of a symmetrical polyester into an unsymmetrical ether.

D) The cleavage of a bisymmetric chain into two identical fragments.

Suitable starting compounds containing the repeating unit -OCH ₂ CH ₂ - are commercially available. Examples include oligoethylene glycols containing from 2 to 6 repeating units. Other suitable compounds with identical end groups are the corresponding dicarboxylic acids and diamines.

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

Pharmaceutical compositions and their preparation

According to the present invention, pharmaceutical compositions are formulated from formulations known in the art but containing our novel conjugates. The compositions may be in the form of lyophilized particles, sterile or aseptically prepared solutions, tablets, ampoules, and 1.1. They may contain carriers such as water (preferably buffered, at physiological pH values, such as phosphate buffered saline (PBS)), or other inert solid or liquid materials.

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, with suitable additives and adjuvants. It is necessary that the carriers and conditions do not adversely affect the activity of the conjugate. Water itself is also included among the carriers.

Use and methods of use

Conjugates are usually solid and are used in pre-prepared doses, each containing an effective amount of conjugate, which, according to the results presented, is from 10 µg to 50 mg. The exact dose varies from one case to another, and depends on the weight and age of the patient, the route of administration, the nature of the disease, the antibody, the superantigen, the bridge (-B-), etc.

The route of administration is in accordance with those known in the art, i.e. effective to lyse the target cell. The amount of conjugate or therapeutically effective amount of conjugate according to the invention is contacted with the target cells. In the above cases, this usually means a parenteral route of administration, such as injection or injection (subcutaneously, intravenously, intraarterially, intramuscularly) into the body of a mammal, e.g. a human. The conjugate may be administered locally or systemically to the subject.

An amount effective for killing target cells is understood to be an amount that effectively activates and directs CTL to kill target cells.

The invention is illustrated by several embodiments, which in no way limit the invention. Section I of the experimental part shows the chemical synthesis of conjugates, and Section II shows the activation of T-cells using 4 conjugates as an example for targeting cells.

EXPERIMENTAL PART. CHAPTER I

Obtaining omega-amino-DEG-carboxylic acid

Isopropyl 8-hydroxy-3,6-dioxa-octanoate (I)

Sodium flakes (23 g, 1.0 mol) were added portionwise to diethylene glycol (500 ml) under a nitrogen atmosphere. After the sodium had completely reacted, the mixture was cooled to room temperature, and bromoacetic acid (76 g, 0.5 mol) was added with stirring. The reaction was carried out for 18 hours at 100°C, after which the excess diethylene glycol was distilled off at a pressure of about 4 mm Hg. Isopropyl alcohol (400 ml) and acetyl chloride (51 g, 0.65 mol) were then added portionwise. The mixture was stirred for 16 hours at 65°C, then cooled to room temperature and neutralized with sodium acetate (3.5 g, 0.15 mol). The mixture was filtered, the filtrate was evaporated to dryness, and the residue was dissolved in water (200 ml). The aqueous phase is extracted with 1,1,1-trichloroethane (3 times 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 leave an oil (55 g).

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

Sodium (23 g, 1.0 mol) in the form of flakes was added portionwise to triethylene glycol (700 ml) under a nitrogen atmosphere. After the sodium had completely reacted, the mixture was cooled to room temperature and bromoacetic acid (76 g, 0.5 mol) was added with stirring. The reaction was carried out for 18 h at 100°C, after which the excess triethylene glycol was evaporated by distillation at about 4 mm Hg in a column of LT 3909 B. Isopropyl alcohol (400 ml) and acetyl chloride (51 g, 0.65 mol) were then added portionwise. After stirring for 18 h at 65°C, the mixture was cooled to room temperature and neutralized with sodium acetate (3.5 g, 0.15 mol). The mixture was filtered, the filtrate was evaporated almost to dryness, and the residue was dissolved in water (200 ml). The aqueous phase is extracted with 1,1,1-trichloroethane (3 times 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 give an oil.

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-dioxa-octanol (3)

8-Chloro-3,6-dioxa-octanol (365 g, 2.2 mol, obtained from triethylene glycol and SOC1 ₂ ) is dissolved in dimethylformamide (400 ml) and potassium phthalimide (370 g, 2.0 mol) is added with stirring. After stirring for 16 h at 110°C, the dimethylformamide is evaporated under reduced pressure. The residue is suspended in toluene (1.5 l) at 40-50°C and the potassium chloride is filtered off. The product is crystallized by cooling (-10°C). A second fraction is obtained from the residue by concentrating it 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) To a solution of 8-(N-phthalimidoyl)-3,6-dioxa-octanol (3) (8.5 g, 36 mmol) and trifluoromethane sulfuric anhydride (10.2 g, 36 mmol) in dichloromethane was added dropwise, with stirring at a temperature of about -5°C, pyridine (2.8 ml, 35 mmol) in dichloromethane (30 ml). After about 30 min, the organic phase was washed with 0.5 M hydrochloric acid and water. After drying with sodium sulfate and filtration, isopropyl-8-hydroxy-3,6-dioxa-octanoate (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 for 20 hours. The reaction mixture was filtered and the filtrate was evaporated. The residue was partitioned between 1,1,1-trichloroethane and water. Evaporation of the organic phase gave an oil (13 g).

^X 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-pentaoxaheptadecanoate (4) (13 g) was dissolved in tetrahydrofuran (50 ml) and hydrochloric acid (conc., 50 ml). The reaction was carried out for 16 hours at room temperature, then the solution was diluted with water (200 ml), and the tetrahydrofuran was evaporated under reduced pressure. The aqueous phase was washed with toluene (1 time) and extracted with dichloromethane (2 times). It was dried over sodium sulfate and the organic phase was evaporated to give the product as 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 (p.p., 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) was dissolved in 150 ml of ethanol and 3 ml of hydrazine hydrate. The solution was stirred for 16 h at room temperature, then HCl (100 ml, 3 M) was added, and the solution was heated to reflux for 3 h. After cooling to room temperature and filtering, it was basified to pH 9 by adding NaOH, and the filtrate was evaporated to near dryness, then acidified with HCl to pH 4 and evaporated to dryness. The product was treated with isopropanol (100 ml) and acetyl chloride (2 ml) at room temperature overnight, and the solution was evaporated. The residue was dissolved in water and extracted with dichloromethane at a basic pH (7-11). Evaporation gave the product (3.3 g).

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

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

FORMULAS OF SYNTHESIZED AMINO-DEG-CARBOXYCIC 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 Preparation of 17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoic acid N-hydroxysuccinimide ester

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

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

II o

Isopropyl-17-amino-3,6,9,12,15-pentaoxa-heptadecanoate (see Experimental Section I) (1.1 g, 3.2 mmol) was dissolved in 3 ml of 1 M sodium hydroxide solution and left at room temperature for 30 minutes. 1.6 ml of 8 M hydrochloric acid was added and the mixture was evaporated to dryness. The residue was dissolved in dichloromethane and filtered, and after evaporation of the solvent, 545 mg of 17-amino-3,6,9,12,15-pentaoxa-heptadecanoic acid was obtained. 460 mg (1.39 mmol) of this compound was dissolved in 10 ml of borate buffer, pH 8.4. The solution was deaerated with nitrogen gas. A solution of 432 mg (1.52 mmol) of N-succinimidyl-2-iodoacetate in 5 ml of dioxane was added dropwise over 1 minute, maintaining pH at 8.4 by adding 5 M NaOH. The reaction mixture was stirred for 15 minutes under nitrogen. According to thin layer chromatography (eluent: CH ₂ Cl ₂ -MeOH, 50:35), the reaction was complete within a few minutes. After 15 minutes, it was acidified to pH ₃ , the solution was frozen and lyophilized. The reaction mixture was fractionated on a reversed-phase column PHP-RPC HP 30/26 (Farmacia, Biosystems AB) using a gradient of acetonitrile from 0 to 13% in the presence of 0.1% trifluoroacetic acid, followed by separation with 13% acetonitrile, 0.1% TFAR (trifluoroacetic acid). The fractions of the desired peak were combined and lyophilized 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 vol.

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

II

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

II

II

(B)

II

Hydroxysuccinimide (4.5 mg, 39 pmol) is weighed into the reaction vial. 17-iodoacetylamino-3,6,9,12, 15-pentaoxaheptadecanoic acid (A) (18.3 mg, 39 pmol) is dissolved in 0.55 ml of dry dioxane and poured into the vial. The vial is deaerated with nitrogen gas, then 0.8 mg (39 pmol) dicyclohexylcarbodiimide in 0.15 ml of dry dioxane is added dropwise. The vial is filled with nitrogen gas, stoppered and placed in the dark. The reaction mixture is stirred for 3.5 hours, the precipitate formed is removed by filtration. NMR analysis showed that the product B obtained in the filtrate constitutes 89%.

Example Preparation of (17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecamoylamino)-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

The 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 is added to the vial. 146 μΐ of a dioxane solution containing 3.6 mg (6.4 pmol) of 17-iodoacetylamino-3, 6, 9,12,15-pentaoxaheptadecanoic acid N-hydroxysuccinimide ester (B) is added to the buffer solution and the reaction is carried out to completion with stirring at room temperature for 25 minutes. The reaction vial is covered with foil to prevent light from entering. Excess reagent B is removed by fractionation on a Sephadex G 25 K 26/40 column using 0.1 phosphate buffer, pH 7.5, containing 0.9% NaCl as eluent. Fractions containing the desired product C are collected and pooled. The solution (22 ml) is concentrated on an Amicon grid through a YM 30 filter to a volume of 8 ml. The concentration and degree of substitution are determined by amino acid analysis. These are 4.7 mg/ml and 18 spacers/monAK C215, respectively.

B. Monoclonal antibody monAK C242

According to the methodology described in Example 2A, the reaction is carried out with an immunoglobulin Ig GI class monoclonal antibody (monAK C242) with 15-, 20- and 22-fold molar excesses of 17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoic acid N-hydroxysuccinimide ester (B), yielding nona-, dodeca- and tetradeca(17-iodoacetylaminoLT 3909 B

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

C. Monoclonal antibody monAK C

According to the methodology described in Example 2A, a monoclonal antibody of the immunoglobulin class Ig G 2a (monAK C) is exposed to 17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoic acid N-hydroxysuccinimide ester 14- and

in 18-fold molar excess, yielding tetra- and hepta (17-iodoacetylamino-3, 6, 9,12,15-pentaoxaheptadecanoylamino)-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 obtained from Toksin technology inc.) (2 mg, 72 nmol) was dissolved in 722 μΐ water and placed in a 15 ml isopropyl tube. 100 μΐ 0.1 M borate buffer, pH 5.6 was added, followed by 2160 nmol Eu ³⁺ complex with chelates (Farmacia Vallac Oi) in 178 μΐ milli-Q. The reaction was allowed to complete overnight at room temperature. Excess reagents were removed by fractionation of the reaction mixture on a Sephadex G 25 PD 10 column (Farmacia Biosystems AB) using 0.1 M phosphate buffer, pH 8.0 as eluent. Fractions containing the desired product D were pooled. The solution (3 ml) of conLT 3909 B was centrifuged on an Amicon grid 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 determined by comparison with a standard EuC1 ₃ solution was 0.8 Eu ³⁺ /SEA.

Obtaining SEA (E)

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

3+ (Eu -SEA-(NHCCH ₂ CH ₂ SS

II

Oh

) 3-4 (E)

(1.24 mg, 44.5 nmol) was placed in a 15 ml isopropyl alcohol tube and 0.75 ml 0.1 M phosphate buffer pH 8.0 was added to the tube. 35 μΐ (180 nmol) was added to the tube.

Eu ³⁺ -SEA buffer, ) 1.6 mg

N-succinimidyl-3-(2-pyridylthio)-propionate in one milliliter of ethanol solution, and the reaction mixture is stirred for 30 min at room temperature. The resulting product E is not isolated before it is reduced to product F _x .

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

II

To the above reaction mixture, 20 μΐ of 0.2 M Eu ³⁺ citrate solution and 50 μΐ of 2 M acetic acid were added to adjust the pH to 5.0. Then, 3.1 mg of dithiotrentol (Merck) in 0.1 ml of 0.9% sodium chloride solution was added and the reaction mixture was stirred for 20 min at room temperature. The total volume was then adjusted to 1 ml by adding 50 μΐ of 0.9% sodium chloride solution. The reaction solution was placed on a column (1 ml) filled with Sephadex G25 NAP 10 (Farmacia Biosystems AB) and the desired product F _x was eluted 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 isopropyl tube and immediately used for the synthesis of product G to avoid oxidation of the disulfide product.

B2. Production of 2-mercaptopropionylaminostaphylococcal enterotoxin A (SEA) (F ₂ )

SEA(NHCCH ₂ CH ₂ SH) _lr g (F ₂ )

II o

Natural SEA (lyophilized product, Toksin Technology Ine.), or recombinantly produced SEA (rSEA) is treated with a twofold molar excess of N-succinimidyl-3-(2-pyridylthio)propionate according to the procedure described in Example 3BI.

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

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⁺ -labeled SEA (F) , described in Example 3B, was added octadeca (17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)monAK C215 (C) (4 mg) in 1.2 ml of 0.1 M phosphate buffer, 0.9% sodium chloride, pH 7.5. The reaction was completed overnight at room temperature. Unreacted iodoalkyl groups were blocked by adding 5 μΐ (1.2 μιηοΐ) of a mercaptoethanol solution obtained by dissolving 20 μΐ in one ml of water. The reaction solution was left for 4 hours at room temperature, after which it was filtered. The filtrate is 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 pooled and analyzed. The protein content is 0.22 mg/ml according to amino acid analysis. The degree of substitution, determined by the Eu ³⁺ content, 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-pentaoxaheptadecanoylamino)monAK C215 (C) is treated with 2-mercaptopropionylamino-rSEA (F ₂ ) in a 1.8-fold molar excess according to the procedure described in Example 4A. The composition of the conjugate is analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (DSN-PAGE) using a PhastGel gradient of 4-15 and the bands scanned on a Phast

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

In another experiment, a 2.7-fold molar excess of _F2 is used, yielding a conjugate with the following composition: 15% monAK C215 with three SEAs, 25% with two SEAs, 34% with one SEA, and 26% unmodified monAK C215.

C. Conjugates of rSEA with monAK C242

According to the procedure described in Example 4A, tetradeca (17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)monAK C242 (C) is treated with a 3.2-fold molar excess of 2-mercaptopropionylamino-rSEA (F ₂ ). The composition of the conjugate is analyzed as described in Example 4B, giving the following results: 4% monAK C242 with four SEAs, 12% with three SEAs, 28% with two SEAs, 36% with one SEA and 20% unmodified monAK C242.

The same reaction is performed, but before fractionation on the column, the product is treated with 0.2 M hydroxylamine for 4 hours to remove unstable bonds between monAK C242 and the spacer and between SEA and the mercaptopropionyl group. The resulting conjugate has the following composition: 1% monAK C242 with four SEAs, 12% with three SEAs, 27% with two SEAs, 36% with one SEA and 24% unmodified monAK.

C2. According to the procedure described in Example 4A, dodeca (17iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)monAK C242 is treated with a three-fold molar excess 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% unmodified monAK C242.

C3. According to the procedure described in Example 4A, nona(17iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)monAK C242 (C) is treated with a three-fold molar excess 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% unmodified monAK C242.

D. rSEA conjugates with monAK C

DI. According to the procedure described in Example 4A, hepta(17iodo-acetylamino-3,6,9,12,15-pentaoxa-heptadecanoylamino)monAK is treated with a 5.4-fold molar excess of 2-mercaptopropionylamino-rSEA (F ₂ ). The composition of the conjugate is analyzed as described in Example 4B, giving the following results: 15% with four SEAs, 24% with three SEAs, 29% with two SEAs, 19% with one SEA, 3% unmodified monAK C and 10% dimer.

The same reaction is carried out in the presence of 0.2 M hydroxylamine to remove the unstable bonds between monAK C and the spacer and between 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% unmodified monAK C and 17% dimer.

D2. According to the procedure described in Example 4B, tetra (17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)monAK C is treated with a 5.7-fold molar excess of 2-mercaptopropionylamino-rSEA (F ₂ ). The conjugate has the following composition: 8% monAK C with four SEAs, 18% with three

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

Example: Preparation of /17-(3-mercaptopropionylamino)-3,6,9,12,15pentaoxaheptadecanoylamino/-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 is added to a solution of 3.65 mg (128 nmol) of rSEA in one 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. 100 μΐ is taken for analysis of the degree of substitution. The remainder is stored frozen until its reduction to the product.

The degree of conversion is determined by removing salts from 100 μΐ of the 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(1978)723-737/. 2.7 spacer is coupled to rSEA.

B. Preparation of /17-(3-mercaptopropionylamino)-3,6,9,12,15-pentaoxaheptadecanoylamino/-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. Reduction occurred within 30 min. The reaction mixture was applied to a column of Sephadex G 25 NAP 10 (Farmacia, Biosystems AB) and eluted with 1.5 ml of 0.1 M phosphate buffer, pH 7.5, containing 0.9% sodium chloride, and then used immediately for the synthesis of product J in Example II.

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-pentaoxaheptadecanoylamino)monAK C242 (B) (4.2 mg, 27 mmol, 0.1 mL 0.1 M phosphate buffer, pH 7.5, containing 0.9% sodium chloride) was reacted with /17-(3-mercaptopropionylamino)-3,6,9,12,15pentaoxaheptadecanoylamino/-rSEA (I) (1.17 mg, 42 nmol, 0.1 mL 0.1 M phosphate buffer, pH 7.5, containing 0.9% sodium chloride) for 43 h in the dark under nitrogen.

1 ml of the buffer specified above). Then add

1.14 μηεοΐ mercaptoethanol. After another 1 h, the reaction mixture is separated on a Superdex 200 HR 16/55 column. The product is eluted 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% unmodified monAK C242.

EXPERIMENTAL SECTION II

Effects of superantigen-antibody conjugates on cells

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

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

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

To determine the cytotoxicity induced by the conjugated SEAC215 monAK and unconjugated SEA and C215 monAK against colon cancer cells that do not express Class II PASKA or that express low and undetectable levels of Class II PASKA, we used various human T-cell lines treated with SEA as effector cells and colonic epithelial cells and Raji cells expressing Class II PASKA (Class II ⁺ PASKA) as target cells. The colon cancer cell lines Colo 205, SW 620 and WiDr do not express Class II PASKA, as determined by staining for monAK against HLA-DR, HLA-DP and HLA-DQ and fluorescence-activated cell analysis. T-cell lines treated with SEA were obtained from peripheral blood by weekly restimulation with mitomycin C II ⁺ PASKA lymphoma BSM cells precoated with SEA in the presence of recombinant IL-2 (20 units/ml). These T-cell lines showed high cytotoxicity against Raji or BSM cells coated with SEA, but not against uncoated cells or cells coated with staphylococcal enterotoxin B (SEB). This inducible killing of SEA depends on the interaction of SEA on the target cell with Class II PASKA, as determined using blocking antibodies to HLA-DR, mutant Raji cells not expressing Class II PASKA (Class II~ PASKA) and L-cells transfected with HLA-DR /Doglsten et al., Immunology 71(1990)96-100/. These T-cell lines were shown to be activated by the C215-SEA conjugate to kill C215 ⁺ Class II PASKA colon cancer cells. In contrast, unconjugated SEA and C215 monAK were unable to elicit more than marginal T-cell killing of C215 ⁺ Class II PASKA colon cancer cells. The cell-induced cytotoxicity mediated by the staphylococcal enterotoxin-antibody conjugate is dependent on the binding of the SEA-C215 monAK conjugate to C215 ⁺ cancer cells. The specificity of this binding was demonstrated by the fact that excess unconjugated monAK C215, but not C242 and W8/32 monAK, inhibited the lysis of colon cancer cells. CD4 ⁺ and CD8 ⁺ T-cells kill C215 ⁺ colon cancer cells treated with SEA-C215 but do not lyse cells treated with SEA. Apparently, the interaction of T-cells with the SEA-C215 monAK conjugate bound to the tumor cell Class II PASKA involves interaction with specific sequences of the beta-chain of the variable region of the T-cell receptor in a manner analogous to the previously demonstrated SEA-mediated killing of Class II ⁺ PASKA cells. This was demonstrated by the interaction of a SEA-specific, but not an autologous SEB-specific T-cell line with the C215-SEA conjugate. The conjugates C242 monAK and Thy-1.2 monAK show activity analogous to that of the C215 monAK conjugate.

Chromium labeling and incubation of target cells with SEA

0.75 x 10 ⁶ target cells and 150 µCi ⁵¹ Cr (American Corp., Arlington Heights, England) were incubated in a volume of 100 μΐ for 45 minutes. The cells were maintained in complete medium containing RPMI-1640 medium (Gibko, Paisley, 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% fetal calf serum (VES, Gibko, Paisley, UK). After incubation, the cells were washed once in complete medium without VES, incubated at 37°C for 60 minutes, washed and resuspended in complete medium containing 10% VES. 5x10 ³ target cells were added to each well in 96-well U-bottom microliter plates (Costar, Cambridge, USA).

Cytotoxicity test

Effector cells/target cells were added to the wells in varying ratios. The final volume in each well was 200 μΐ. Each test was repeated three times. The plates were incubated at 37°C for 4 h, after which the released chromium was collected. The amount of ⁵¹ Cr was determined using a gamma counter (Cobra, Auto-gamma, Paccard). The percentage of cytotoxicity was determined according to the formula:

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

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

Results

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

Human T-cell lines lyse SW620, Colo 205 and WiDr Class ^II PASKA cells in the presence of SEA-C215 monAK conjugate, but not in the presence of unconjugated SEA and C215 monAK (Fig. I). Colon cancer cell lysis occurred at 10-100 ng/ml SEA-C215 monAK conjugate concentrations. High lysis rates at various effector/target ratios were observed for SEA-215 monAK relative to SW620 (Fig. I). In contrast, unconjugated SEA or C215 monAK did not induce cytotoxicity against SW620 cells at all effector-target ratios tested. This suggests that the ability to lyse Colo 205 Class II PASKA cells is conjugate-related and not elicited by unconjugated SEA and C215 monAK. SEA and the conjugate SEA-C215 monAK, but not C215 monAK, induce T-cell killing of Class II ⁺ PASKA Raji cells and interferon-treated Class II ⁺ PASKA Colo 205 cells.

To demonstrate that SEA-C215 monAK conjugate-induced lysis is driven by specific binding of the conjugate to the C215 monAK molecule on the target cell, an inhibition assay was performed with excess unconjugated C215 monAK and C242 monAK, which bind to a foreign antigen on colon cancer cells (with respect to C215 monAK binding). Addition of monAK C215 strongly inhibited cytotoxicity, whereas C242 monAK showed no effect (Fig. 2). Similarly, SEA-C242 monAK conjugate lysis was specifically inhibited by excess unconjugated C242 monAK, but not C215 monAK.

The ability of the SEA-C215 monAK conjugate to induce T-cell-dependent lysis of Class II PASKA cells from the SW620 colon cancer line was observed in both CD4 ⁺ and CD8 ⁺ T-cell populations. SEA does not activate either of these T-cell subclasses and does not induce cell death of SW620 cells, but does lyse Class II ⁺ PASKA Raji cells (Table 2).

The SEA-C215 monAK conjugate induces lysis of SW620 and Raji cells by T-cell lines exposed to SEA, but not by T-cell lines exposed to SEB (Fig. 3). The selectivity of SEA- and SEB-lines is indicated by their selective response to SEA and SEB, respectively, when exposed to Raji cells (Fig. 4). This indicates that the SEA-C215 monAK conjugate retains specificity for the beta-chain of the variable region of the T-cell receptor, analogous to the specificity of unconjugated SEA.

Figure description fig. SEA-C215 monAK conjugate directs CTL against colon cancer Class II PASKA cells. The upper left graph shows the effect of SEA-sensitive CTL on SW620 cells at various effector/target ratios in the absence (-) or presence of SEA-C215 monAK conjugate, SEA, C215 and a mixture of C215 and SEA (C215+SEA) · at a concentration of 1 µg/ml of each additive. The other graphs show the ability of SEA-C215 monAK conjugate and SEA to direct SEA-sensitive CTL against colon cancer lines SW620, Colo205 and WiDr, Class II C215 ⁺ PASKA cells treated with interferon Colo 205 Class II ⁺ PASKA C215 ⁺ cells and Raji II ⁺ PASKA C215' cells. The effector/target ratio of EfekLT 3909 B is 30:1. Addition of unconjugated C215 monAK at some concentrations does not direct CTL against these cell lines. Fluorescence-activated cell assays using monAK against HLA-DR, -DP, -DQ on SW620, Colo 205, and WiDr cells did not detect any surface expression of Class II PASKA, whereas abundant expression of HLA-DR-, -DP, and -DQ was observed on Raji cells, and HLA-DR and -DP on interferon-treated Colo 205 cells. Colo 205 cells were treated with 1000 units/ml recombinant gamma-interferon for 48 hours before use in the CTL assay.

Fig. Dependence of CTL targeting against colon cancer cells induced by SEA-C215 monAK and SEA-C242 monAK conjugates on antigenic selectivity of monAK. Lysis of Colo 205 cells sensitized by 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 target cells 10 min before the conjugates.

Fig. Lysis of colon cancer cells coated with SEA-C215 monAK conjugate induces CTLs responsive to SEA, but not to SEB. Autologous SEA- and SEB-selective T-cell lines were used, with an effector/target ratio of 10:1, against the target cells SW620 and Raji, in the absence (control) and in the presence of SEA-C215 monAK conjugate, and, respectively, 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 LT 3909 B cell targets (Colo 205 tumor cells and EL-4 tumor cells, respectively).

Table I

Binding of SEA-C215 monAK conjugate to colon cancer C215 ⁺ cells and to Raji Class II ⁺ PASKA cells

Reagent 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 plus a control (bovine serum albumin) for 30 min on ice, washed and processed as previously described. The staining of C215 monAK and C242 monAK bound to Colo205 was determined using FITC-labeled rabbit I g

against mouse antigens. Staining of SEA bound to Raji cells was determined using rabbit antisera against SEA followed by FITC-labeled porcine Ig against rabbit antigens. Staining of SEA-C215 monAK conjugate bound to Colo205 and Raji cells was determined using procedures previously described for C215 monAK and SEA. Fluorescence-activated assay was performed on a Becton and Diccinson instrument. Staining for the second and third steps was used to determine background.

Table

Characterizing the C215-SEA conjugate in colon cancer cell lysis, CD4 ⁺ CTL and CD8 ⁺ CTL effects

Effector* Target % Cytotoxicity

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

with 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.