PT98395A - PROCESS FOR THE PREPARATION OF A CONJUGATED SUSBTANCIA AND A REAGENT FOR ITS SYNTHESIS - Google Patents

Description

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Description relating to the patent of invention of KABI-PHARMACIA AB, industrial and commercial sue, established in S-751 82 Uppsala, Sweden, (inventors: Hubert Agback, Leif Shrgren, Martin Haraldsson and Eva Akerblom, resident in Sweden); for the preparation of a conjugated substance and of a reagent for its synthesis "

The term conjugate is a common designation for substances prepared by covalently bonding different or identical organic compounds in combination so that the properties of the compounds pass into the conjugate. The conjugate substance, reagent and novel polyesters of the present invention have as common structural feature ethoxy groups linked in sequence, i.e. with the structure - (OCH2 CH2) n -, wherein n is an integer equal to or greater than 1.

The conjugate of the invention obeys the general formula A-B-A 'wherein A and A' comprise residues of organic compounds F and F ', respectively. At least one of the compounds is a polymer, its polymeric structure also being present in the residue. B represents an organic bridge linking A and A 'together and comprising the structure -OCH 2 CH 2) n-. A and A 'may comprise several identical residues originating from the compounds to be conjugated (F and F')

For the preparation of the conjugates, hetero- or homobifunctional reagents of the type Z-B'-Z8 are often used, wherein Z and Z " are reactive functional groups which are inert with respect to the reaction with one another (in pairs they are nucleophilic or electrophilic) and B 'is an inert bridge. By inert term is meant that the bridge is stable and has no groups which are capable of neutralizing the reactivity of Z

and / or Z1.

To prepare those sets wherein one of the compounds (P or F ') is a polymer, it is difficult to conjugate uniform conjugate substances. The substances obtained will often consist of a mixture of more or less similar conjugated molecules. The common varieties of the individual molecules of a given conjugate are: different numbers of A attached to one and the same A 'and vice versa, different binding positions, B exist as inter and / or intramolecular crosslinks, etc. The length and the bridge structure -B- are very important so as to impart properties of the compounds to the conjugate. For compounds that are frankly soluble in water, a hydrophobic bridge may result in conjugates that are insoluble and / or frankly active. If the bridge is too short and the biologically active compounds are conjugated, the activity will often be impaired. In the extreme case a wrong or too short bridge may completely impair the activity. The structure - (OCH 2 CH 2) n 'is present in the polyethylene glycol (PEG). The molecular weights of PEGs are given as mean values, that is, PEG is usually a mixture of several molecules in which the integer n varies. It appears that PEGs have unique amphiphilic properties that can be used when they are attached to biopolymers. The structure - (OCH 2 CH 2) n - is also present in some amino-PEG-carboxylic acids. For example the compounds NH 2 CH 2 CH 2 (OCH 2 CH 2) n O (CH 2) 2 COOH with η = 1 to 10 (Houghton and Southby, Synth. Commun. 19 (18 (1989) 3199-3209) were suggested as starting materials for the preparation of polyether amides (OCH 2 CH 2) 2 OCH 2 COOH as a feedstock for macrocyclic-based ethers (Julien et al., Tetrahedron Letters 29 (1988)) was used as the starting material for the m- 3803-06.

Amino-PEG-carboxylic acids of the formula NH2CH2CH2 (OCH2CH2) nOCH22COOH (n = 1-10) have recently been reported (EP-A-410,280) (20.1.91). Some of their reactive derivatives and conjugates of proteins prepared therefrom.

The conjugates having the - (OCH 9 CH) - structure on the bridge were synthesized prior to this prioritization date z 2 n 2 Î ©

I

in. Slama and Rando attached cholesterol to a monosaccharide via hydrophilic and hydrophobic bridges (biochemistry 19 (1980) 4595-4600 and Carbohydrate Research 88 (1981) 213-221; a heterobifunctional α-coplating reagent). For their objectives the best conjugates had the bridge:

(NHCH-CHO (OCH-CEL) OCHO-C0-), where η = 1 and n '= 1 or 2. Slama and Rando did not change the structure -OCH2CH2 - increasing the integer n. For unknown reasons they have preferred to duplicate the complete structure NHCH 2 C (OCH 2 CH 2) n OCH 2 CO- making n '= 2. The enzyme-antibody-conjugate conjugates containing the - (OCH 2 C 3) -alkyl wherein n may be integers are known ( EP-A-254,172 and FR-A-2,626,373). In the latter application commercially available polyethylene glycol (PEG) was used as one of the raw materials for the coupling reagent. This made it difficult to obtain conjugate substances uniform in relation to the length of the bridge. Oxaalkylene structures, for example -OCE3 CE3 -, have been suggested in heterobifunctional reagents for the selective coupling to aldehyde groups and thiol groups respectively (EP-A-240 200 and WO-A-89/12624).

In addition to the abovementioned publications, the Patent and Trademark Office of Sweden quoted in " International Type Search Report "; EP-A2-345,789, WO-Al-88/03412, Biochem. Biophys. Comm. 164 (1989-11): 3, as publications of particular interest in relation to priority claims.

The present invention provides conjugated substances in which the spectrum of individual molecules have greater uniformity and good availability, particularly in aqueous hydrophilic media. This object is achieved by insertion of an amphiphilic bridge structure of uniform and defined length. The conjugates of the invention are particularly adapted for in vivo and in vitro diagnostic uses as well as for therapeutic uses (drugs). The conjugate of the invention is characterized in that the bridge -B- comprises the structure -SRRCONHCH2CH2 (OCH2CH2) n O (CH2) COY- (I) m

The free valences in formula (I) bind A and A1, respectively. This takes place either directly or through

from other divalent inert structures which are comprised within the bridge -B-. The length of -B- is generally smaller than 180 atoms, for example less than 100 atoms but longer than 13, preferably longer than 16. n is an integer greater than 0, for example 1 to 20, and is such that n is uniform for bridges which bind identical positions together on individual molecules of the conjugate (conjugate). m is one or 2. From the synthetic point of view, n is preferably 10 or <10. In order for the conjugate to express the unique amphiphilic properties of PEG, the integer n must be greater than 2, preferably greater than 3 or greater than 4. Thus based on different combinations of criteria the ranges for integer n may be 1-20 , 1-10, 1-9, 2-20, 2-10, 2-9, 3-20, 3-10, 3-9, 4-20, 4-10, 4-9, 5-20, 5 -10, and 5-9.

Sr is directly attached to a saturated carbon atom at each of its valences, r = 1 or 2, i.e. represents a disulfide group or a thioether group. If Sr is directly bonded to A, one of the sulfur atoms may be from F. Y is -NH-, -NHNH- or -NHN = CH- which at its left-hand ends is attached to the CO group shown at the right-hand terminal in formula I and at its right ends to a saturated carbon atom or to a carbonyl group (only when Y is equal to -NHNH-). The atoms which bind to the right-hand end of Y are not shown in formula I. R is preferably alkylene (having 1 to 4 carbon atoms, often 1 or 2 carbon atoms), which is possibly substituted by one or more to 3, in the preferred case less than 2) hydroxy (OH) groups. At most one oxygen atom is attached to one and the same carbon at R. With regard to the bio availability of the hydrophilic media R may in many cases be equivalently substituted by a higher alkylene selected from the group comprising linear, branched and cyclic alkylene with condition of the higher alkylene having hydrophilic substituents which compensate for increased hydrophobicity.

A further condition is that for r = 1, B can have 1-aza-2,5-dioxo-cyclopentan-1,3-diyl group which in its 4-amino-

position 3 is attached to the sulfur atom and in its 1-position to R, or the 1,4-diyl analogue group which in its 4-position is attached to the sulfur atom.

According to a preferred embodiment the bridge -B- must not contain any aromatic ring. The polymer may be a synthetic biopolymer or a biopolymer. The term polymer represents a compound wherein 3 or more, preferably more than 10, repeating monomer units bind sequentially to one another. The term polymer also encompasses inorganic polymers, such as glass and other polyaryl silicates.

Examples of synthetic polymers are poly (hydroxyalkyl (meth) acrylate), poly ((meth) acrylamide), polyvinyl alcohol, etc. and forms derived from these polymers. The term biopolymer means a polymer in which the basic backbone is of biological origin. The term also encompasses biopolymers which have been derived. Biopolymers typically have a nucleic acid or polysaccharide structure and / or polypeptides. Proteins including polypeptides (for example albumin and immunoglobulins) and polysaccharides, for example soluble and insoluble (dextran, starch, hepatic cellulose, etc.) are important.

Polymers of immediate interest (for example those having a polypeptide and / or polysaccharide structure) generally have functional groups such as hydroxy (-OH), carboxy (-COOH), amino (primary or secondary) and / or mercapto (-SH). When a given polymer lacks the appropriate functional group, a chemical modification can be made as a rule for the group to be inserted.

Compounds F and F &quot; are chosen according to the properties that they must confer to the conjugate. The compounds may thus be: vehicle-type compounds, bioaffinity compounds, analytically detectable compounds, compounds which are insoluble or soluble in aqueous media, therapeutically active compounds (drugs), enzymes, immunological stimulators, toxins, etc. Particularly important toxins are the peptide cytotoxins which exert their effect intracellularly and thus comprise a peptide segment responsible for cell wall penetration and another segment for intracellular toxicity (toxin, diphtheria, ricin, exotoxin- Pseudomonas, etc. Another type of peptide toxins are those which exert their effect through immunological stimulation (for example by activation of cytotoxic T cells by simultaneous binding to T cells and cells containing antigen- and MHC class II). An example of this latter type is the whole Staphylococcus toxin A.

Bioaffinity compounds, i.e. compounds which exert a biospecific activity, are particularly interesting, since they may be used as part of a conjugate as a leader because of their bioaffinity parts. Examples are the anti-bodies and their active anti-body fragments and corresponding antigen / hapten, Fc / Fc fragments of immunoglobulins (Ig) and the corresponding receptors (for example IgG binds to Protein A and G), avidin / strepavidine and biotin, lectins and the corresponding carbohydrate structures, enzymes and the respective substrates, coenzymes, cofactor, and co-substrate, etc. Antibodies of various classes (in particular IgG) and subclasses and various specificities may constitute a part of the conjugate according to the invention. The specificities of the antibodies (and their fragments) may for example be tumor cells and / or antigens / tumor haptens, hormones, hormone receptors, etc.

Particularly interesting insoluble polymers are those which are used as absorbents in connection with creatography, immunoassays, blood fractionation, etc.

Within the field of in vitro and in vivo diagnostics, the conjugates between an ana lytically detectable compound and a (targeting) compound which reveals a biospecific affinity to detect and localize the counterpart of the router are of great importance. Analytically detectable compounds may be radioactive, enzymatically active (including enzyme sub-tracts, co-substrates, co-enzymes, etc.), fluorogenic, chemiluminogenic, biochemiluminogenic particles (e.g. latex), etc. 6

For therapeutic purposes the bioactivity compounds can be conjugated with medicaments and with other substances exerting a therapeutic effect.

For the synthesis of the conjugate of the invention a new heterobifunctional reagent of the Ζ-Β '-ΖΖ type was developed. This reagent complies with the following general formula (II): ## STR3 ## in which m and m have the same meaning as above of formula (I) ie the reagent substance is not a mixture of compounds having different values of m and n values different R is an alkylene group having the same meaning as above Z 2 is an electrophilic group reactive with HS thiol (-SH) or protected thiol (for example AcS-), provided that a thiol group and a hydroxy group are not attached to one and the same carbon atom in R. Examples of HS-reactive electrophilic groups are: (i) halogen which is attached to a (ii) is a saturated carbon atom, preferably in the form of an alpha-haloalkylcarbonyl group (for example Z 2 -C 2 CO-, wherein Z 2 is preferably bromo or iodo), (ii) activated thiol, preferably called a reactive disulfide (-SSR que which is attached to a saturated carbon atom; (iii) 3,5-dioxo-1-aza-cyclopent With respect to reactivity against thiol groups, it is possible to use in an equivalent manner other double carbon-carbon linkages which form pi-electron systems conjugated to a carbonyl group, a nito group or a cyano group instead of 3,5-dioxo-1-aza-cyclopent-3-en-1-yl;

Reactive disulfides so well known in the context of synthetic chemistry (See EP-A-063,109, 064,040, and 128,885). R ê is defined by the chemical reaction between -S-S-R e and HS-releasing R -SSH which is thermodynamically stabilized to be withdrawn from other thiol-disulfide exchange reactions. Many thiol compounds (R 2, SH) correspond to this criterion by spontaneous tau-tomerization to a thione form in aqueous solutions, i.e. the thione form is more stable than the corresponding thiol form. One of the preconditions for this type of tautomerization may be that the sulfur atom of group 7

thiol is attached to a carbon atom which forms a part of an aromatic ring which (a) is heterocyclic having the sulfur atom of thiol located at a distance from an even number of atom of a heteroatom in the ring, or (b) is not heterocyclic and is substituted with electron-absorbing groups.

Examples of 5-nitro-2-pyridyl, 5-carbo xy-2-pyridyl, 2-pyridyl, 4-pyridyl, 2-benzothiazolyl, 4-nitro-3-carboxyphenyl and the N-oxides of the above-mentioned pyridyl groups.

1 is carboxy-activated, i.e. an electrophilic group. Examples are the carboxylic acid halides (-COCl, -COBr, and -COI), mixed carboxylic acid anhydrides (-COOOR3), recrystalline esters, such as N-succinimidyloxycarbonyl, -C (= NH) -OR2, 4- nitrophenylcarboxylate (-C0-C6H4NO2) etc. R 2 and R 2 may be lower (1-6C) alkyl and R 2 is also benzyl or (2-3C) alkylene with one of its valences replacing H in NH (yielding cyclic structures such as oxazolin-2-yl which may may be substituted with lower (1-6C) alkyl or benzyl in its 3- and / or 4-position. The Z 2 'terminus may be reacted selectively with a compound F' having a nucleophile group selected from: (i) -NHR 3 ', such as in primary and secondary amines

(R6 is selected from hydrogen and lower alkyl (1-6C) and in hydrazine / hydrazide, i.e. NH2 NH2 and compounds in which NH2 NH- and NH2 NH- CO- are attached to an aliphatic, preferably saturated, carbon atom. (II) -OH, such as in an alcohol. The chemical reaction at the Z 2 'terminus of the reagent (II) with a compound F' means that F 'becomes covalently bound to the formula (II) through an amide group or a (-CONHNH- and -CONHN * C-, respectively for groups according to (i) above and through an ester group for groups according to (ii) above. Where appropriate, Z- can be converted (reduced) to a thiol group in a thiol-disulfide exchange reaction In the latter case F 'becomes thio-side The Z-terminus may be reacted selectively with a compound F having a thiol group (SH ) or a reactive electrophile group with HS If F has a thiol group the reaction can take place directly. is attached to R in the formula (II) via the thioether (-S-) or disulfide (-S-S-) group. The use of the reagent (II) is carried out in known manner. The reaction system is chosen such that the side reactions of Z 2 and Z 2 'are avoided. When F and / or F1 are biopolymers it is preferable to carry out the reaction in an aqueous medium only, and in order to achieve selectivity, the pH value should be 8 to 9.5 for the reaction in Z1 ' The apotric systems are often inert against Z2 which means that in general they are preferred. The result obtained will be a conjugate in which F and F * are connected together through a bridge obeying formula I.

Extensive bridges may be introduced by reacting the reagent of formula (II) at either its Z 1 or Z 2 terminus with suitable bifunctional compounds. For example, if Z 1 is reacted with an alkylene diamine, alkylene dihydrazine, alkylene dihydrazide, etc. and only one of its H 2 N- groups is consumed, the remaining free NH 2 groups may be used for reaction with other compounds, for example F or F '(with carboxy, optionally after activation). The elongation of the bridge may also be achieved by reacting the F and / or F 'compounds with suitable bifunctional reagents of the Α-Β'-Ζ tipo type (see page 1) before linking them together by the use of the reagent of the invention. B 'may be chosen from the same R group as above. If each of the compounds F and F 'are reacted initially with the same reagent Z-B1-Z1 in the group Z' and then bonded together through the group Z thus introduced the group B 'appears twice in the conjugate the tip).

Known techniques encompass a large number of bifunctional reagents which are useful for elongation of the chain, either starting from a reagent of formula (II) or one of compounds F and F '. Specific examples are N-succinimidyl 4- (N-maleinimidyl) butyrate N-succinimidyl iodideacetate 9

N-succinimidyl S-acetyl-2-mercaptoacetate, and 3- (pyridyl-2-dithio) propiorate, N-succinimidyl, alkylene diamine, alkylene diacylhydrazide, etc. Alkylene has the same meaning as above.

A vicinal diol, for example in a carbohydrate structure, may be oxidized in a known manner for the synthesis of conjugates to two aldehyde groups and subsequently reacted with a -CONHNH 3 group to give the group CONHN = C -. Important F and F 'compounds having carbohydrate structures are the glycoproteins. The -CONHNE group may be present in a bifunctional group Ζ-Β '-Ζ', optionally following reaction with a polymer. The elongation of the chain starting from a reagent of the present invention may result in conjugates wherein -B- φ and: (1) -COR'-Sr-RCONHCH2 CH2 (OCH2 CH2) nO (CH2) m COY-; CO is attached to NH, (2) -COR'-S-RCONHCH-CH- (OCH_CH) 0 (CHO) CONHNR &quot; NHN =; R 2 is - N = is usually attached to a carbon atom hybridizing to the sp in A which means that the structure -N = is part of the structure -C = N-. This structure can be formed by the reaction between an aldehyde and a group NH2-NHCO-, (3) -OCO (CH2 CH2) n CH2 CH2 NCH2 NH2 R'-Sr- (cont.) -RC0NHCHo CHO (OCHO CHO) 0 (CHO) COY- ; (4) -CO (CHO) 0 (CHO CHOO) CHO CH_NHCO R '-S (cont'd) z m z z xi. z z r -RC0NHCH_CHo (OCH_CH3) 0 (CH3) CONHNR &quot; NHN =; 2 2 2 2 n 2 m - N = is connected in the above-mentioned manner in (2), Further variations are possible, and r have the same meaning as above.

By varying the reagents that are used, different chain stretches can be constructed starting from the Y-terminus. R and R 'are alkylenes chosen in the same way as R in formula (I). 10 f

The reactant of formula II may be prepared from compounds which comply with the formula III wherein n is 1 or 2. n is equal to an integer from 1 to 20 , such as 2 to 20. The synthesis of some compounds which obey the formula III with m = 1 and 2, and η = 1 to 10 was previously described (Julien et al., Tetrahedron Letters 29 (1988) 3803-06 (1989) 3199-3209, EP-A-410,280 (published 20.1.91), and Slama and Rando, Biochemistry 19 (1980) 4595-4600 and Carbohydrate Research (Houghton and Southby, Synth. 88 (1981) 213-221).

Reagents conforming to formula II may be synthesized by reacting a compound of formula (III) with a bifunctional reagent of the formula: wherein Z = Z, B Which is as defined above, and z '= activated carboxy. See above for appropriate reagents. After the reaction the -COOH function is converted to an activated carboxy group, for example Z1 is an activated ester such as N-succinimidyloxycarbonyl, 4-nitrophenyloxycarbonyl, 2,4-di- nitrophenyloxycarbonyl, etc.

Compounds of formula III (m = 1 and n = 2 to 20) and derivatives having the NH4- and / or -COOH groups are substituted with groups which can be easily transformed into N4- and -COOH groups, respectively , are novel and refer to a separate aspect of the present invention.

This aspect provides for a number of different bifunctional substances having pronounced amphiphilic properties, i.e. the property of being simultaneously soluble in water and in organic and lipid solvents.

This is a desirable property for bridging reagents that are used in the preparation of biomolecule-binding conjugates.

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

XCH "CH (OCHO CH -) OCHOYL (L)

n is an integer from 2 to 20, preferably 3 to 20. X is H 2 N - including its protonated form (+ H 3 N-) or H 2 N- substituted in the form of H 2 N-, preferably by hydrolysis or reduction.

Examples are unsubstituted amino (H2 N-); nitro; amido (carbamido), for example acylamido (formylamido, acetylamido ... ... hexanoylamido) including acylamido groups

which have electron-absorbing substituents on the acyl carbon alpha of the acyl radical and then particularly CF3 CONH2, CH3 COCH2 CONH2, etc.; phthalimidyl which is possibly substituted on a ring; carbamate (in particular R³CONONH- and (R³CONO) (R2 'OCOâ,,N), such as N- (t-butyloxycarbonyl) amino (Boc), N- (benzyloxycarbonyl) amino and di (N- (benzyloxycarbonyl- )) amino (Z and diS, respectively) which are possibly substituted on the ring; wherein the carbon atom which is attached to the nitrogen atom is alpha to an aromatic system such as N-monobenzylamino and dibenzylamino, N-tritylamino (triphenylmethylamino) etc. including analogous groups in which the methyl carbon atom (including the benzylic carbon atom) is replaced with a silicon (Si) atom such as N, N-di tert-butylsilyl) amino, and 4-oxo-1,3,5-triazin-1-yl including those which are substituted with lower alkyl at their 3- and / or 5- positions.

Above and hereinafter R &quot; and R'2 represent lower alkyl, in particular secondary and tertiary alkyl groups, and a methyl group which is substituted with 1 to 3 phenyl groups which may be substituted for the ring. The lower alkyl and lower acyl groups have 1 to 6 carbon atoms. Y is carboxy (-COOH including -COO) or a group which is carboxy-transmissible, preferably by hydrolysis or oxidation. The most important groups are ister groups wherein the carbonyl carbon atom or the corresponding atom in the ortho esters is attached to the methylene group at the right-hand end of formula (I). Examples are alkyl esters groups (-COOR '); (C1 -C4) alkyl groups and reactive ester groups such as N-succinimidyloxycarbonyl, 4-nitrophenyloxycarbonyl, imidatoalkyl groups (-C (= NH) OR4 ') including 5-membered cyclic oxazolin- 2-yl) with or without lower alkyl. its 4 and / or 5 positions has the above given significance for 12

Other Y groups are -CHO, CN, -CONR2 R2, wherein R1 and R2 are as defined above, and -CONH2 O The compound of the invention may be synthesized from known materials by combining processes which are known . Suitable synthetic routes are: A. Chain formation. B. Transformation of terminal functional groups. C. Transformation of a symmetrical polyether in a non-symmetrical ether.

Division of a bissimetric chain into two identical fragments.

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

Suitable starting materials having different terminal groups are the monocarboxylic omega-hydroxy acids in which the end groups are separated by a pure ethylene oxide polyolefin. These compounds having up to 5 repeating units have been described in the prior art. (Nakatsuji, Kawamura and Okahara, Synthesis (1981) p.42). A. Formation of the chain

The synthesis of Willi amson ethers can be applied to the synthesis of chains having the repeating unit -OCH 2 CH 2 - and identical or different terminal groups. The process means the alkylation of an alcohol (QZ * * + HOQ '-> Q-0-Q * or otherwise QOH + Ζ'Ό' -> Q-OQ '). Choosing (i) Q = X'CH 2 (OCH 2 CH 2) p - wherein X 1 is XCH 2 - or a group which in one or more phases is convertible to XCH 2 - and is stable under the conditions for the synthesis of Williamson ether, and (ii) Wherein Y 'is Y or a group which in one or more phases is Y-transformable and is stable under the conditions applicable. .per are 0 or positive integers such that p + r = n. The Williamson ether synthesis can also be applied to the synthesis of the bisimetric chain type which is described in part D.

In the case where Y 'is not Y, Y is preferably selected from groups which are stable to strong bases. For example, Y 'may be -CE 2 OR 3, -CH = CR 2 -R 2, wherein R 4 is selected from lower (1-6C) alkyl groups, preferably secondary or tertiary alkyl groups having up to 5 atoms carbon atoms and possibly substituted at their alpha positions with at most three phenyl groups which are possibly substituted, and R 1 and R 2 'are hydrogen, lower (1-6C) alkyl or phenyl which is possibly substituted.

In the above formulas Z "is a substitutable group with a moderate to high reactivity and may be halogen, alkanesulfonate, arenesulfonate, preferably toluene sulfonate (tosylate), and perfluoroalkane sulfonate, preferably trifluoromethanesulfonate etc. The synthesis of Wil liamson ether can be carried out in inert solvents and usually in the presence of bases often in strong bases, for example sodium hydride etc. By combining the appropriate length components, in principle, any chain may be developed by a sequence of stretching steps. The chain may also be elongated with an OCH2 CH2 moiety by the addition of Michael of HOQ or HOQ for the compound X * '- C (X' '' HCHgr where Q and Q ', respectively, have the same meaning as above. The groups X'1 and X'11, respectively, have to be chosen in such a way that -OQ and OQ 'are directed to the carbon atom of the compound X "-C (X' '1) = CH3 and so that the terminal groups in Q and Q", X '' may be hydrogen and X '' may be a group which is easily convertible to an amino or substituted amino group, for example nitro, alternatively X '' and X '', respectively , may be groups which upon transformation give rise to a group X '' - C (X '' ') CH 2 - which may be converted to the group HOOCCE 2 - or a derivative thereof. methylsulfinyl and X11 'is methylthio, and this requires the hydrolysis of the product obtained in the addition phase, said hydrolysis giving an aldehyde which can be further oxidized to the

carboxylic acid. The conditions for Michael addition are similar to Williamson's ether synthesis.

A third alternative for chain elongation is reduction of thione ester using Nickel of Ra-ney or corresponding reagents having a high affinity for sulfur. Suitable thione ethers have the formula: X'CH2 (OCH2CH2) gOCH2CS- (OCH2CH2) s -CH2Y 'or wherein X 'and Y' have the above significance and qs are 0 or positive integers such that q + s = nl, where n have the above significance.

Thione esters may be synthesized by rearrangement of the corresponding thiol esters by the action of an alkylating agent, for example methyl iodide, dimethyl sulfate or diazo methane. Thiol esters can be synthesized by reaction with a carboxylic acid halide with a thiol compound. Another alternative is the reaction of a carboxylic acid ester with a sulfur transfer reagent which is selective for the double bond oxygen when present in the carboxylic acid ester. Examples of such reagents are phosphorus pentasulfide or preferably Lawesson's reagent (2,4-bis (4-methoxyphenyl) -1,2,3,4-dithiaphosphetane-2,4-bis-sulfide). In this arrangement the functional end groups should not contain a carboxy oxygen. B. Transformation of terminal groups.

A compound which obeys the formula: wherein X 'is XCH2 - or is a group which in one or more steps is trans-formable in And Y 'is Y or a group which in one or more steps is Y-convertible to reaction conditions so as to give the desired transformation and the final product or where appropriate produce an intermediate product which can be used in a fa. if coupling. - 15 -

Examples of X '(in addition to XCH2 -) are hydroxymethyl, R4' OCH2 -, CRg 'Rg' = CH-, where R4 ', Rg1 and Rg1 have the above scheme. In the case of X 'being hydroxymethyl the conversion may take place, for example by reaction with sulfonyl halide in the presence of a base followed by the reaction with ammonia and, if necessary, other reagents to give the appropriately substituted amino group as for example phthalimido. In the case of X'- is R4 'OCH2' the group R1 'is removed, for example by acid catalyzed hydrogenolysis. The exposed hydroxymethyl group is then converted into a group containing a nitrogen atom as above. In the case where X 1 is CR 2 R 3 = = CH-, the = CH- group of the alkenylene group is oxidized to an aldehyde group, for example by ozonization. The aldehyde group is then converted to an aminomethyl group by reductive amination.

Examples of -Y '(in addition to -C (Y)) are hydroxymethyl, -CH 2 OR 4', -CH = CR, - R g ', wherein R 4', R 5 'and R 6' have the same meaning as above. In the case of Y 'being hydroxymethyl the reaction may take place by oxidation to a carboxy group (-COOH). In the case of Y 'is -CH 2 OR 3', R 4 'is removed, for example by acid catalyzed hydrogenolysis, and if R 4' contains at least one phenyl group in the alpha position, the removal may be effected by hydrogenolysis catalyzed with acids. The exposed hydroxymethyl group may then be transformed according to the above. Alternatively, the group is oxidized directly to the corresponding carboxy group (-COOH). In the case where -Y 'is -CH = CRg'Rg', -CH = of the alkenylene group can be oxidized to a carboxy group (-COOH), possibly via an aldehyde of the intermediate (for example by ozonization) which may be still oxidized. The oxidations may be carried out in the presence of a suitable tertiary alcohol, for example t-butanol, so that the corresponding ether is directly obtained. C. Transformation of a symmetrical polyether X, CH3 (OCH3 -CH3) OCH3 Xn to a non-symmetrical ether,

The symmetrical ethers may be synthesized from shorter ethylene polyoxide ethers by elongation of the chain, for example using the Wil ether synthesis. - 16 -

liamson. The transformation of one of the two identical groups X 1, wherein X 1 is X 1 or Y 'as hereinbefore referred to another group may be effected by partial reaction of the desired type and subsequent separation of the non-symmetrical product from the unreacted starting material and the double side reaction product. Examples of such transformations are: a) When X1 is a carboxy dicarboxylic acid is converted to the corresponding di (acyl halide) which in turn can be reacted with a deficient amount of ammonia followed by hydrolysis of the acyl halide groups ings. The formed carboxylic acid amide is separated from the reaction mixture and then its amide function is selectively reduced to an aminomethyl group. Alternatively, the separation is effected after the reduction step by means of a sequence of ion exchange separation phases. b) When it is aminomethyl the diamino group is reacted with a deficient amount of an acylating carboxylic acid derivative such as the carboxylic acid chloride or the corresponding anhydride. The monoacylated product is then separated from the starting material and the diacylated product, and then its aminomethyl group is oxidized, for example through the corresponding aldehyde, to a carboxy group, and its acyl group is removed by hydrolysis. A particularly useful variation of this process is the reaction with cyclic anhydride. In the latter case the ion exchange separation will be facilitated since the intermediate product is an amino acid, and the starting material is a diamine, and the side product is dicarboxylic acid. (c) If X is a moderate to high lipophilicity group, for example trityloxymethyl or allyloxymethyl, the partial removal or transformation of one of the groups will facilitate the net partitioning due to a significant difference between the partition coefficients for the product, for the starting material and for the by-product. Analogous criteria are valid if the appropriate lipophilic group has been introduced into a symmetrical polyether having hydrophilic groups X 1, for example oligoethylene glycol which is monosubstituted with trityl. 17

d) When it is hydroxymethyl the reaction can be carried out by a synthesis of Williamson ether, for example by the addition of an excess of a haloacetic acid. After the reaction the mixture is esterified with a suitable lower (1-6C) alcohol, for example isopropanol. The choice of the appropriate alcohol, the differences between the partition coefficients (water and organic solvents) of the components of the reaction mixture will become more pronounced. This will facilitate the separation of the product from the starting material and the disubstituted side product. For example, a diester will be transferred from an aqueous phase to an organic solvent in neutral solution, while the monoester will be transferred to an acidic pH value. For the relatively volatile compounds, most of the starting material can be removed by distillation.

Examples are diethylene glycol or triethylene glycol. D. Division of a bissimetric chain into two identical fragments.

A special case relates to a compound of the formula:

A) X'-O- (OCH 2 CH 2) n -CH 2 CH 2 CH = CHCH 2 - (CH 2 CH 2) n -CH 2 Y '

where X 'and Y', respectively, have the same meaning as above. The division is effected by oxidation of the double bond; in case A directly for two identical carboxy groups and in case B for two identical aldehyde groups which in turn are converted to aminomethyl groups by reductive amination.

The starting compounds

(CHOCHOO) -CHO-CHO-CHO-CHO-CHO-CHO-CHO-CHO-CHO-CHO-

2 Z Zn 2 2 ZZnZ are respectively and preferably synthesized through a Williamson ether synthesis by reacting HOCH 2 CH = CHCH 2 OH with two equivalents of Χ '' - (OCH 2 CH 2) n -Z "and Y 1 CH 2 - (OCH 2 CH 2) Z11 respectively, wherein X ', Y', Z'1 and n have the same meaning as above. An alternative route is to react Z 1 '-CH 2 CH = CHCH 2 -Z "with two equivalents of X'CH 2 - (OCH 2 CH 2) n OH and Y' CH 2 - (OCH 2 CH 2) n -OH, respectively. There are also available ana-

logos. For example, compounds in which the group -CH 2 CH = CHCH 2 "is substituted by the group -CH 2 CH-

THE

in which A is possibly substituted lower alkylidene (preferably 1-6C), and preferably dimethyl methylene. In this case the group A is removed by acid hydrolysis, for example by the peroxide and subsequent transformation of the aldehyde group formed into an aminomethyl group or into a carboxy group in known manner. The invention is defined by the appended claims which form a part of the specification. A three-part exemplification is given below: Part 1 illustrates the synthesis of amino-PEG-carboxylic acids which conform to formula IV (m = 1). Part II illustrates the synthesis of heterobifunctional reagents corresponding to formula II and of conjugates having a bridging structure according to formula I, and

Part III summarizes the results obtained for the conjugates between the T-cell (superantigen) immune stimulator of enterotoxin A (SEA) and antibodies directed against tumor necrosis.

Examples 5 and 6 (Part 2) illustrate the synthesis of a conjugate between an immunoglobulin and a peptide. Comparative experiments have shown that, provided there is a bridge according to the invention, the solubility of the conjugate will be increased and therefore also the bioavailability of the peptide in aqueous medium. Comparison was made against a conjugate comprising the same peptide and antibody, but having a small hydrophobic bridge which is not according to the invention. EXPERIMENTAL PART. PART 1. * Isopropyl 8-hydroxy-3,6-dioxaoctanoate (1). 1 (23 g, 1.0 mole) was added under nitrogen

(500 ml) under nitrogen atmosphere. When all of the sodium had reacted, the mixture was cooled to room temperature and bromoacetic acid (76 g, 0.5 mol) was added with stirring. After 18 hours at 100 ° C, excess diethylene glycol was removed by distillation at 4 mm Hg. Isopropyl alcohol (400 ml) and acetyl chloride (51 g, 0.65 mol) were then added. After stirring for 18 hours at 65 ° C, the mixture was air-cooled to room temperature and neutralized with sodium acetate (3.5 g, 0.15 mol). The mixture was filtered and the filtrate was evaporated to near dryness, and then dissolved in water (200 ml). The aqueous phase was extracted with 1,1,1-trichloroethane (3 x 50 ml). The combined organic phases were washed with water (20 ml). The product was extracted with the combined aqueous phases with dichloromethane (50 ml) which after evaporation provided an oil (55 g). isopropyl 11-hydroxy-3,6,9-trioxa-undecanoate (2).

Sodium (23 g, 1.0 mole) was added as chips and portionwise to triethylene glycol (700 ml) under a nitrogen atmosphere. When the sodium completely reacted, the mixture was cooled to room temperature and bromoacetic acid (76 g, 0.5 mol) was added with stirring. After 18 hours at 100 ° C, the excess of diethylene glycol was distilled off at about 4 mm Hg. Isopropyl alcohol (400 ml) and portions acetyl chloride (51 g, 0.65 mol) were then added. After stirring for 18 hours 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 and the filtrate evaporated to near dryness, and then dissolved in water (200 ml). The aqueous phase was extracted with 1,1,1-trichloroethane (3 x 50 ml). The combined organic phases were washed with water (20 mL). The product was extracted with the combined aqueous phases with dichloromethane (50 ml) which upon evaporation gave an oil. 1 H-nmr (CDCl3); 1.26 (d, 6H), 3.07 (s, 2H), 3.6-3.8 (m, 12H); 4.11 (s, 2H), 5.09 (m, 1 H), 20- (N-phthalimidoyl) -3,6-dioxa-octanol (3).

8-Chloro-3,6-dioxa-octanol (365 g, 2.2 mol, prepared from triethylene glycol and SOC4) in dimethylformamide (400 ml) was dissolved and was added with phthalimide potassium (370 g, 2.0 moles). After stirring for 18 hours at 110 ° C, dimethylformamide was distilled off under reduced pressure. The residue was suspended in toluene (1.5 L) at 40-50 ° C and potassium chloride was collected by filtration. The product crystallizes by cooling (-10 ° C). A second fraction of the mother liquor is available by concentrating it and repeating the crystallization procedure. CHCl3; 2H), 3.60-3.38 (m, 6H), -3.73-3.78 (t, 2H), 3.89-3.94 (t, 2H), -7.70-7.89 (m, (m, 4H). Isopropyl 17- (N-phthalimidoyl) -3,6,9,12,15-pentaoxoheptadecanoate (4).

A solution of pyridine (2.8 mL, 35 mmol) in dichloromethane (30 mL) was added dropwise with stirring at about -5 ° C to a solution of 8- (N-phthalimidoyl) -3,6- (8.5 g, 36 mmol) and trifluoromethanesulphonic acid anhydride (10.2 g, 36 mmol) in dichloromethane. After about 30 minutes the organic phase was washed with 0.5 M hydrochloric acid and water. After drying (Na 2 SO 4) and filtration was added isopropyl 8-hydroxy-3,6-dioxa-octanoate (1) (12 g, 48 mmol) and Na 2 SO 4 (6.54 mmol), and the mixture was vigorously stirred for 20 hours at room temperature. 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 afforded an oil (13 g). 1 H-nmr (CDCl 3); &amp; 1.26 (d, 6H), -3.58-3.76 (m, 18H), -3.90 (t, 2H), 4.11 (s, 2H), -5.09 (m, 1H), -7.70-7.89 (m, 4H). 17- (N-phthalimidoyl) -3,6,9,12,15-pentaoxaheptadecanoic acid (5)

The isopropyl 17- (N-phthalimidoyl) -3,6,9,12,15-pentaoxoheptadecanoate (4) (13 g) was dissolved in tetrahydrofuran (50 ml) and hydrochloric acid (conc. 50 ml) . After 16 hours at ambient temperature the solution was diluted with water (200 ml) and the tetrahydrofuran was removed under reduced pressure. The - 21 -

The aqueous phase was washed with toluene (1x) and extracted with dichloromethane (2x). Drying (Na 2 SO 4) and evaporation of the organic phase gave a product as an oil (8.5 g) 1 H-nmr (CDCl 3): δ 3.57-3.76 (m, 18H), 3.91 (t, 2H), Isopropyl 17-amino-3,6,9,12,15-pentaoxoheptadecanoate (6), â € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒ

17- (N-Phthalimidoyl) -3,6,9,12,15-pentaoxaheptadecanoyl acid (5) (8.5 g) was dissolved in 150 ml of ethanol and 3 ml of hydrazine hydrate. The solution was stirred at room temperature for 16 hours, then HCl (100 mL, 3M) was added and the solution was then refluxed for 3 hours. After cooling to room temperature and filtration, the pH (pH 9, NaOH) was adjusted and the filtrate evaporated to near dryness. Water was added and the re-evaporation was carried out almost to dryness, and then the pH of the solution was adjusted (pH 4, HCl) followed by evaporation to dryness. The product was treated with isopropanol (100 mL) and acetyl chloride (2 mL) at room temperature overnight and evaporated. The residue was taken up in water and extracted into dichloromethane at an alkaline pH value (7-11). Evaporation resulted in a desired product (3.3 g)

1 H-NMR (CDCl 3): δ 1.26 (d, 6H), 3.17 (t, 2H), 3.65-3.80 (m, 18H), 4.16 (s, 2H), 5.07 (m, 1H) 7-Triphenyl-3,6-dioxaheptanol (7)

Triphenylmethyl chloride (28 g, 0.1 mol) was added to a solution of pyridine (8 ml, 0.1 mol) in diethylene glycol (100 ml, 0.94 mol) and the mixture was stirred at 50 ° C for 24 hours. The crystals formed were filtered off and washed with water. There were obtained 31 g M.p. 103-105 ° C 1 H-nmr (CH 3 OD): δ 2.42 (br, 1H), -3.25 (t, 2H), 3.55-3.72 (m, 6H), -7.20-7.72 (m, 9H); 7.44-7.53 (m, 6H) 10,10,10-Triphenyl-3,6,9-trioxa-decanol (8).

Triphenylmethyl chloride (187 g, 0.67 mol) was added to a solution of pyridine (54 ml, 0.67 mol) in triethylene glycol (1000 ml, 7.3 mol) and the mixture was stirred at 60 (1000 mL) and dichloromethane (250 mL) was stirred at room temperature for 16 hours and partitioned between four portions and each portion was shaken (= 250 mL). The organic phases were combined and evaporated. About 259 g were obtained. 13,13,13-Triphenyl-3,6,9,12-tetraoxa-tetradecanol (9).

Triphenylmethyl chloride (129 g, 0.46 mol) was added to a solution of pyridine (38 ml, 0.48 mol) in tetraethylene glycol (800 ml, 4.2 mol) and the mixture was stirred at 80 ° C for 20 hours. Water (800 ml) was added and the mixture was extracted with dichloromethane (3 x 200 ml). The combined organic phases were washed with water (150 ml), then the product was obtained as a syrup after evaporation. Small amounts of disubstituted and non-reactive materials were present in the product. 200 g was obtained. 16,16,16-Triphenyl-3,6,9,12,15-pentaoxahexadecanol (10).

Triphenylmethyl chloride (2.8 g, 10 mmol) was added to a solution of pyridine (1 ml, 12 moles) in pentaethylene glycol (25 g, 0.1 mol) and the mixture was stirred at 80 ° C for 2 hours . Water (100 ml) was added and the mixture was extracted with dichloromethane (40 ml). The aqueous phase was evaporated using azeotropic distillation in the presence of toluene and ethanol. The residue was treated with pyridine (1 ml) and triphenylmethyl chloride (2.5 g) in the same manner as above. The same procedure was applied one more time. The three organic phases were combined and evaporated. Small amounts of disubstituted and non-reactive materials were present in the product. There were obtained 10 g. 19,19,19-Triphenyl-3,6,9,12,15,18-hexaoxa-nonadecanol (11).

Triphenylmethyl chloride (9.7 g, 34 mmol) was added to a solution of pyridine (2.8 mL, 12 mmol) in hexaethylene glycol (100 g, 36 moles) and the mixture was stirred at 80 ° C for 3 hours . Water (400 ml) was added and the mixture was extracted with dichloromethane (150 ml). The aqueous phase was once again treated as described above, but the amounts and conditions of the reaction were as described in this example. The 23

The organic extracts were evaporated and washed with water (100 ml) and then extracted with dichloromethane (50 ml). Small amounts of disubstituted and non-reactive materials were present in the product. 41.6 g. 7,7,7-triphenyl-3,6-dioxaheptyl-4-methylbenzenesulfonate (12)

P-Toluenesulfonyl chloride (5 g, 26 mmol) was added to a solution of 7,7,7-triphenyl-3,6-dioxahep-tanol (7) (3.5 g, 10 mmol) in pyridine ( 10 ml). The mixture was

stirred at room temperature for 30 minutes, after which water (1 ml) was added and stirring was continued for another 10 minutes. Dichloromethane (100 ml) was added and the mixture was extracted with hydrochloric acid (1 M) until the pyridine had been completely removed. Thereafter the organic phase was washed with saturated NaHCO 3. After drying (Na2 SO4) and evaporation, the product crystallized from diethyl ether / hexane. There were obtained 2 g. 10,10,10, -triphenyl-3,6,9-trioxa-decyl 4-methyl-benzenesulfonate (13).

To a solution of 10,10,10-triphenyl-3,6,9-trioxa-decanol (8) (4.8 g, 12 mmol) in p-toluenesulfonyl chloride (5 g, 26 mmol) was added pyridine (10 ml). The mixture was stirred at room temperature for 1 hour, after which water (1 ml) was added and stirring was continued for another 10 minutes. The mixture was diluted with dichloromethane (50 ml) and washed with hydrochloric acid (1 M) (2 x 100 ml), water (50 ml) and NaHCO3 (saturated, 50 ml). The organic phase was evaporated and the product purified on silica gel (toluene: ethyl acetate, 19: 1), after which the dichloromethane / ether product crystallized. 4.2 g. CDCl3: Î'2.20 (s, 3H), 3.24 (m, 2H), 3.60-3.80 (m8H), 4.20 (m, 2H), -7.20-7.90 (m, 19H). 8- (N-phthalimidoyl) -3,6-dioxaoctyl 4-methylbenzenesulfonate (14).

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

(14).

8- (N-phthalimidoyl) -3,6-dioxa-octanol (3) (82.2 g, 0.3 mol) was dissolved in pyridine (30 ml, 0.37 mol) and p- (56.9 g, 0.3 mole) for 1 hour and stirring at 0 ° C. The mixture was then stirred at 10 ° C for 16 hours. A solid cake was formed which was added ice (0.5 kg) mixed with HCl (concentrated, 20 ml) and the mixture was stirred at 50 ° C for 4 hours, after which ethyl acetate (200 ml) ). Filtration of the complete reaction mixture resulted in a product in a solid form. 97 g was obtained.

(m, 8H), 3.87 (t, 2H), 4.09 (q, 2H), 7.31-7.86 (m, 8H), 17- (N-phthalimidoyl) -3,6,9,12,15-pentaoxaheptadecanol (15).

10.10.10-triphenyl-3,6,9-trioxa-decanol (8) (0.78 g, 2 mmol) and dissolved in dimethylformamide (15 ml) was added dropwise to sodium hydride (80% , 0.24 g, 8 mmol, washed with 2x hexane). The mixture was heated at 30øC for 30 minutes after which 8- (N-phthalimidoyl) -3,6-dioxaoctyl-4-methylbenzenesulfonate (14) (0.87 g, 2 mmol) was added in form solid. After stirring overnight at room temperature, acetic acid anhydride (5 ml) was added, and the mixture was allowed to stand for an additional 3 hours at room temperature. Water (2 ml) was added and after 30 minutes the product was partitioned between toluene (25 ml) and a solution of NaHCO 3 (50 ml). The aqueous phase was evaporated and contained about 1 g of a substance which was taken up in dichloromethane and treated with 0.2 ml of trifluoroacetic acid and about 0.2 ml of water at room temperature for 10 minutes. The reaction mixture was then evaporated. The product obtained was purified on a silica (chloroform-methanol, 19: 1) column. About 0.7 g was obtained. 1 H-nmr (CDCl3): δ 3.59-3.67 (m, 20H), 3.71-3.75 (m, 4H), 3.90 (t, 2H); , 7.71-7.86 (m, 4H).

T-butyl 17- (N-phthalimidoyl) -3,6,9,12,15-pentaoxoheptadecanoate (16)

Pyridinium dichromate (0.64 g, = 4 eq.), Acetic acid anhydride (1 ml = 20 eq.) And t-butyl alcohol (1 ml = 30 eq.) Were added in the order presented to a solution of 17- (N-phthalimidoyl) -3,6,9,12,15-pentaoxaheptadecanol (15) (190 mg) in dichloromethane (5 ml). The reaction mixture was stirred at room temperature for 3 hours, and then ethyl acetate (25 ml) was added. After 10 minutes the liquid phase was passed through a column containing silica gel (= 5 cm x 5 cm O) and the column was eluted with more ethyl acetate. After evaporation the residue was purified on a silica gel column (chloroform: methanol, 19: 1). About 0.1 g was obtained. 1 H-nmr (CDCl 3): δ 1.47 (s, 9H), 3.58-3.76 (m, 18H), 3.90 (t, 2H), 4.02 (s, 2H), -7.70-7.87 (m, 4H) 14,14-Trifenll-4,7,10,13-tetraoxane-tetradecene (17).

Allyl bromide (2 ml, 1.1 eq.) And 10,10,10-Triphenyl-3,6,9-trioxa-decanol (8) (8.5 g, 22 mmol) were dissolved in dimethylformamide (25 ml ) and added dropwise to sodium hydride (1 g) at 0 ° C for 30 minutes. After stirring for 3 hours at room temperature, methanol was added until the solution became clear. Toluene (100 ml) and water (100 ml) were added and the reaction mixture was extracted. The toluene phase was washed with a saturated saline solution and was then dried (Na2 SO4 &gt; 4). The product was obtained as a syrup (10 g) after evaporation of the solvent. 3,6,9-Trioxa-11-dodecen-1-ol (18).

14,14,14-triphenyl-4,7,10,13-tetraoxane-tetradecene (17) (5.5 g, 13 mmol) was dissolved in dichloromethane (50 ml) and then trifluoroacetic acid (1, 2 ml, 15.6 mmol) and water (1 mL, 55 mmol) and the reaction mixture was stirred vigorously for 10 minutes. The product (1.5 g) was obtained after purification on a silica gel column (CHClâ,ƒ: MeOH, 9: 1). 26 3,6,9,12,15,18-Hexaoxa-20-heneicosen-1-ol (19).

3,6,9-trioxa-11-dodecen-1-ol (18) (2.07 g, 10.9 mmol) and 10,10,10-triphenyl- 6,9-trioxa-decyl (13) (5.95 g, 10.9 mmol) were dissolved in dimethylformamide and added dropwise with sodium hydride over 10 minutes at room temperature. After two hours dichloromethane and water were added. The phases were separated, and trifluoroacetic acid (1 mL, 13 mmol) and water (1 mL, 55 mmol) were added to the organic phase. The mixture was evaporated to dryness after vigorous stirring for 1 hour. Methanol (70%) was added and then the tri-phenylmethanol (2 g) was filtered off and the solvent evaporated. The product (2.5 g) was obtained after purification on a silica gel column (CHCl3 · MeOH, 97: 3). 1 H-nmr (CDCl 3): δ 3.56-3.74 (m, 24H), 4.00 (m, 2H), 5.20 (m, 2H), 5.89 (m, 1H) 21-Heptaoxa-23-tetracosenoic acid (20)

Bromoacetic acid (0.15 g, 1.1 mmol) and sodium hydride (0.15 g, 5 mmol) were added to a solution of 3,6,9,12,15,18-hexaoxa-20- heneicosene-l-01 (19) (0.33 g, 1 mmol) in tetrahydrofuran. The mixture was stirred at room temperature for 3 hours, after which water was added to consume excess reagent and the pH value was adjusted to pH 1 (HCl). The mixture was washed with diethyl ether (50 ml) and then extracted with dichloromethane (2 x 50 ml). Drying (sodium sulfate) and evaporation of the solvent led to the product (0.26 g). 23- (N-tert-butoxycarbonylamino) -3,6,9,12,15,18,21-heptaoxatricosanoic acid (21)

Ozone was slowly bubbled through a solution of 3,6,9,12,15,18,21-heptaoxa-23-tetracosenioic acid (20) (0.21 g, 0.55 mmol) in methanol ( 30 ml) for 30 minutes at about -60 ° C. The mixture was allowed to stand for 30 minutes, then air was bubbled through this solution for 30 minutes, after which dropwise,

was added dropwise dimethyl sulphide (50 μL, 0.65 mmol) dissolved in methanol (5 mL). The solution was brought to room temperature for 1 hour, then a solution of ammonium chloride (0.2 g, 3.7 mmol) in water (5 ml) and sodium cyanoborohydride , 2 g, 3.1 mmol). After 16 hours at ambient temperature the pH of the solution was adjusted to about 1 (HCl), and then the solvent was distilled off. The dried solid phase was extracted with dichloromethane which was then evaporated. Dissolution in sodium hydroxide (1 M), extraction with dichloromethane and evaporation afforded an oil (0.25 g). A pure product was obtained by separating the t-butoxycarbonyl derivative from the amino function; 0.17 g of sodium hydroxide oil (0.4 g) in water (3 ml) and di-t-butyl carbonate (0.12 g) dissolved in dioxane (5 ml) were dissolved. After stirring for 16 hours the dioxane was evaporated and the aqueous phase was washed with n-hexane (2 x 10 ml) and the pH was adjusted to about 4 (HCl), after which the mixture was extracted with dichloromethane ( 5x50 ml). Drying (sodium sulfate) and evaporation of the solvent gave an oil which was purified on a silica gel (CHCl3: MeOH: AcOH, 45: 4: 1) column. 0.15 g. 1H NMR (D2O, 500 MHz): Î'1.31 (s, 9H), 3.14 (t, 2H), 3.48 (t, 2H), 3.59 (m, 24H), 3.84 (s, 2H)

11- (N-phthalimidoyl) -3,6,9-trioxaundecanoic acid (22)

Sodium hydride (3.8 g, 80%, 130 mmol) and bromoacetic acid (7.6 g, 55 mmol) dissolved in tetrahydrofuran (5 mL) were added in the order presented to a solution of 8- (N-phthalimidoyl) -3,6-dioxa-octanol (3) (10.1 g, 36 mmol) in tetrahydrofuran (150 ml). After stirring for 4 hours at ambient temperature acetic acid anhydride (40 ml) was added and the mixture was stirred for an additional 18 hours, after which the mixture was filtered and evaporated in the presence of some water. Purification of the product on silica gel (CHCl3: MeOH, 3: 2) gave the product. 1.2 g. 11-amino-3,6,9-trioxa-undecanoate (23).

Hydrazine hydrate (0.7 ml, 14 mmol) was added to a solution of 11- (N-phthalimidoyl) -3,6,9-trioxa-undecanoic acid (22) (1.2 g, 3.5 mmoles) in ethanol (25 ml) and aq.

The mixture was stirred at room temperature for 18 hours, after which hydrochloric acid (3.7 ml, conc.) and water were added. The mixture was refluxed for 2 hours and then evaporated almost to dryness after which was added water (25 ml) and the pH adjusted to about 9 (NaOH). After evaporation to dryness, methanol (200 ml) and acetyl chloride (2 ml) were added and the mixture was stirred at room temperature for 66 hours. The formed methyl ester was purified on a silica gel column. (M, 13H), 4.20 (s, 2H), 3.20 (s, 2H), 3.20 (s, 2H).

3,6,9,12,15,18,21,24,27-Nonaoxa-29-trieneenoic acid (24)

Sodium hydride (50 mg, 1.6 mmol) was added to a solution of 3,6,9,12,15,18-hexaoxa- 20-henoicosen-1-ol (19) (0.6 g, 9 mmol) and 7,7,7-triphenyl-3,6-dioxaheptyl-4-methylbenzenesulfonate (12) (0.9 g, 1.8 mmol) in dimethylformamide (10 ml). The mixture was stirred at room temperature for 56 hours, and then water (10 ml) and dichloromethane (15 ml) were added. The mixture was stirred and the organic phase was evaporated and purified on a silica gel column (CHCl3 MeOH, 9: 1). The product was dissolved in dichloromethane (20 ml) and acid (3 drops) and water (5 drops) were added, and then the mixture was stirred for 4 hours and evaporated. Extraction of the residue with methanol: water (70:30) and filtration gave after evaporation an oil which was dissolved in tetrahydrofuran (10 ml) to which were added sodium hydride (ca. 5 eq.) And bromoacetic acid (about 2 eq.), after which the mixture was stirred at room temperature for 16 hours. Water was added to destroy excess sodium hydride and the mixture was evaporated and then purified on a silica gel column (CHCl 3 MeOH 3: 1). There was obtained 0.14 g. (M, 2H), 5.20 (m, 2H), 5.91 (m, 2H), 3.50-3.78 (m, 1H) 29- (Nt-butoxycarbonylamino) -3,6,9,12,15,18,21,24,27-nona | oxa-29-nonacosanoic acid (25). Ozone was bubbled slowly through

of a solution of 3,6,9,12,15,18,21,24,27-nonaoxa-29-tri-lithium (24) (0.11 g, 0.23 mmol) in methanol (25 ml) for 30 minutes at about 70 ° C. After 30 minutes, air was bubbled through the solution for 30 minutes, and then dimethylsulfide (50 μl, 0.65 mmol) was added. The solution was brought to room temperature over a period of 1 hour, and then a solution of ammonium hydrochloride (0.1 g, 1.9 mmol) in water (5 ml) and sodium cyanoborohydride ( 0.1 g, 1.6 mmol). After 16 hours at room temperature the pH was adjusted to about 1 (HCl), and the solvent was evaporated. The dried material was extracted with dichloromethane and was then evaporated. The residue obtained was an oil (0.1 g). A pure product was obtained by preparing a t-butoxycarbonyl derivative of the amino function; the oil was added sodium hydroxide (0.4 g) dissolved in water (2 ml) and t-dibutyl dicarbonate (0.16 g) dissolved in dioxane (5 ml). After stirring for 16 hours the dioxane was evaporated and the aqueous phase was washed with n-hexane (2 x 10 mL) and the pH was adjusted to about 4 (HCl), after which the product was extracted with dichloromethane. Formulas of the acids amino-PEG-carboxylic acids. (CH 2) 2 2 n 2 3 2 1 n-2 2 n-3 Ft N-CH 2 CH 2 - (OCH 2 CH 2) 20 H 3

FtN-CH2CH2- (OCH2CH2) 4OCH2 16 ch2 = chch2- (och2ch2) 30Tr 17

THE

FtN-CH 2 CH 2 - (OCH 2 CH 2) 4 OCH 2

X OCH (CH 3) 2 CH = CHCHO- (OCHO CH 2) 2 2 2 2 n 18 n-3 19 n-6

To

(CH 2) 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 O (CH 2)

H 2 N -CH 2 CH 2 - (OCH 2 CH 2) 4 OCH 2 OH OH O 2 NHCH 2 CH 2 (OCH 2 CH 2) 6 OCH 2 OH 6 6 30 30

Tr- (OCH 2 CH 2) n OH H F N N -CH 2 CH 2 - (OCH 2 CH 2) 2 OCH 2 OH 7 n-28 8-39 n- 4 10 n- 5 11 n-

(CH2) 2 - (OCH2 CH2) 2 OCH2 23 Ϊ CH2 = CHCH2 - (OCH2 CH2) gOCH3 - OH

FtN-CH 2 CH 2 - (OCH 2 CH 2) 2OT 3 14 vi ϊ Ϊ

S, 'O', NHCH 2 CH 2 (OCH 2 CH 2) gOCH 2, NDH 25

FtN-CH 2 CH 2 - (OCH 2 CH 2) 4 OCH 2 CH 2 OH

PREPARATION OF BIFUNCTIONAL REAGENTS AND COCCULATION PRODUCTS

The structural formulas are presented on a separate page.

Example 1

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

Isopropyl 17-amino-3,6,9,12,15-pentaoxaheptadecanoate (1.1 g, 3.2 mmol) was dissolved in 3 ml of 1M solution of isopropyl hydroxide sodium hydroxide solution and allowed to stand at room temperature for 30 min. 1.5 ml of 6M hydrochloric acid 545 mg of 17-amino-3,6,9,12,15-pentaoxaheptadecanoic acid was added for 1 minute and the pH was maintained at 8.4 by addition of 5M NaOH. The reaction solution was stirred for 15 minutes during the introduction of a-zoto gas. According to thin layer chromatography (eluent: CH2 Cl2 -MeOH 60:35) the reaction was considered complete in only a few minutes. After 15 minutes the pH of the reaction mixture was

adjusted to 3 and the solution was frozen and lyophilized. The reaction mixture was pooled on a reversed phase column. PEP-RPC HR 30/26 (Pharmacia Biosystems ΔΒ) using a gradient of 0 to 13% acetonitrile with 0.1% trifluoroacetic acid followed by isocratic separation at 13 % caetonitrile, 0.1% TFA. The desired peak fractions were combined and lyophilized to give 351 mg of acid (A).

Yield: 76%. The structure of the product was established by the aid of its NMR spectrum.  € ƒâ € ƒâ € ƒNMR (D2 O) expressed in values of: â € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒ1CH2C 4.23 s, 0CH2C0H 3.76 s0.0 -0CH2CH2O- 3.71-3.76, -NHCH2CH2O- 3.65 t, -NHCH2CH2O- 3.41 B. Preparation of 17-N-hydroxysuccinimide ester -iodo-acetylamino-3,6,9,12,15-pentaoxaheptadecanic acid (B)

Hydroxysuccinimide (4.5 mg, 39 mmol) was weighed into a reaction vial. The 17-iodoa-cetylamino-3,6,9,12,15-pentaoxaheptadecanoic acid (18.3 mg, 39 mmol) was dissolved in 0.55 ml of dry dioxane and added to the reaction flask. deaerated with nitrogen gas and then a solution of 8.0 mg (39 mmol) of dicyclohexylcarbodiimide in 0.15 ml of dry dioxane was added dropwise to the reaction flask.The flask was filled with nitrogen gas, sealed The precipitate formed was removed by filtration The percentage of product B formed in the filtrate was determined by NMR analysis and gave a value of 89%.

Example 2

Preparation of (17-iodoacetylamino-3,6,9,12,15-pentaoxahepta-decanoylamino-immunoglobulin (C) A. Monoclonal antibody Mab C215 An anti-

immunoglobulin IgG2a monoclonal antibody (Mab C215) (34 mg, 0.218 / mol) dissolved in 17.7 ml of 0.1 M borate buffer pH 8.1 containing 0.9% sodium chloride. 146 ul of a dioxane solution containing 3.6 mg (6.4 mmol) of 17-iodoacetylamino-3,6,9,12,15-pentaoxoheptadecanoic acid N-hydroxysuccinimide ester (B) were injected into the buffer solution and the reaction was quenched with stirring for 25 minutes at room temperature. The reaction flask was covered with a sheet to prevent light. Excess reagent (B) was removed by fractionation on a Sephadex G 25 K 26/40 column using a 0.1 M phosphate buffer pH 7.5 containing 0.9% sodium chloride as eluent. Fractions containing the desired product C were combined. The solution (22 ml) was concentrated in an Amicon cell through a YM 30 filter to 8 ml. The concentration and degree of substitution were determined with amino acid analysis and gave the value of 4.7 mg / ml and 18 spacers per Mab C215 respectively. B. Mab C242 Monoclonal Antibody

A monoclonal antibody (Mab C242) from the immunoglobulin class IgG 1 was reacted with a 15, 20 and 22-fold molar excess of 17-iodoacetylamino-3,6,9,12,15-N-hydroxysuccinimide ester (17-iodoacetylamino) -3,6,9,12,15-pentaoxaheptadecanoylamino) -Mab C242 (C) -acetylamide was prepared according to the procedure described in example 2A to give the nona, dodeca and tetradeca

C. Mab C monoclonal antibody

A monoclonal antibody (Mab C) from IgG 2a class immunoglobulin was reacted with a 14- and 18-fold molar excess of 17-iodoacetylamino-3,6,9,12,12-pentaoxaheptadecanoic acid N-hydroxysuccinimide ester (B) respectively according to the procedure described in example 2A to give the tetrahepta (17-iodoacetylamino) -3,6,9,12,15-pentaoxaheptaecanoylamino) -Mab C. (C) Example 3

Preparation of enterotoxin Ά staphylococcus 33

3 labeled with 2-mercaptopropionylamino-Eu (SEA) 3+ A. Preparation of Eu (D) -labeled SEA

SEA (Lyophilized product from Toxin Technology Inc.) (2 mg, 72 nmoles) was dissolved in 722 μl of milliliter water and was added to 15 ml of a polypropylene tube. 100 Âμl of 0.1 M borate buffer at pH 8.6 and then 3+ of 2160 nmoles of Eu-Chelate reagents (Pharmacia Wallac)

Oy) at 178 Jul of Milli-Q. The reaction was quenched at room temperature overnight. Excess reagent was removed by fractionation of the reaction solution on a Sephadex G 25 PD 10 column (Pharmacia Biosystems AB) using a 0.1 M phosphate buffer pH 8.0 as eluent. The fractions with the desired product D were combined. The solution (3 ml) was concentrated in an Ami con cell through a YM5 filter to a volume of 0.8 ml. The concentration was determined with amino acid analysis and gave the value of 1.7 mg / ml. The degree of substitution was determined by comparing a standard solution of EuCl 3 to 0.8 Eu by SEA.

(SEQ ID NO: 1) Preparation of SE A labeled with 3- (2-pyridyldithio) propionyl-3 + 3-aminoethylamino and SEA labeled with 3-mercaptopropionylamino Eu (F) 3+

Eu-SEA (1.24 mg, 44.8 mmol) was added in 0.75 ml of a phosphate buffer pH 8.0 to 15 ml of a polypropylene tube. A solution of 1.6 mg of N-succinimidyl 3- (2-pyridyldithio) propionate in 1 ml of ethanol was added to the tube and the reaction solution was stirred for 30 min. at room temperature. The obtained product E was not isolated before being reduced to the product F. To the reaction mixture obtained above was added 20 ml of a 0.2 M solution of ethyl acetate and 50 ml of acetic acid 2M to adjust the pH value to 5. Then a solution of 3.1 mg of dithiothreitol (Merck) in 0.1 ml of a 0.9% solution of boron chloride was added and the reaction solution was stirred for 20 min. at room temperature. The total volume was then adjusted to 1 ml by addition of 50 μl of 0.9% sodium chloride solution. The reaction reaction (1 ml) was placed on a Sephadex G25 NAP-10 column (Pharma-

Biosystems AB) and the desired product F was eluted by adding 1.5 ml of pH 7.5 phosphate buffer containing 0.9% sodium chloride. The eluted product F was collected in a 15 ml polypropylene tube and used immediately in the synthesis of the product G to avoid reoxidation to a disulfide-type compound. B2. Preparation of 2-mercaptopropionylamino of staphylococci in terotoxin A (SEA) (F2)

Native SEA (lyophilized product from Toxin Technology Inc.) or recombinant prepared SEA (rSEA) with a two-fold excess of N-succinimidyl 3- (2-pyridyldithio) -propionate was reacted according to the procedure described in example 3B1 . The degree of substitution was determined by UV analysis according to the procedure of Carlsson and co-workers giving the value of 1.9 mercaptopropionyl groups by SEA.

Example 4

Preparation of SEA-Monoclonal Antibody Conjugate (GlOch G2) A. Conjugates between Eu2 + SEA and Mab C215 (Gl) The solution of 4-mercaptopropion-3-nylamino Eu (F) -labeled SEa solution described in example 3B were 1.2 ml of a solution of octadeca (17-iodoactylamino-3,6,9,12,15-pentaoxaheptadecanoylamino) Mab C215 (C) (4 mg) in 0.1 M phosphate buffer pH 7.5 containing 0.9% sodium chloride. The reaction was quenched by standing at room temperature overnight. The unreacted iodoalkyl groups were then blocked by addition of 5 .mu.l (1.2 .mu.mol) of a solution of 20 .mu.l mercaptoethanol in 1 ml. Of water. The reaction solution was allowed to stand for 4 hours at room temperature and then filtered. The filtrate was then fractionated on a Superose 12 HR 16/50 column (Pharmacia Biosystems AB) using as an eluent a 0.002 M phosphate buffer at pH 7.5 containing 0.9% sodium chloride. The fractions with the desired product G were combined and analyzed. The protein content was 0.22 mg / ml determined by. amino acid analysis. The degree of substitution was 1 SEA per 35 ~ 3+ -

The product was also studied for the determination of the immunostimulating properties and ability to bind to antibodies.

By increasing the amount of compound (F) relative to compound (C) a greater degree of substitution was obtained. B. Conjugates between rSEA and Mab C215 (G2)

Octadeca (17-iodoactylamino-3,6,9,12,15-pentaoxaheptadecanoylamino) Mab C215 (C) was reacted with a 1.8 molar excess of 2-mercaptopropionylamino-rSEA (F2) according to the procedure described in example 4A. The conjugate composition was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a Phas®-Gel 4-15 gradient and the ligations were screened with Phast IMAGE (Phar soft Biosystems AB). The conjugate obtained was composed of 6+ Mab C215 with three SEA. 15% with two SEAs, 28% with an SEA and 51% not substituted with Mab C215.

In another experiment a 2.7 fold molar excess of F2 was used obtaining a conjugate with the composition of 15% Mab C215 with three SEA, 25% with two SEA, 34% with an SEA and 26% not substituted with Mab C215 . C. Conjugates between rSEA and Mab C242

Cl. (17-iodoactylamino-3,6,9,12,15-pentaohexadecanoylamino) Mab C242 (C) was reacted with a 3.2 molar excess of 2-mercaptopropionylamino-rSEA (F2) according to the procedure described in example 4A. The conjugate composition was assayed as described in Example 4B and was found to be 4% Mab C242 with four SEA, 12% with three SEA, 28% with two SEA's, 36% with an SEA and 20% not substituted with Mab C242.

The same reaction was conducted but the reaction product was treated for 4 hours with 0.2 M hydroxylamine prior to fractionation of the column to remove non-stable bonds between Mab C242 and the spacers and between SEA and the mercaptopionyl group. The conjugate formed had the composition 1% Mab C 242 with four SEA, 12% with three SEA, 27% with two SEA and 24% not substituted with Mab. C2. (17-Iodoactylamino-3,6,9,12,15-penta

(C) with a 3-molar excess of 2-mercaptopropionylamino-rSEA (F2) according to the procedure described in example 4A. The conjugate formed had the composition 6% Mab C242 with three SEA, 26% with two SEA, 36% with an SEA and 31% not substituted with Mab C242. C3. (17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino) -Mab C242 (C) was reacted with a 3-molar excess of 2-mercaptopropionylamino-rSEA (F2) according to the procedure described in example 4A. The conjugate obtained had the composition 13% Mab C242 with two SEA, 39% with an SEA and 46% not substituted with Mab C242. D. Conjugates between rSEA and Mab c

Dl. (17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino) -Mab C242 (C) was reacted with a 5.4-fold molar excess of 2-mercaptopropionylamino-rSEA (F2) according to The procedure described in example 4A. The conjugate composition was analyzed as described in Example 4B 15% Mab C242 with four SEA, 24% with three SEA, 29% with two SEA, 19% with an SEA and 3% not substituted with Mab C and 10% of a form dimetric.

The same reaction was conducted with 0.2 M hydroxylamine present to remove unstable bonds between Mab and the spacer and between SEA and the mercaptopropionyl group. The conjugate formed had the following composition 11% Mab C with three SEA, 24% with two SEA, 30% with an SEA, 18% unsubstituted with Mab C and 17% in a dimetric form. D2. (17-iodoacetylamino) -3,6,9,12,15-pentaoxaheptadecanoylamino) Mab C was reacted with a 5.7-fold molar excess of 2-mercaptopropionylamino-rSEA (F2) according to the procedure described in example 4B . The conjugate had the following composition: 8% Mab C2 with four SEA, 18% with three SEA, 30% with two SEA, 26% with an SEA and 5% unsubstituted with Mab C and 12% in a dimeric formula.

Example 5

Peptide sequence coupling 145-165 derived from aloanti-

human HLA-A2.1 genotype to monoclonal antibody Mab C215 (H)

(17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino) -imunoglobulin G2a (C) (5.4 mg, 34.6 nmoles) dissolved in 1.6 ml of 0.1% phosphate buffer M at pH 7.5 containing 0.9% sodium chloride was added to a 5 ml reaction flask. The solution was deaerated with nitrogen gas and then the HLA-A2.1 peptide sequence 145-165 His-LysTrpGluAlaHisValAlaGluGlnLeuArgAlaTyrLeuGluGlyThrCysVal (2.5 mg, 0.8 jumoles) in the solid state was added in small portions while stirring the solution. The pH value of the reaction solution was confirmed at 7.4. Before closing the flask, more gaseous nitrogen was bubbled into the solution. The flask was covered with a sheet and the reaction solution was stirred overnight at room temperature. To block the unreacted iodoalkyl groups was added 10.5 Âμl (1.5 jumoles) of a solution of 10 Âμl of mercaptoethanol in 1 ml of water and the reaction solution was stirred for 4 hours. The reaction solution was fractionated and filtered on a Superose 12 HR 10/50 column (Pharmacia Biosystems AB) using a 2 mM phosphate buffer at pH 7.5 containing 0.9% sodium chloride as eluent. Fractions were combined with the desired product (H). Protein concentration and degree of modification were determined by amino acid analysis as being 176 μg protein per ml and 11 peptides per IgG.

In a similar synthesis the peptide was added in an amount of 5 mg (1.7 jumoles) giving a product with 17 peptides per IgG.

Example 6

Acplication of the peptide sequence 93-113 derived from the human HLA-A2.1 alloantigen of monoclonal antibody Mab C215 (1).

(17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino) -imunoglobin G2a (4 mg, 25.6 nmoles) dissolved in 1.6 ml of 0.1 M phosphate buffer at pH 7.5 containing 0.9% sodium chloride was added to a 5 ml reaction flask. (This compound was prepared in a similar manner to compound C, 38

Example 2A, using less excess of reagent B). The solution was deaerated with nitrogen gas. The HLA-a2.1 peptide MetTyrGlyCys AspValGlySerAspTrpArgPheLeuArgGlyTyr (4.7 mg, 2.1 mmol) was suspended in 0.2 ml of acetonitrile and dissolved by the addition of 0.1 ml of a 0.1 M phosphate buffer at pH 7.5 containing 0.9% sodium chloride. This solution was added dropwise from the reaction flask. The pH value was checked and was 7.5.

Gaseous nitrogen was bubbled through the reaction solution before the vial was closed. The flask was covered with a sheet and the reaction solution was stirred overnight. To block the unreacted iodoalkyl groups were added 10æl (1.4 μmol) of a solution of 10 μl of mercaptoethanol in 1 ml of water. The reaction solution was stirred for an additional 4 hours, filtered and then fractionated on a Superose 12 HR 10/50 column using a 2 mM phosphate buffer at pH 7.5 containing 0.9% sodium chloride as eluent . The fractions with the desired product (I) were combined. The protein concentration and degree of modification were determined by amino acid analysis and gave the value of 196æg of protein per ml and 7 peptides per IgG respectively.

Example 7

Preparation of 17- [3- (2-pyridyldithio) propionylamino] -3,6,9,12,15-pentaoxaheptadecanecarboxylic acid N-hydroxysuccinimide A. Preparation of 17- [3- ( 2-pyridyldithio) propionylamino] -3,6,9,12,15-pentaoxaheptadecanoic acid (J)

17-Amino-3,6,9,12,15-pentaoxaheptadecanoic acid (66 mg, 0.2 mmol) was dissolved in 3.5 ml of 1 M borate buffer at pH 8.4. The pH value decreased to 8.1. N-Succinimidyl 3- (2-pyridyldithio) -propionate (69 mg, 0.22 mmol) dissolved in 0.8 ml of dioxane was added to the previous solution. The pH of the reaction solution decreased to 7.6. The reaction was completed within 10 minutes as demonstrated by thin layer chromatography (Eluent: CH 2 Cl 2 -MeOH 60:35). - 39 -

After 30 minutes the pH of the reaction solution was adjusted to 4.5 with 5M hydrochloric acid and the solution was frozen and lyophilized. The reaction mixture was fractionated on a PEP RPC HR16 / 10 reverse phase column (Pharmacia Biosys tems AB) using a gradient of 0.17% acetonitrile with 0.1% TFA followed by 17% isocratic separation of acetonitrile with 0.1% TFA. The fractions with the desired compound J were combined and lyophilized. Fractionation was repeated 13 times. There were obtained: 39 mg. The structure of product J was established with the aid of its NMR spectrum.

B. Preparation of 17- [3- (2-pyridyldithio) proplonylamino] -3,6,9,12,15-pentaoxahepta-decanoic acid N-hydroxysuccinimide ester (K)

Hydroxysuccinimide (1.87 mg.

16.3 μmol) in a 5 ml reaction flask. 17- [3- (2-pyridyldithio) propionylamino] -3,6,9,12,15-pentaoxaheptadecanoic acid (J) (8.0 mg, 16.4 μmol) was dissolved in 0.5 ml of dry dioxane and added to the flask. Gaseous nitrogen was bubbled through the solution. A solution of dicyclohexylcarbodiimide (6.8 mg, 32.8 mole) in 200 ml of dioxane was added to the reaction portion and more nitrogen was bubbled through the reaction solution before the vial was closed. The reaction was allowed to proceed for 24 hours and the formed preservative was removed by filtration. The NMR analysis of the filtrate revealed that the reaction was almost complete to compound K. Example 8

Preparation of di (17- [3- (2-pyridyldithio) propionylamino] -3,6,9,12,15-pentaoxaheptadecanoylamino) immunoglobulin G1 (L)

An immobilized monoclonal antibody of IgG1 class imu-globulin (Mab C242) (6 mg, 38 mmol) dissolved in 2.23 ml of a 0.1 M borate buffer at pH 8.0 containing 0.9% NaCl was added to a 5 ml reaction flask. The solution was diluted with 0.77 ml of the above buffer to a final concentration of i2 mg protein per ml. A solution of 17- [3- (2-pyridyldithio) propionyl] -4- (2-pyridyldithio)

(K) (0.26 mg, 447 mmol) dissolved in 100 μl of dioxane was added to the solution within the reaction range. The reaction solution was stirred for 25 minutes at room temperature and then placed in a refrigerator overnight. The reaction solution was partitioned onto a Superdex 75 HR 10/30 column (Pharmacia Biosystems AB) using a 0.1 M phosphate buffer at pH 7.5 containing 0.9% NaCl as eluent. The fractions containing the desired product (L ) were combined (7 ml) and concentrated in an Amicon cell through a YM 30 filter to 1.5 ml. The concentration and degree of substitution were determined with amino acid analysis revealing the value of 3.51 mg protein per ml. ml and 2 spacers per IgG respectively.

Example 9

Recombination of two monoclonal antibody molecules for the product (N) A. Preparation of (17- / 3-thiopropionylamino / -3,6,9,12,15-pentaoxaheptadecanoylamino) immunoglobulin G1 (M)

To a tube of Ellenman was added di (17- [3- (2-pyridyldithio) propionylamino] -3,6,9,12,15-pentaoxahep-tadecanoylamino) -imunoglobulin G1 (M) (2.5 mg, mmoles) dissolved in 0.7 ml of 0.1 M phosphate buffer at pH 7.5 containing 0.9% sodium chloride. The pH value was adjusted to 4.7 with 1 M acetic acid. Then, 100 Âμl (2.3 mg) of a solution of 6.9 mg of dithiothreitol in 300 Âμl of a solution of sodium chloride 0.9%. The reaction solution was allowed to stand at room temperature for 25 minutes and then desalted on a Sephadex G25 NAP 10 column (Pharmacia Biosystems AB). 0.1 M phosphate buffer at pH 7.5 containing 0.9% sodium chloride and 2 mg EDTA per ml as eluent was used. The product M was eluted in a volume of 1.5 ml and used immediately in the synthesis of the product (N) to avoid reoxidation of the free mercapto groups. B. Preparation of the crosslinked monoclonal antibody (N) To a 5 ml reaction flask were added 0.7 ml (1.25 mg) of the solution of (17- [3-thiopropionylamino] -3-

6, 9, 12, 15-pentaoxaheptadecanoylamino) immunoglobulin G1 (M) described in example 9A and 0.35 ml of a solution of tri (17-iodoa-cetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino) immunoglobulin G1 (1.26 mg). (This compound was prepared in a similar manner to compound C using a minor excess of reagent B, and monoclonal antibody Mab C242).

Gaseous nitrogen was bubbled through the reaction solution. The vial was then closed, covered with a sheet to prevent light and left overnight at room temperature. The unreacted iodoalkyl groups were blocked by the addition of 2.5 μl (0.6 μmol) of a solution of 20 μl of mercaptoethanol in 1 ml of water. The reaction solution was allowed to stand at room temperature for 6 hours and then fractionated on a Superose 12 HR 10/30 column (Phar soft Biosystems AB) using a 5 mM phosphate buffer at pH 7.5 containing sodium chloride 0.9% as eluent. The fractions were combined with the dimeric product (N).

Example 10.

Preparation of [17β- (3-mercaptopropionylamino) -3,6,9,12,15-pentaoxaheptadecanoylamino] -ESAA (P) A. Preparation of [17β- (2-pyridyldithio) propionylamino] -3,6,9 , 12,15-pentaoxaheptadecanoylamino / -rSEA (O)

A solution of 17- [3- (2-pyridylthio) propionylamino] -3,6,9,12,15-pentaoxaheptadecanoic acid N-hydroxysuccinimide ester (0.53 mg, 896 nmoles) in 43 μl of dioxane) in a solution of 3.67 mg (128 nmoles) of rSEA in 1 ml of 0.1 M phosphate buffer pH 7.5 containing 0.9% sodium chloride. The reaction was completed in 30 min. at room temperature. 100 p.1 were taken for analysis of the degree of substitution. The remaining fraction was stored in a frozen form until reduced to product P. The degree of substitution was determined by desalting 100 μl of the reaction solution on a Sephadex G50 NICK column (Pharmacia Biosystems AB) and analyzing the eluate by UV spectroscopy according to Carlsson et al. (Biochem., J. 173 ((1978) 723-737) 2.7 spacers were coupled to rSEA.

B. Preparation of [17β-mercaptopropionylamino) -3,6,9,12,15-pentaoxaheptadecanoylamino] -ESA (P) The pH value of the reaction solution with product 0 (0.9 ml) was adjusted was treated with 2N HCl at pH 4.4 and 2.9 mg of dithiothreitol dissolved in 75 μl of 0.9% sodium chloride were added. The reduction was completed in 30 min. The reaction solution was added to a Sephadex G25 NAP 10 column (Pharmacia Biosystems AB) and eluted with 1.5 ml of 0.1 M phosphate buffer at a pH of 7.5 with 0.9% NaCl and immediately used in the synthesis of the product Q in example 11.

Example 11. Preparation of SEA- (monoclonal antibody Q double spacer

(17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino) Mab C242 (C) (4.2 mg, 27 mmol) in 1.0 mL of 0.1 M phosphate buffer pH 7 , 5 with 0.9% NaCl) for 43 h with N-17- (3-mercaptopropionylamino) -3,6-

(1.17 mg, 42 nmoles in 1 ml of the abovementioned buffer) in the dark and in a nitrogen atmosphere. Then 1.14 pmols of mercaptoethanol were added. After the reaction solution was fractionated on a Sephadex 200 HR 16/65 column. The product was eluted with 2 mM phosphate buffer at a pH of 7.5 with 0.9% NaCl. The fractions with the desired product Q were combined and analyzed as described in Example 4B. The conjugate consisted of 9% Mab C242 with two SEA, 25% with an SEA and 66% of Mab C242 not subtitled.

ICHnCNHCH-CH- (OCH "CH-).

O

ICH0CNHCELCEL (OCH3 CH3 OCH

Ml 0 2 2 '2 2 4 4 0 1 0 0 Jr

IgG- (NHCCHLO (CHâ,ƒCHâ,,O), CHO CHâ,NHCHC CHâ,,I) Z ZZZZ ZnO IIn- 4,7,9,12,14,18-43 -

SEA-NHCNH3 -CH2 -N.

/ \ "R \

N

N (Eu3 + -SEA) r r n coo coo - Eu wa

COO COO eu3 + -sea- (nhcch2ch2ss / /) 3-4 0 3+

I-SEA- (NHCCELCELSH). \\ Z Z O-4

SEA (NHCCHLCH-SH). II 2 2 1.9

O 3+

/ (NHCCH-OCHO CHOO) .CH "CHONHCCH0SCHOCHOCNH-Eu-SEA) τ« 2 2 2 4 2 2, | 2 2 2 O O m

O (NHCCH "O (CH" CHO ") .CH_CHNHCCH" SCH "CHOOH) __ || Z Z Z i Z Z ι Z Z Z Z "Itl

O O

n-m

- 44 -

CO Ή 1 Ά Ά Ά Ά Ά t t t t t t t t rei β Ά Ά β β β β β β β β β β β β β β β β β β β β β β β β. tt Η C ti E EHβ ω aβ rH Ο> 1 rH Ο Μ η η η η η η η η η η η η η η η η η η η η η η η

β Ico rH a o

CN

M u ao

(1): wherein R 1 is hydrogen or C 1 -C 4 alkyl, and R 1 is hydrogen or C 1 -C 4 alkyl; co> 4 0 <Eh cn I-IM 01> 4 U> 1 H CN 0-0 ffi IU

CO

What's up

M

CN CNC CNC CN CN CN CN CNC CNC CNC CN CNC CNC CNC CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN auuuu Cd td CN CN CN CN CNUuu Cd Cd Cd Cd oouuuouo - 'CN - CN CN' Ν 'Ν' CN CN a a -. CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN

O

O to CNC CNC CNC B CNC B CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC CNC

the tr &gt; H

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CNcq or 0 Cd 3 3 O O tn H-45 -

46

EXPERIMENTAL PART II

Effects of superantigen-antibody conjugates on cells The bacterial toxin used in the following experiments was Staphylococcus enterotoxin A (SEA) obtained from Toxin Technologies (WI, USA) or produced as a recombinant E. coli protein.

Antibodies were C215, C242 and Thy-1.2 mAb. C215 is an IgG2a mAb raised against human colon carcinoma cell lines and reacts with a 37kD protein antigen on several human colon cell lines. References to these mAbs have been referred to above. The conjugates were prepared as described in the previous section.

Prior to the priority date, only studies with SEA-C215 mAb conjugates labeled with 3+

I . During the priority year the results were checked with unlabeled SEA-215, SEA-242 and SEA-Th2-mAb conjugates. Results now incorporated refer to unlabeled conjugates.

To determine the cytotoxicity mediated by the SEA-215 mAb conjugate and the unconjugated SEA and C215 mAb against colon carcinoma cells lacking MHC Class II or expressing small but undetectable amounts of MHC Class II, several T cell lines expanded with human SEA as effector cells and a panel of colon carcinoma cells and MHC Class II + Raji cells as target cells. Colo205, SW620 and Widr colon carcinoma cell lines did not all have MHC Class II as determined by development with mAb against HLA-DR, HLA-DP and HLA-DQ and FACS analysis.

SEA-expanded T cell lines were established from the peripheral blood by weekly restimulations with SEA pre-coated mitomycin C-treated MHO Class II + BSM cells in the presence of the recombinant IL-2 (20 units / ml ). These T cell lines were strongly cytotoxic against SEA-coated Raji or BSM cells but not uncoated cells or staphylococcal enterotoxin B (SEB) coated cells. This SEA-induced extermination is dependent on the interaction of SEA with MHC Class II in the target cell as is ~

terminated by HLA-DR blocking antibodies, MHC Class II Raji mutant cells, and HLA-DR transfected L cells (Dohlsten et al., Immunology 71 (1990) 96-100) .These T cell lines could be activated to exterminate cells of the C215 + MHC Class II colon carcinoma by the C215-SEA conjugate In contrast the unconjugated SEA and C215 mAb were unable to induce more than a marginal extermination of T cells against MHC Class II C215 colon carcinoma cells. staphylococcal enterotoxin antibody dependent on cell-mediated cytotoxicity depended on the binding of the SEA-C215 mAb conjugate to C215 + tumor cells. Specificity in this binding was demonstrated by the fact that the excess of unconjugated C215 mAb but not C242 and w6 / 32 mAb inhibited lysis of colon carcinoma cells. CD4 + and CD8 + T cells demonstrate extermination of C215 colon carcinoma cells treated with SEA-C 215, but did not lysate SEA-treated cells. The interaction of T cells with the SEA-C215 mAb conjugate bound to MHC Class II tumor cells appears to involve interaction with specific sequences of V-beta TCR in a manner similar to that already demonstrated for SEA-induced extermination of MHC Class II +. This was indicated by the interaction of a specific SEA but not for a specific SEB of T cell lines with the C215-SEA conjugate. Conjugates C242 mAb and Thy-1.2 mAb demonstrated activity in analogy with the C215 mAb conjugate.

Chromium labeling and incubation of target cells with SEA Î ± g

0.75xl target cells and 150æC: were incubated. 51 chromium (Amersham Corp., Arlington Hights, England) for 45 minutes at 37 ° C in a volume of 100 Âμl. Cells were maintained in complete medium containing RPMI-1640 medium (Gibco, Pais-ley, Great Britain ) supplemented with 2.8% (v / v) 7.5% NaHCC &gt; 3.1% sodium pyruvate, 2% 200 mM L-glutamine, 1% 1M Hepes, 1% gentamicin 10 mg / ml and 10% fetal calf serum (FCS, Gibco, Paisley, Great Britain). After incubation the cells were washed once with complete FCS-free medium and incubated for 60 minutes at 37Â ° C and resuspended in 10% FCS containing complete medium. 5x104 target cells were added to each well of round-shaped U-shaped round-hole 96-well rounding plates (Costar, Cambridge, United States of America).

Cytotoxicity assay

Effector cells were added to the wells for various ratios of effector / target cells. The final volume in each bore was 200 μl. Each assay was performed in triplicate. The plates were incubated for 4 hours at 37 ° C a-post, which released chromium was collected. The amount of Cr was determined on a gamma counter (Cobra Auto-gamma, Pac kard). The percentage cytotoxicity was calculated by the formula% cytotoxicity = (XM) / (TM) * 100, where X is the release of chromium in cpm in the test sample, M is the punctate release of chromium from the target cells incubated with the medium, and T is the total release of chromium obtained by incubating the target cell with 1% sodium dodecylsulfate.

Results obtained

SEA-C242, SEA-C215 and SEA-anti-Thy-1.2 mAb conjugates bind to cells expressing the important epitopes of mAb, respectively, and MHC Class II + cells. Unconjugated SEA binds on the other hand to only MHC Class II + cells. Unconjugated cells C215, C242 and Thy-1.2 mAb bind to important cells but not to Raji cells. (Table 1).

Human T cell lines lysed MHC Class II SW620, Colo205 and Widr cells in the presence of the SEA-C215 mAb conjugate but not in the presence of unconjugated SEA and C215 mAb (Fig. 1). Lysis of colon carcinoma cells was observed at 10-100 mg / ml of the SEA-C215 MAb conjugate. High levels of lysis were observed for various effector to target ratios with the SEA-215 mAb conjugate against SW620 (Fig. 1). In contrast, SEA or C215 mAb did not mediate cytotoxicity against SW620 cells for all assayed effector to target ratios. This indicates that the ability to lyse MHC Class II Colo205 cells is restricted to the set and can not be induced by SEA and C215 mAb unconjugated. The SEA-SEA-C215 conjugate 49

mAb but not C215 mAb mediated the extermination of T cells from MHC Class II Raji cells and interferon-treated MHC Class II Colo205 cells (Fig. 1).

To demonstrate that the SEA-C215 mAb conjugate mediated the lysis involved in the specific binding of the C215 mAb conjugate to the target cells, blocking studies were performed with excess C215 mAb and non-conjugated mAb C242, which bind to a irrelevant antigen from carcinoid cells of the celon (relative to binding to C215 mAb). Addition of mAb C215 strongly blocked cytotoxicity, whereas C242 mAb had no influence (Fig. 2). Similarly, if carried out by a conjugate of SEA-C242 mAb, it was specifically blocked by excess C242 mAb unconjugated but not by C215 mAb. The ability of the SEA-C215 mAb conjugate to induce T cell dependent lysis of the MHC Class II SW620 colon carcinoma cells was observed in CD4 + and CD8 + T cell populations (Table 2). SEA did not activate any of these T cell subsets to mediate extermination of SW620 cells but induced lysis of MHC Class II + Raji cells (Table 2).

The SEA-C215 mAb conjugate induced lysis of SE620 and Raji cells by an SEA-expanded T cell line, but not an SEB-expanded T cell line (Fig. 3). The specificity of the 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 mAb conjugate retains a specificity similar to V-beta TCR as for unconjugated SEA.

Picture's description

Fig. 1. SEA-C215 mAb conjugate directs CTL against MHC Class II colon carcinoma cells. The upper left panel demonstrates the effect of SEA-responsive CTLs against SW620 cells at various effector to target ratios in the absence (-) or presence of the SEA-C215 mAb conjugate, SEA-C215 and a mixture of C215 and SEA ( C215 + SEA) to a concentration of 1æg / ml of each additive. The other panels demonstrate the ability of SEA-C215 mAb conjugate, and SEA to target SEA-responsive CTLA to SEA against carcinoid cell lines of the C215 + MHC Class II-SW620, Colo205 and WiDr colon, and treated Colo 205 cells with interferon and C215 MHC Class II + C215 + cells and C215-MHC Class II + Raji cells. The ratio of effector to target was 30: 1. Addition of unconjugated C215 mAb at various concentrations did not induce any displacement of CTL against these cell lines. FCS analysis on SW I 620 cells, Colo205 and Widr cells using mAb against HLA-DR, -DP, -DQ did not detect any surface expression of MHC Class II while abundant expression of HLA-DR, -DP, -DQ in Raji HLA-DR and -DP cells in interferon-treated Colo205 cells. Colo205 cells were treated with 1000 units / ml of the recombinant interferon-gamma for 48 hours prior to use in the CTL assay.

Fig. 2. The SEA-C215 mAb conjugate and the SEA-C242 mAb conjugate induced CTL shift against colon carcinoma cells that depend on the antigen selectivity of the mAb. Lysis of the Colo205 cells by an SEA-responsive CTL line in the presence of the SEA-C215 mAb conjugate and SEA-C242 mAb (3 μg / ml) is blocked by the addition of C215 and C242 non-conjugated mAb (30 μg / ml ), respectively. Unconjugated mAbs or control medium (-) were added to target cells 10 minutes prior to conjugates.

Fig. 3. Lysis of the colon carcinoma cells coated with the SEA-C215 mAb conjugate is mediated by CTs responding to SEA but not to SEB. Selective and autologous T SEA and SEB cell lines were used at a ratio of effector to 10: 1 target against SW620 and Raji target cells in the absence (control) or in the presence of the SEA-C215 mAb conjugate, a mixture unconjugated C215 mAb and SEA (C215 + SEA) and C215 mAb and unbound SEB (C215 + SEB) at a concentration of 1 μg / ml of each additive. 51

Fig. 4. Cytotoxicity induced by the SEA-C242 mAb conjugate and SEA-Anti-Thy-1.2 mAb conjugate against its target cells (Colo 205 tumor cells and EL-4 tumor cells, respectively).

The SEA-C215 mAb conjugate binds to C215 + colon carcinoma cells and MHC Class II + Raji cells

Reagent Cell Analysis of SEA-C215 mAb Colo205 Pos Raji Pos C215 mAb Colo205 Pos Raji Neg SEA-C242 mAb Colo205 Pos Raji Pos C242 mAb Colo205 Pos Raji Neg SEA-anti-Thy-1.2 mAb EL-4 Pos Anti-Thy-1.2 mAb EL-4 Pos SEA Colo205 Neg Raji Pos control Colo205 Neg Raji Neg EL-4 Neg

The cells were incubated with the various control additives (PBS-BSA) for 30 minutes on ice, washed and processed as described below. The development of C215 mAb and C242 mAb bound to Colo205 and anti-Thy-1.2 cells bound to EL-4 cells was detected using 1 g of rabbit anti-mouse labeled FITC. Development of SEA to Raji cells was detected using a rabbit anti-SEA serum followed by 1 g anti-rabbit pig FITC. The development of the SEA-C215 mAb conjugate to Colo205 and Raji cells was detected using the procedures described above for C215 mAb and SEA. The FACS analysis was performed on a FACS device &quot; star plus &quot; of Becton and Dickinson. Disclosure with the second and third phases was only used to define the background noise.

Table 2

The CD4 + and CD8 + CTLs lysate the colon carcinoma cells presenting the C215-SEA conjugate. % cytotoxicity

(A) CTL (SEA-3) was used in target effector ratios of 30: 1 in the absence of (control) or in the presence of SEA and -SAA at 1æg / ml.

- 53 -

Claims (1)

A and A 'comprising residues of organic compounds F and F' respectively, at least one of said residues being a polymer (carrier) and having said properties having properties retained by the conjugate, and -B- is a bridge element which is covalently bonded to A and A ', characterized by one of the two compounds F and F' comprising a primary or secondary amino group, while the other comprises a thiol group I is a reactive group with thiol, and one of the compounds F and F 'is reacted with a heterobifunctional reagent of the formula: wherein n is an integer, wherein R 1, R 2, example 1 to 20, m is an integer 1 or 2, preferably 1, Z is a thiol group when one of the compounds F and F 'contains a thiol-reactive electrophilic group and a thiol-reactive electrophilic group when one of the compounds F and F 'contains a thiol group, R is alkylene having 1 to 4 carbon atoms, preferably &lt; 2 carbon atoms, possibly substituted with one or more (1 to 3, in the preferred case &lt; 2) hydroxyl groups (OH), Z 1 is carboxy activated, under conditions which allow the selective reaction at any of the Z 2 or Z 2 groups to form a -Sr bond wherein r is an integer 1 or 2 group or for the formation of a -CONH- group, respectively, in which group -CONH- the -CO- part is attached to (CH 2) of the heterobifunctional reagent of formula (III) and the part -NH- is a residue of said primary or secondary amino group, and then the product obtained is selectively reacted with the other part of said compounds F and F1. 2. A compound according to claim 1, wherein the group Z1 is (i) halogen which is attached to a saturated carbon atom, preferably in the form of an alpha-haloalkylcarbonyl, (ii) activated thiol, preferably a compound designated generally by reactive disulfide which is attached to a saturated carbon atom, or (iii) 3 , 5-dioxo-1-aza-cyclopent-3-en-1-yl. 3. A process according to any one of claims 1 to 2 characterized in that the group Z.sup.1 is chosen from carboxylic acid halides (-COCl, -COBr, and -COI), mixed carboxylic acid anhydrides (-COOOCR 3, reacting esters such as N-succinimidyloxycarbonyl, -C (= NH) -OR 2 and 4-nitrophenylcarboxylate (-CONO-C g NH 4 NO 2), wherein R 1 and R 2 may be lower (1-6C) alkyl and R 2 is also benzyl or A process as claimed in any one of claims 1 to 3 wherein the reaction medium is aqueous and the terminal reaction is carried out at a value of pH of less than 8 and the Z-terminal reaction is carried out at a pH in the range of 8 to 9.5. The process according to any of claims 1 to 4, characterized in that the polymer is a biopolymer which has a polypeptide structure or a saccharide polysaccharide structure. 6. A process according to any one of claims 1 to 5 wherein the polymer is an antibody. 7. A process according to any one of claims 1 to 6, characterized by n> g. 8. A process for the preparation of a heterobifunctional reagent characterized in that an amino-PEG-carboxylic acid of the formula NH 2 CH 2 CH 2 (OCH 2 CH 2) n O (CH 2) m COOH, wherein n is an integer from 1 to 20 in â € ƒâ € ƒâ € ƒwherein Z is a thiol group, a protected thiol group or a thiol-reactive group, B 'is an alkylene group having 1 to 4 carbon atoms possibly substituted with 1 to 3 hydroxy groups, and Z 'is a carboxy group, under conditions which allow amide formation between Z' and the NH 2 group of the PEG-carboxylic acid. 9. A process as claimed in claim 8 wherein Z is (i) halogen which is attached to a saturated carbon atom, preferably in the form of an alphahaloalkylcarbonyl (e.g. (ii) activated thiol, preferably a compound generally referred to as reactive disulfide which is attached to a saturated carbon atom, or (iii) 3,5-dioxo-1- aza-cyclopent-3-en-1-yl. A process according to any one of claims 8 to 9, characterized in that n> 2. A process according to any one of claims 8 to 10, characterized in that m = 1. The applicant claims the priorities of the Swedish patent applications filed on July 20, 1990, under Nos. 9002484-5 and 9002490-2. Lisbon, July 19, 1991 60