IE921898A1 - Tri- and tetravalent mono specific antigen binding proteins - Google Patents

Abstract

The invention provides a tri- or tetra-valent monospecific antigen-binding protein comprising three or four Fab fragments bound to each other covalently by a covalently hy a connecting structure, which protein is not a natural immunoglobuline. Further provided are novel connecting structures for use in assembling the protein of the invention are useful, for example, in the treatment and diagnosis ofcancer.

Description

TRI- AND TETRA-VALENT MONOSPECIFIC ANTIGEN-BINDING PROTEINS The present invention relate· to tri- and tetra-valent 5 Monospecific antigen-binding protein· and to Methods for their production as veil as to tri- and tetra-valent ligand· for their construction. The invention relates in particular, but not exclusively, to the use of recombinant DNA technology to produce such tri- and tetra-valent Monospecific antigen-binding proteins.

There has been such interest in recent years in antibodies and thair- fragments.-- It is veil known that complete antibody Molecules are made up of heavy chain and light chain heterodimers. For instance an IgG Molecule comprises four polypeptide chains, tvo heavy-light chain heterodimers.

Bach light chain consists of two domains, the N-terminal domain being known as the variable or VL domain and the Ctermine1 domain being known as the constant or CL domain. Each heavy chain consists of four or five domains, depending on the class of the antibody. The N-terminal domain is known as the variable or VH domain. This is attached at its C-terminal end to the N-terminal and of tha next domain, which is known as ths first constant or CHI domain. The next part of each heavy chain ia known as the hinge region and this is then followed by the second, third and, in some oases, fourth constant or CH2, CH3 and CH4 domains respectively.

In an assembled antibody, the VL and VH domains associate together to form an antigen binding site. Also, the CL and CHI domains associate together to keep one heavy chain associated with one light chain. Two heavy-light Chain heterodimers associate together partly by interaction of the CH2, CH3 and, if present, CH4 domains of the tvo heavy chains and partly because of interaction between tha hinge regions on the two heavy chains.

Sach heavy chain hinge region includes at least one, end 5 often several, cysteine residues. in the aeeembled antibody, the hinge regions of the heavy chains are aligned so thet in ter-chain disulphide bonds can be forned between the cysteine residues in the hinge regions, covalently bonding the two heavy-light chain heterodimers together.

Thus, fully aeeeahled antibodies are at least bivalent in that they have at least two antigen binding sites.

It haa..bean-known .for atone long tine that if the disulphide bonds in an antibody’s hinge region are broken by mild reduction, it is possible to produce a monovalent antibody coaprising a single heavy-light chain heterodiner.

It has also been known for soae long tiae that treatment of antibodies with certain proteolytic ensymee leads to the production of various antibody fragments. For instance, if an antibody is cleaved close to the N-terminal side of each hinge region, two antigen binding fragaents (Fab) and one constant region fragment (Fc) are produced. Each Fab fragment comprises the light chain associated with a truncated heavy chain comprising only the VH and CHI domains. The Fo portion comprises the remaining domains of the heavy chains held together by the hinge region. alternatively, the antibody may be cleaved close to the C30 terminal side of the hinge. This produces a fragment known as the F(ab')2 fragment. This essentially comprises two Fab fragments but with the CHI domains still attached to the hinge regions. Thus, the F(ab*)2 fragment is a bivalent fragment having the two antigen binding sites linked together by the hinge region. The P(ab')3 fragment can be cleaved by reduction to produce a monovalent Fab* fragment. This can be regarded ae being a Fab fragment having on it a hinge region.

It has also proved to be possible, by careful control of digestion conditions, to claavs an antibody between the VL and CL and between the VH and CHI domains. This gives rise to two fragaents known aa Fv fragaents. Bach Fv fragment comprises a VL and a VH domain associated with one another. Bach Fv fragment is monovalent for antigen binding.

Studies of the amino acid sequence of individual variable 10 domains has shown that there are three areas in each variable domain where the sequence varies considerably.

These areas have been termed hypervariable regions or complementarity determining regions (CDRsy. The location of these CDRs has been published [Rabat, 2.A. et al., in Sequences of Proteins oi Immunological Interest, US Department of Health and Human Services, NTH, USA, 1987 and Hu, T.T. and Rabat, 2.A., J. Exp. Med., 112, 211-250, 1970].

Structural studies on crystallised Fv fragments and molecular modelling studies have shown that each variable doaain consists of three loop regions supported on βpleated sheet framework regions. In the case of hapten antigen binding the loop regions appear to form a pocket for receiving the antigen.

There is considerable overlap between the CDRs, as determined by sequence analysis, and the loop regions, as determined by structural analysis. However, it is generally accepted that the CDRs, possibly in combination with some extra residues present in the loop region, are primarily involved in determining the antigen binding specificity of the antibody.

In more recent years, there has been much interest in producing antibodies or their fragments by use of recombinant DMA technology. The patent literature is replete with disclosures in this area. Recombinant DNA technology has been used not only to reproduce natural antibodies but also to produce novel antibodies. For instance, it ia now possible to produce ahimerio antibodies, wherein tha variable domains frcm one species are linked to constant dona ins from another species.

It is also possible to produce modified antibodies, in which the residues in the CORs and, if necessary, a number of other residues in the variable domains have been changed so that a different antigen can be bound. This is a useful procedure in that it allows a specificity from, for instance, a mouse monoclonal antibody (Mhb) to be created in a human antibody without altering the essentially human nature of the antibody.' This has advantages where Itie desired to use the antibody io vivo. A further discussion is given in W0-A-91/09967.

WO-A-90/09195 and WO-A-90/09196 relate to cross-linked antibodies and processes for their preparation. Cross linked antibody conjugates are described which have at least one non-disulphide (S-S) interchain bridge optionally containing a reporter or effector molecule. The bridge may be the residue of a homo- or hetero-functional crosslinking reagent and is located away from the antigen binding domains of the antibody. The antibody conjugates have an enhanced binding capacity, in vivo have good blood clearance and, in the presence of a tumour, high tumour : blood and tumour : bone ratios. The conjugates are of use in the diagnosis and therapy of tumourb.

They may be prepared by reaction of a cross-linking reagent with an antibody or a fragment thereof. The cross-linking reagent nay react either with thiol groups on the antibody molecules or with the side chains of amino acid residues such as glutamic acid, aspartic acid, lysine or tyrosine residues.

However, we have found that while cross linked antibodies as described in HO-A-90/09195 and WO-A-90/09196 have improved properties over natural immunoglobulins and in particular exhibit highly successful binding to tumour cells and good clearance from the blood, they are subject to high uptake by the kidneys and are retained in this tissue. This creates a toxicity problem, particularly when the antibody ie radiolabelled for use in therapy and radioimaging. What ie required is therefore an antibody molecule which retains the superior binding and clearance properties of cross-linked antibodies but which is not taken up or retained by kidney IQ tissue and thus avoids kidney toxicity problems.

W0-A-91/03493 relates to bi- or tri-valent multispecific Fab conjugates. Thaxuanjiigates which are described comprise three or four Fab' antibody fragments linked together using orthophenylenedimaleimide bridging structures. The disclosed trimeric conjugates comprise either two Fab' fragments of a first specificity and one Fab' fragment of a second specificity or three different Fab' fragments each of different specificities. Thus, the triaeric conjugates are either bi-or tri-specific. In a similar fashion, the disclosed tetraneric conjugates are at least bispecific and may be tri- or tetra-specifio. it is reported in WO-A-91/03943 that, in certain circumstances, a population of T lymphocytes can be induced to kill target cells, such as tumour cells, by treatment with a bispecific dimeric conjugate, wherein one specificity is directed at a specific antigenic structure on the Tlymphocyte population and the other specificity is directed at an antigen on the target cells. This effect is referred to as redirect cellular cytotoxicity (RCC).

The invention disclosed in WO-A-91/03493 is based on the assertion that RCC can be significantly improved by use of trimeric or tetrameric multispecific conjugates. use of such conjugates also allows the range of T lymphocyte antigens which can be specific to be increased. It is thus essential to the invention claimed in WO-A-91/03493 that the tri- or tetra-merie conjugates should be at least biepecific. λ more detailed discussion of the invention disclosed in W05 A-91/03493 is found in Tutt, A. et al., Eur. J. Immunol., 21. 1351-1358, 1991, which confirms thet it is essential, in order to enhance RCC, to use tri- or tetra-serie conjugates which are at least biepecific. However, it should bs noted that nowhere in WO-A-91/03493 or Tutt et al., supra, are the clearance properties of tri- or tstra-seria Fab conjugates discussed.

It has been .suggested.,. An Tour copending international Patent Specification No. W091/19739, that multivalent antigen15 binding Fv fragments will be of use in imaging or treating tumours in vivo.

A further requirement for multivalent antigen binding protein* such as those discussed above is for a cross linking molecule capable of cross linking antibody fragments together. Xn addition to its cross linking function, such a cross linking molecule can advantageously provide for the introduction of effector or reporter molecules to the antibody conjugate.

A number of cross-linking molecules have been deeoribed. For example, European Patent specification No. 0446071 (Hybritsch Incorporated) discloses the production of trifunctional cross linkers for use in the production of bi30 specific trimeric antibody-like molecules. The application of such tris-maleinide compounds to the production of bi- or tri-specific trivalent antibody-like compounds is disclosed in European Patent Application 0453082 (Hybriteah Incorporated). The clearance properties of the antibody conjugates disclosed are not referred to. A distinct drawback of the disclosed linkers is that it is difficult to attach a functional group such as a radioisotope thereto.

In particular a aacrocyole aroee-linking group ia not easily incorporated into such linkers.

The present invention ie based on the discovery that triS and tetra-valent Monospecific Fab-like proteins are particularly suitable for anti-cancer therapy. These proteins demonstrate the superior binding and clearance properties of cross-linked antibodies but are not taken up and/or retained by non-tumour tissues, including kidney tissue. In addition, the present invention provides novel linker Molecules which greatly facilitate the attaohaent of reporter or effector groups to tri- or tetra-valsnt Fabliks proteins.· , - -Therefore, according to the present invention, there is provided a tri- or tetra-valent Monospecific antigenbinding protein comprising three or four Fab fragments bound to each other by a connecting structure, which protein is not a natural immunoglobulin.

The Multivalent antigen-binding proteins of the invention are referred to herein as TFM (tri-Pab) and QFM (tetraFab). It will be understood that the expression Fab is used herein to include optionally Modified Fab and Fab' antibody fragaents derived froa natural antibodies or synthesised, either cheMically or by recosbinant DMA technology. By optionally modified" is Meant that the Fab or Fab* fragaent aay contain a number of insertions, deletions or changes in the aainc acid sequence, as long as the binding ability of the fragaent is not adversely affected.

Preferably, in compounds according to the invention the Fab fragaents are bound together covalently by the use of a single linker Molecule. surprisingly, it has been observed that TFM and QFM have markedly superior character let ice to whole antibodies, Fab, F(ab')2 and monospecific cross-linked derivatives of these fragments. While Fab, F(ab')2 and their cross-linked counterparts are relatively specific for tumour cells when used in vivo, TFM and QFW show a greatly increased avidity compared therewith. At the same time, they are eliminated from the blood much more efficiently than whole antibodies. Furthermore, in contrast to previously described monospecific cross-linked Fab and P(ab’)2 fragments, TFM and QFM do not accumulate in the kidney. This gives rise to a decrease in undesirable side-effects, particularly where the antibody molecule is conjugated to a toxin or a radioisotope for anticancer therapy.

Preferably, the multivalent Fab-like proteins of the invention are specific for a tumour-associated antigen. Advantageously, therefore, at least the CDRs of the Fab fragments are derived from a tumour-specific monoclonal antibody (MAb). Alternatively, the CDRs may be synthetic. It will be appreciated that any tumour-specific antigen may be targetted by the Fab-like proteins of the present invention.

The TFM or QFM compounds of the invention may be labelled by one or more reporter or effector groups, for example the types described below. The label may be incorporated on the Fab portion of the TFM or QFM molecule, and/or on the connecting structure linking the Fab portions to each other. Where the Fab portion itself is labelled, the label will generally be located such that it does not interfere with the binding site of the fragment. Methods of labelling antibodies with a reporter or effector group are well known, and are described in our published patent specifications EP 238196, EP 384624, BP 385601, WO88/O5433, W089/01475, W089/01476 and W090/01475. Where it is desired to include a reporter or effector group in the connecting structure, this may be achieved by reaction of the reporter or effector group with a reactive functional group present in the connecting structure, for example in analogous fashion to that used for th· labelling of th· Fab fragment, or the reporter or effector group may be advantageously built in to the connecting structure, for example as described below.

Preferably, in the TFM and QFM compounds of the invention, the Pab monomer· are cross-linked together by a crosslinker. The cross-linker may be any chemical capable of linking the Fab fragments together. Preferably, however, the cross-linker1 is a specifically designed chemical compound such as the maleimide compounds described in SP« A—0446071 and 1P-A-O453O82, although it will be understood that any structure having three or four functional groups reactive, with.any reactive amino^acid fmind on'an antibody chain may be used. in one preference the connecting structure In the compounds of the invention is a poly lysine linker.

According to a second aspect of the invention, therefore, we provide a cross-linking agent of formula (1); R1CH(R2)HHCOR3 (1) wherein R1 is a carboxyl (-co2H) or esterified carboxyl (25 OO^R) group or a group -COA where A is an effector or reporter molecule attached to the -CO group either directly or via a spacer group to form a carbon-carbon, or carbonhetero atom linkage; R2 and R3, which may be the same or different, is each an optionally substituted straight or branched alkylene, alkenylene or alkynylene chain [optionally interrupted by one or more -o- or -S- atoms, or -N(R4) (where R* ie a hydrogen atom or a Cj_6 alkyl group) , -M(R4)C0-, -CON(R*)-, Cs_e cycloalkylene, c$_12 arylene or C5-io ^dt^foarylene groups) containing one or more reactive functional groups such that the total number of reactive functional groups in R2 and R3 together is three cr more.

In the compound· of formila (l), the tern effector group" is to be understood to mean any group capable of eliciting a change in, or a response from, a biological system and which also confers this property to the compound of formula (1). The term reporter group is to be understood to mean any group which ie detectable by analytical means in vitro and/or in vivo and whioh confers this property to the compound of formula (1)10 Zffactor groups Include, for example, any physiologically active substance, antibacterial, antiviral or antifungal compound. Particular physiologically active substances includa_antinaoplastJ.c agent·, toxins (such as enzymatically active toxins of bacterial or plant origin and fragments thereof e.g. rioin and fragments thereof), enzymes, antiflasmatory compounds and substances active as cardiovascular, e.g. fibrinolytic, and central nervous system, agents.

Particular antineoplastic agents include cytotoxic and cytostatic agents, for example alkylating agents, such as nitrogen mustards (e.g. chlorambucil, melphalan, machlorethamine, cyclophosphamide, or uracil mustard) and derivatives thereof, trlathylenephosphoramide, triethylenethiophosphoramide, busulphan, or cisplatin; antimetabolites, such as methotrexate, fluorouracil, floxuridine, cytarabine, aercaptopurine, thioguanine, fluoroacetic acid or fluorocitric acid, antibiotics, such as bleomycins (e.g. bleomycin sulphate), doxorubicin, daunoruhicin, mitomycins (e.g. mitomycin C), actinomycins (e.g. dactinomycin) plioamyoin, calichaemicln and derivatives thereof, or esperamicin and derivatives thereof; mitotic inhibitors, such as stopoaide, vincristine or vinblastine and derivatives thereof; alkaloids, such as elliptioine; polyols such as taxicin-I or taxicin-li; hormones, suah as androgens (e.g. dronostanolone or testolactone), progestine (e.g. megestrol acetate or medroxyprogesterone acetate), estrogens (e.g. dimethylstilbestrol diphosphate, polyestradiol phosphate or eatramuetine phosphate) or antiestrogens (e.g. tamoxifen); anthraquinones, such as mitoxantrone, ureas, such as hydroxyurea; hydrazines, such as procarbazine; or imidazoles, such as dacarbazine.

Particularly useful effector groups are oalichaemicin and derivatives thereof (see for example South African Patent Specifications Nos. 85/8794, 88/8127 and 90/2839).

Suitable reporter groups include chelated metals, fluorescent compounds or compounds which may be detected by nmr or.ppectrgscppyZ’ Chelated metals include chelates of di- or tripoeitive metals having a coordination number from 2 to 8 inclusive. Particular examples of such metals include technetium (Tc), rhenium (Re), cobalt (Co), copper (Cu), gold (Au), silver (Ag), lead (Pb) bismuth (Bi), indium (In), gallium (Ga), yttrium (¥), terbium (Tb), gadolinium (Gd), and scandium (Sc). In general the metal is preferably a radionuclide. Particular radionuclides include *Tc, l66Re, 188Re, seCo, WCO, e7Cu, 195Au, 199au, noAg, 203Pb, 206Bi, 307Bi, luln, *7Ga, wGa, eeY, »°Y, lwTb, ls3Gd and «7Sc.

The chelated metal may be for example one of the above types of metal chelated with any suitable polydentate chelating agent, for example cyclic polyamines, polyethers, (e.g. crown ethers and derivatives thereof); polyamides; porphyrins; and carbocyclic derivatives.

In general, the type of chelating agent will depend on the metal in use. One particularly useful group of chelating agents in conjugates according to the invention, however, are acyclic and cyclic polyamines, especially polyaminocarboxylie acids, for example diethylenetriaminepentaacetic acid and derivatives thereof, and ma cro eye lie amines, e.g. cyclic tri-aza and tetre-aza derivatives; and polyamides, especially desferrioxamine and derivatives thereof.

Examples of particular macrocyclic amines include compounds of foraula (2)s I -CH J, CCHiX' V/1 I ft <2> (wherein L ie a substituent containing a reactive group, B is a C2-i4 alkylene chain interrupted by one or tvo optionally substituted nitrogen atone; W1 end W2, which nay be the ease or different, is each an optionally substituted nitrogen atom; p is zero or an integer 1 and q is zero or an integer 1 or 2 with the proviso that when p is zero, q is an Integer 1 or 2}. It will be appreciated that the group L provides an attachment point for the macrocycle to the rest of the compound of formula (1}. Typical groups include for example amine (-NH?) containing groups.

Preferred amines of formula (2) include tri-aza derivatives of formula (3): (3) [wherein νχ and Wa which may be the same or different is each a group -F(where r is zero or an integer 1 to 6 and R1 ie an alkyl, alkoxyalkyl, -COaH, -SO3H, -PO3Ha or aryl group) and B is a group -CHa(CHa)eF(R) (where e and t, which nay be the ease or different is eaah zero or an integer 1, 2 or 3; and R represents -(CHj)rRl where r and Rl are ae just described) ]; and tetra-aza derivatives of formula (4); £> (4) (wherein w1 -and v? -which-, say be the sane or different ie each a group -N( (CH2)rRl]- (as just defined) and B is a group -CH2(CH2)BM(R)CH2(CH2)iN(R) (CH2,tCH2- (where d is zero or an integer 1, 2 or 3 and e, t and R are as just defined]. λ particularly useful amine of fornula (3) is the oompound of formula (5) •CHiCOx H L (5) A particularly useful anine of formula (4) is the compound of fornula (6): HOjCHiC CH2COxH (6) Preferred chelated metals in conjugates according to the Invention include indium chelated by a compound of formula (3), particularly the compound of formula (5); or yttrium chelated by a compound of formula (4), particularly the compound of formula (β). X11ln and ^Y are particularly preferred.

The effector or reporter group may in general be attached to the remainder of the compound of formula (1) via any suitable carbon atom or heteroatom, e.g. nitrogen, oxygen, sulphur or phosphorous atom, present in it, either directly to form a compound A-C0CH(R2)HBC0R3 or indirectly to form a compound- A^ep~eec£ fa^ifHcOR3 where Sp is a spacer group attached independently to A and to -CO- group through a carbon-carbon or carbon-heteroatom linkage as just described. Suitable spacer groups include acylic or cyclic aliphatic or aromatic residues in particular alkylene [e.g. ethylene, propylene, butylene], alkoxyalklene [e.g. methoxymethylene, ethoxymethylene, ethoxyethylene], arylene [e.g. phenylene] aralkylene [e.g. phenalkylene such as phenethylene] or cycloaIkylalkylene [e.g. cyclohexylmethylene] groups.

The linkage between A and the group -CO- or A and the spacer group may if desired be chosen so as to be cleavable, such as by proteolytic enzymes, for example as described in European Patent Specification Ho. 175617.

Esterified carboxyl (-COjR) groups represented by R1 in compounds of formula (l) include those groups wherein R is an organic group, for example an acyclic aliphatic group, or an aromatic or heteroaromatic group.

Thus R may be an optionally substituted straight or branched Cj.jq alkyl, (e.g. methyl, ethyl, n-propyl, i-propyl, spropyl, n-butyl, i-butyl, β-butyl, t-butyl), or C2_2o alkynyl group optionally interrupted by one or more -0* or -S- atoms; or a C5_B cycloalkyl (e.g. cyclopentyl or cyclohexyl), Cs_e cycloalkyl ci-« alkyl (e.g.cyclopentylmethyl, cyclohexylmethyl), Cg_12 aryl (a.g. optionally substituted phenyl or naphthyl) ce-ia or ci-« alkyl (e.g. optionally substituted benzyl, phenethyl, or naphthylnethyl), C5_10 heteroaryl (e.g. furanyl, pyridyl, thienyl) or cs_10 heteroaryl Cx_6 alkyl (e.g. furanylmethyl, pyridylmethyl, or thieny lmethyl) group.

The reactive Functional group In compounds of formula (1) nay in general be any group capable of reacting with a thiol, anino, carboxyl, hydroxyl, aldehyde, aromatic or heteroaromatic group. Aromatic groups include, for exaaple, phenolic grouper. Meter oar onatic groups include, for exaaple, imidazolyl groups.

Thus, the reactive functional group may be, for example, a halogen atom, for example a chlorine, bromine or iodine atom, or a group selected from -SH, -S-S-Het (where Het is an optionally substituted heterocyclic group, e.g. an optionally substituted pyridyl group), -NH2, hydrazine (NHNH2) or a derivative thereof, [for example -N(CH3)NH2, NHCONHNHj, -NHCSNHNHj or phenyl hydrazine], haloacetamide (e.g. iodoacetamide or bromoacetamide) -NCO, -NCS, -COR1®, [where R1® is a halogen atom such as a chlorine or bromine atom, or a K3, alkoxy, e.g. methoxy, C6_12 aryloxy (e.g. nitrophenyloxy or dinitrophenyloxy) imidyloxy (e.g. succinimidyloxy) or imidazolyoxy group], imide, e.g. maleimide, a vinyl group of formula -Hetl-C(Het2)CHj (where Het1 and Het2, which may be the seme or different, is each a nitrogen containing heterocyclic group, e.g. a pyridyl group or Het1 is a nitrogen containing heterocyclic group and Het2 is a hydrogen atom), for example a vinyl pyridyl group of formula Ο e.g. «ethyl, group). especially or a dione of formula (where R11 is a c,„4alkyl, groups, imide.

In general, compounds of fomula (1) in which the reactive functional groups are the sane are preferred, although for sone uses it nay be preferable to have «ore than one type of reactive functional. -$eoup. Particularly preferred functional groups are those capable of reacting with thiol groups. Groups of this type include imide (particularly maleimide), haloacetanide (particularly iodoacetamide), SH, Het-S-S-, -Het1(Het2)-CH2 or especially naleinide, groups are particularly useful.

The compounds of formula (1) may contain three or more reactive functional groups, depending on their intended use. Useful compounds include those containing three or four reactive functional groups, particularly three of four thiol-reactive groups, e.g. three or four maleimide groups, although if desired five, six, seven or eight such groups may be present.

The reactive functional groups nay be distributed in the groups R2 and R3 is any desired way. Thus, for example, each of R2 and R3 nay contain 1, 2, 3 or more reactive functional groups (providing the total number in both is three or more). Alternatively, the reactive functional groups may be in one of R2 or R3 only.

Th· groups St3 and R3 in coapounds of formula (1) fore a template to which th· reactive functional group· are attached and may ha varied within any desired size and composition. Thus, on· particularly preferred, hut not limiting, group of compounds of the invention has the formula (l) wherein R1 is as defined above and R2, which may ba the same or different is each an optionally substituted straight or branched C1-25 alkylene (e.g. C2_le alkylene such as methylene, ethylene, propylene, butylene, pentylene, hexylene or heptylene), C2.23 elkenylene or C2_20 alkynylene chains, [optionally interrupted by one or more -o- or -satoms, -N(R*)- (where R4 _ie a hydrpgenatoa or a alkyl group such-4), C5_8 cycloalkylene (e.g, cyclopentylene or cyclohexylene), 15 c6-ia aryiene (β·9· phenylene or substituted phenylene) or cs-io heteroarylene (e.g. furanyl, thienyl or pyridinyl groups)] containing one or more reactive functional groups such that the total number of reactive functional groups in R2 and R3 together is three or aore.

Optional substituents present in the groups R2 and R3 include carboxyl (-CO^H) and esterified carboxyl (-CO2R) [where R is as defined above] and amino (-OT^) or substituted amino (NR^R7) [where R6 and R7, which may be the same or different, is each a hydrogen atom or a C1_6 alkyl group, or a group -COR8 where R8 is as defined for R2, providing that when one of R6 and R7 is a hydrogen atom, the other is not, and when one of R6 and R7 ie a group -COR®, the other is a hydrogen atom].

It will be appreciated that when one, or both of R2 and R3 contains a substituent -NHCOR8 this allow for further reactive functional groups to be built into the compound of formula (.1).

Particularly useful groups R2 or R3 may have a structure 8) (CHj)B»HCO(CH2)(Where m, n and p, which may be tbe same or different ia each an integer 1, 2 or 4 and Z ia a reactive functional group as defined above.

Particularly useful groups of compounds of the invention (8) Another preferred cross-linking agent hae the formula (10): R9CH(R2)CONHCH(R2)CONHCH(R2) conh2 (10) where R9 is -NH2 or a substituted amino group, e.g. a group -NHCOA, and A and R2 are as defined for compounds of formula (1). It will be appreciated that in compounds of this type each R2 group may be tbe same or, if desired, different to its neighbour.

The compounds of formulae (1) and (10) are of particular use for cross-linking biological materials, especially proteins, and in particular antibodies, providing the biological material (a) have one or more functional groups capable of reacting with the compound of foraulae (1) or (10). The compounds are particularly useful for producing TIM and QFM compounds according to the invention.

The cross-linking reaction may be achieved using conventional processes, for exaaple by mixing the starting materials, such as Fab fragments and the appropriate linker, in an aqueous solvent, e.g. at ambient temperature. The relative concentrations of the starting materials used vill depend to a large extent on the coapound of formula (1) or (10) and the number- of reactive functional groups r it contains, and the nature of tbe desired product, but generally the biological material(s), e.g. proteins such as an antibodies, e.g. a Fab fragment, will be present in excess concentration.

The compounds of formulae (1) and (10) may be prepared by a number of processes, for example as described In the examples appended hereto. In these processes reactive groups may need to be protected, when it ia desired that they do not participate in a particular reaction. Conventional carboxylic acids may be esterified (for example to generate benzyl esters) and amino groups may be acylated (for example to generate benzyloxycarbonylamino groups) · The protecting groups may be removed using conventional procedures, for example in the case of a benzyl ester by treatment with an acid, e.g* formic acid, and in the case of a benzyloxycarbonylamino group by treatment with a coapound suoh as trimethylsilyl iodide.

Thus, for example, compounds of formula (l) wherein R1 is a group -COA or -CO-SP-A may be prepared by reaction of a corresponding compound wherein R1 is a group -CO2H or an activated derivative thereof (for example a succinimide, e.g. obtained by reaction of the acid with Nhydroxyauccinimide in the presence of Ft dicyclohexylcarbodiimide) with the group A or SP-A, optionally in the presence of a base, in a solvent such as an ether, e.g. a cyclic ether such as tetrahydrofuran. in this reaction the starting material A or Sp-A will require a group capable of reacting with the acid-activated derivative thereof. Such groups Include, for example, amino and hydroxyl groups.

Compounds of formula (1) wherein R1 is a -CO^H group may be 10 prepared by hydrolysis of the corresponding ester (-COjR), using conventional procedures, for example by hydrolysis using an acid e.g. trifluoroacetic_acid, in an inert solvent such as a -halogen^ted'hydrocarbon.

In general the compounds of formula (l) in which R1 is a C02R group may be prepared in a step-wise fashion from an esterified amino-acid starting material of formula (11): ROOCCH(Y)NH2 (11) (where Y is a side chain containing a reactive group [e.g. an amino (-NM2) group] or a displaceable group (e.g. a halogen atom) ] using a series of displacement or condensation reactions involving other intermediates with appropriate reactive groups using conventional procedures. The general synthetic principle may be illustrated by reference to the intermediates and examples described herein where the preparation of certain compounds according to the invention is illustrated using a known starting material.

Other compounds according to the Invention may be prepared using the same approach but with different starting materials and intermediates to introduce other types of groups R2 and R3 containing different reactive functional groups.

Compounds of formula (10) may be prepared in analogous fashlo using displacement and condensation reactions for η n example ae illustrated in the intermediates and examples set out herein.

The performance of any suitable reactive functional group is 5 always subject to the structural constraints placed upon it by the linker molecule itself. It has been found that increased linearity of the linkers facilitates tbe addition of a macrocycle and the chelation of an effector group.

Therefore, the invention comprises novel linkers which have a substantially linear backbone structure and are capable of accomodating a macrocycle group.

Particularly preferred are ligands of the formula: (12) wherein R1 is as described above and Hal is a ma1eimide group.

Preferably, the TPM or QFM of the invention is a tri- or tetra-valent monospecific antigen-binding protein comprising three or four Fab fragments bound to each other by a linker having attached thereto a macrocycle.

It will be understood that although the attachment of the macrocycle to the linkers of the invention is preferred, it is also possible to attach the macrocycle to one or more*of the Fab fragments incorporated into the multi-valent proteins of the invention. This approach is particularly preferred where the linker used does not facilitate the attachment of a macrocycle group.

Preferably, therefore, the TFM or QFM of the invention contains a radiolabel. The radiolabel is chelated by the macrocycle.

In a further preference the Feb fragment in each TFK or QFM compound according to the invention are bound to each other by a connecting structure linked to a thiol group on each Fab fragment.

Particularly preferred are tri- or tetra-valent protein constructs of the invention in which the connecting structureis. one of the following-linkers: MH-VuA (CT998J Or: HWMaA I .

I Z-OH I (CT557) CO pH Or: I - C* & **** (CT558) Naturally-occurring Fab' fragments have a number of thiol groups in tb* hiijga T*gion, typically two, four or even eleven. in an advantageous embodiment of the present invention, however, genetically modified Fab’ fragments are used which have only a single free thiol group in the binge region. Construction by recombinant DNA technology of such Fab* fragments, referred to as 6 cys Fab' fragments, is described in our copending European patent application No. 0347433.

Decreasing the number of cysteine residues in the hinge region of a Fab-like fragment such as a Fab' advantageously decreases the possibilities of incorrect interaction between the Fab-like molecule and the linker molecule.

Normally, purified Fab' fragments produced by recombinant DNA technology are recovered with blocked hinge thiol groups, .in this instance, Fab* fragments are preferably partially reduced before assembly into TFK or QFM compounds of the invention.

Preferably, the Fab* fragments are cross linked using a cross-linker of formula (1) or (10). Most preferably, the cross-linker is one of the structures depicted in the examples attached hereto. Most preferably, the linker is CT998, CT557 or CT558.

P. 27 The tri- or tetra-valent monospecific antigen binding proteins of the invention may be used for la vivo diagnosis or therapy.

Thus the invention also includes tri- or tetra-valent monospecific antigen-binding proteins according to the Invention having attached thereto diagnostically or therapeutically functional effector molecules, atoms or other species. Any of the effector or reporter groups described above may be included.

The proteins of the invention are of use for In vivo di agnostic.or. Jthe^apeutic purposes. Thus, the invention also includes diagnostic or therapeutic compositions for ia vivo use comprising an effective amount of a protein according to the invention in combination with a pharmaceutically acceptable diluent, excipient or carrier.

The composition may comprise other active ingredients.

The composition may take any suitable form for administration, and may, in particular, be in a form suitable for parenteral administration, e.g by injection or infusion, for example by bolus injection or continuous infusion. Where the composition is for injection or infusion it may take the form of a suspension, solution or emulsion of the protein of the invention in an oily or aqueous vehicle and it may contain formulatory agents such as suspending stabilising and/or dispensing agents.

Alternatively, the compositioin may be in a dry form, for reconstitution before use with an appropriate sterile liquid.

The dose at whioh the protein according to the invention may be administered will depend on whether the protein is being used for diagnosis or treatment, on the nature of the condition to be diagnosed or treated, on whether the protein is being used prophylaotioally or to treat an exisiting < * πτυ_ηο τσπ m · ie 071 405 4166 P.28 condition and on th· particular Fab fragment and effector or reporter group selected. Dose will also be selected according to age and condition of the patient. Thu·, for example, doses in the range 0.01 to lOmg/Kg/day may be used.

Advantageously, since the compounds according to the invention are cleared rapidly from the blood, multiple dosing regimes may be used.

Moreover, the invention includes methods of diagnosis or 10 therapy comprising administering an effective amount of a protein of the invention to a human or animal subject.

Most preferably, the method of the*invention is directed to the tratment or diagnosis of cancer.

The Invention further comprise® the use of a tri- or tatravalent protein as described in the preceding aspects of the invention for the treatment of an ailment, preferably cancer. Furthermore, the invention comprises the use of a tri- or tetra-valent protein according to the invention in the aanufacture of a composition for the treatment of the ailment, which is preferably cancer.

The present invention is now described, by way of example only, with reference to the accompanying drawings, In which: figure l is a graph showing an HPLC analysis of a crosslinking reaction as performed according to the following examples; figure 2 shows the results of an antigen-binding ELISA ooaparing monomeric, dimeric, trimeric and tetrameric Fab* proteins; figure 3 is a graph shoving the improved off-rate of the trimeric Fab'-llke proteins (TFM) of the invention; 51- riiN-qo TWH 11:17 071 405 4166 P.29 figure 4 is a graph shoving the blood clearance performance of T7M compared to whole IgG; figure 5 compares the tissue distribution of TFK end IgG 5 administered to tumour-bearing mice; figure 6 depicts the tumour:blood ratio of TFM and IgG adiminietered to tumour-bearing mice; figure 7 shows biodistribution data similar to that shown in figure 5, taken at various time-points after administration of the antibody constructs; figure 9 is a graph showing an BPLC analysis of a cross15 linking reaction for the formation of a QFM (tetra-Fab) molecule; figure 9 shows biodistribution data for QFH constructs; figure 10 compares the biodistribution of site-specific and random TFK constructs using the CT998 linker; figure 11 demonstrates the increased avidity for antigen of TFK over IgG; and figure 12 and figure 13 show the biodistribution performance of A5B7-specific TFK. 1- TIIN-09 TWH 11:17 071 406 4166 P.30 fiXjUBEjLM A. synthesis of linkers The following examples describe the synthesis of linkers according to the invention. In the construction of linkers, a number of intermediate compounds are used. The following abbreviations are used in the Examples: BOC t-butoxycarbonyl 10 Z bensyloxycarbcnyl THF tetrahydrofuran TFA trifluoroacetic acid jp- MAL 15 -CO(CHa)2N^jJj © DMF dimethylformamide BOC-Lys(B-Z) acid (B.O2g) was dissolved in dry THF (80ml) under Nj. The temperature of the reaction mixture was lowered to -20*C and ethylohloroformate (2.29g, 2.02ml) and N-methylmorpholine (2.13g, 2.31ml) were added, maintaining the temperature at -20*C. After 30 min ammonia (16ml of a 2M solution in methanol) was added and the reaction allowed to come to room temperature. The organic layer was added to a saturated sodium bicarbonate solution and the aqueous * * π η» Tint ί 1 · 1 o 071 ίπς d1R8 P. 31 layer extracted with ethyl acetate, dried (MgS04) and evaporated to give Intermediate l (6.5g) as a fine white solid. 1HNMR (CO3OD) S 7.5-7.2 (a) 5H, 5.1 (s) 2H, 4.0 (a) 1H, 3.12 (t) 2H, 1.84-1.32 (a) 15H.

Interwadiafca, a ft** CF^GCO* Intermediate l (4.1g) was dissolved in a 1:1 solution (50al) of TFA and CH^C 12 and the reaction mixture stirred at room temperature for 30 min. The solvent was evaporated and the residue triturated with ether, and dried to give Intermediate 2 (4.1g) as a white solid. 1HNMR (CD30D) g 7.5-7 (m) 5H, 5.1 (e) 2H, 3.85 (t) IH 3.15 (t) 2H, 1.95-1.75 (Β) 2H, 1.6-1.35 (m) 4H.

BOC-Lys(E-Z) acid (4.2ig), Intermediate 2 (4.l5g), ethylchloroformate (l.2lg), 1.06ml) and n-sethylmorpholine (i.l2g, l.2iml} were reacted together as described for Intermediate 1 to yield Intermediate 3 (7.3g). ΓΗΚ!_Γ)Ο TUH 5 1 ‘ 1Q Π71 df)R 41RR P. 32 1HNMR (CD3OD) δ 7.4-7.2 (Sk) 1OH, 5.1 () 4B, 4.35 (q) 1H, 4.0 (q) IB, 3.2 (t) 4H, 1.95-1.30 () 21H. interwedlatAJt io CFaCOO~ HH1 Intermediate- .3- ^4^. 3g)- was treated with TFA/CB^Clj 2 to yield Intermediate 4 (4.3g). 1HNMR (CD3OD) S 7.4-7.2 (m) 10H, 5.1 (β) 4H, 4.35 (t) IB, 3.85 (t) 1H, 3.12 (q, 4H, 1.9-1.3 (m) 12H.

BOC-Lys (£-Z) acid (2.29g), Intermediate 4 (3.76g) ethylchloroformate (665mg, 0.577ml) and M-methylmorpholine (6O7mg, 660μ1) were reacted together in THF (40ml) as described for Intermediate 1 to yield Intermediate 5 (5.1g). 1HNMR (CDjOD) 4 7.5-7.2 (ft) 15H, 5.1 (s) 6H, 4.3 (m) 2H, 4.2 (t) IB, 3.15 (t) 6H, 1,9-1.3 (m) 27H. intermediate 5 rrn r τηπ * « . * Λ mi yinc Λίββ P. ft N*t Intermediate 5 (4.0g) was dissolved in methanol (100ml) and the solution degassed for 15 min with nitrogen. The solution was then hydrogenated at room temperature using 10% Fd/C (180mg) and a hydrogen balloon. The catalyst was filtered off and the solution concentrated in vacuo to give intermediate 6 (2.3g) as a pale yellow oil.

^HNMR (CD-jOD) δ 4.45 -4.25 (m) 2H, 4.0 (b.t) 1H, 2.65 (t) 6H, ... . -2.0’jlrti.r-fje)· 27H. latsraaiiatft-I CcttiX | I 7 Lye (E-Z) benzyl ester (0.5g) was dissolved in dimethylsulphoxide (3.0ml) with slight heating. H-methylraorpholine (0.134g) was then added followed immediately by a solution of bis-Z-Lys N-bydroxysuccinimide ester (0.754g) in dimethylsulphoxide (3.0ml). The reaction mixture was allowed to stand at room temperature for several hours and the reaction monitored by reverse phase HPLC (To: A-70%, 030%, T15: A-0, 0100%, TJ5: A-0, 0100%, A=0.1/Ha0,‘ C—0.1% TTA/CHjCM) product eluted at 20.5 min] until complete. Intermediate 7 was then collected from the reaction solution in 0.1% TFA/CH3car:H2O and freeze dried to yield a fine white powder (750mg).

A A M io Intermediate B 2’Crt WHCOCHUtti Intermediate 7 (0.25g) was dissolved in dry CBaCl3 (100ml) under n2 gae with stirring and then treated with trimethylsilyl iodide (lOpg, 467μΐ) under h3 gae. The resulting pale yellow solution was left stirring at room temperature: overnight^ after which it had turned a dark brown colour. The CB3C13 was evaporated off under reduced pressure to give a dark brown residue which was dissolved in H20 (10ml) and then extracted with ether (3xioml). The agueoue and ether layers were checked for the presence of free amino groups using a ninhydrin spray. The aqueous layer contained all the free amino material and was freeze dried overnight to give Intermediate 7 as a pale yellow residue. n or Sxaarole i XIH Mek THF (10ml) was added to dried N-maleoyl-^-alanine (444ag) and the reaction mixture stirred at -20*C for 10 min. Ethyl chloroformate (286mg, 288μ1) and H-mathylmorpholine (266mg, 288μ1) ,νβχο. then-^ddedabd the reaction~mixture left at 20*c for 30 min. Intermediate 5 (400mg) in dry DMF (10ml) is was added maintaining the reaction mixture at -20 *C. The mixture was left to come to room temperature and then purified using reverse phase HPLC (Dyanamax column C6oA) using the following programme: A C TO 70 30 A * 0.1% TFA/HjO T2Q 50 50 C - 0.1% TFA/CH3CN to yield the desired trimaleimide compound 6 (retention time .7 min, 180 mg) of the Example. lHHHR (CD30D S 6.85 (s) 5H, 4.4-4.3 (Β) 2H, 4.1-4.0 (m) 1H, 3.8 (t) 6H, 3.15 (t) 6H, 2.5 (t) 6H, 1.95 1.3 (m) 27H. t / 4 ΛΛ P.36 Th· compound of Example l (lOOmg) was treated with TFA/CHjClj (1:1, 10ml) as described for tbe preparation of Intermdlate 2. The solvent/acid was evaporated to dryness to give an oil. Ether was added to precipitate the TFA salt, which was then freexe dried for several hours to yield the desired salt 7 (86ag). 1HNMR (CDjOD) S 6,8 {») 6H, 4.4-4.2 (m) 2H, 3.95 (t) 1H, 3.7 (t) 6H, 3.2-3.1 (>) 6H, 2.45 (t) «Η, 1.9510 1.3 (X) 18H.

Exaaplt.3 - ...- . - . .

U&HaA MAC-MM UH CT998 2-(4-Amino)butylperhydro-l, 4, 7, io-tatrazadeeine-1, 4, 7, 10-tatra (2-acetic acid) [Example i(b) in International Patent Speoification No. WO/89/01476] was treated with bis25 (p-nitrophenyl) succinate in dimethylsulphoxide in the presence of N-methylxorpholine at 20 *C for 3h to yield the corresponding active ester 8: which was recovered as a solid (i09»g) and without further purification was dissolved in DMF(5ml). To the resulting solution was added the compound of Example 7 (86mg) in DMF (5ml) followed by N*»ethyl»orpholine (98ml, 90mg). The ρ π reaction mixture was left at 37 *C overnight and the desired product CT998 isolated using reverse phase HPLC (Dynamax column C60k) and the following programme: A c To 70 30 A · 0.1» TFA/HaOT20 50 50 C - 0.1» tfa/ch3cw ^25 0 100 Retention time of CT998 - 15 min 10 Yield = 11 mg 1HNMR (D20) δ 6.8 (β) 6H, 4.3-2.4 (b.m) 59H, 2.0.1.2 (b.m) 24H. _ FAB MASS S?BCI®A P776; My4S* - 1412, 1435 (Ha+ adduct) 1415 (K+ adduct).

HK M«A 2-C.W ii * t co Cri- CT557 Intermediate 7 (O.2g) was dissolved in dimethylsulphoxide and N-methylmorpholine (0.166g) added to the solution followed by succinimidyl maleinido propionate (0.44g) in dimethylsulphoxide (3.0ml). On slight heating of the mixture a pale yellow solution resulted which on standing formed a white precipitate (hydrolysed propionate). The progress of the reaction was monitored using TLC and a ninhydrin apray. After approximately 30 min at room temperature the reaction was ninhydrin negative indicating that all free amino groups had reacted. The mixture was then purified using reverse phase HPLC and the following programme: 00 Ο 100 A - 0.1% TFA/Ο C - 0.1% TFA/CH3CN The product peak eluted at approximately 14.0 min. The 5 product pool was collected in o.i% trifluoroacetic acid/H20:CH3CN and freeze dried overnight to give the compound of Example 4 (CT557) as a fine white material.

Example 5 HM •H^WHoA I MfttUK UN, CT558 The compound of this Example was prepared using a similar 20 series of reactions and reagents to that described for the preparation of CT557 in Example 4, except that Lys benzyl ester was used in pace of Lye(K-3) benzyl ester to react with bis-Z-Lys N-hydroxysucciniaide ester, to yield the appropriate tetra-N-z intermediate which was then deprotected and reacted with succinimidyl maleimido propionate as described for Example 4 to yield CT558.

B. Construction of TFK and fiFX gxoapifc.6 The monoclonal antibody 372.3 is specific for a tumour associated glycoprotein, termed TAC72 (Colcher et al., PEAS 78, 3199-3203). chimeric Fab’ fragments of the antibody 372.3 containing a single hinge thiol group (CB72.3 Fab*S Cys) were prepared as described in International patent specification 8089/01974 and W089/01783. The hinge thiol group of CB72.3 Fab *6 cys is often recovered in a blocked font and partial reduction of the CB72.3 Fab* δ eye must be carried out to allow cross-linking to proceed. This was achieved by incubating the CB72.3 Fab* S eye at 3-7mg/ml in 0.1M sodium acetate/citrate buffer pH 6.0 containing 2mM EDTA with 4.smK 0-mercaptoethylamine for 30 minutes at 37*C. The reducing agent was then removed by desalting on a column of Sephadex G-25 into 0.1M acetate/citrate buffer pH6 containing 2mM EDTA. The extent of reduction was tested on an aliquot of the reduced, desalted material by titration with dithiodipyridine. The protocol typically produced approximately one thiol per OB72.3 Fab* C Cys molecule.

Cross-linking to tri-Fab with the tri-maleimide linker CT557 was then carried out by one of two methods. In the first of these CT557 was dissolved in dry DMF and added to the freshly reduced, desalted Fab* in a five times molar excess of CT557 over Fab1. After incubation at 37*C for 1 hour an •xcess of N-ethylmaleimide was added, a further ten minutes incubation at 37 °C was carried out and the mixture was then desalted on a column of Sephadex G-25 into 0.1M acetate/citrate pH6.0 containing 2mM EDTA. This procedure generated cB72.3 Fab1 δ Cys with CT557 attached. Meanwhile a further batch of freshly reduced and desalted cB72.3 Fab1 δ Cys was prepared as described above and added to the Fab'CT557 in a ratio of 2:1 (Fab* :Fab'-CT557). The reaction mix was maintained overnight at 37 *c and then the extent of cross-linking assessed by HPLC gel filtration and SDS-PAGE. HPLC gel filtration analysis was carried out on a DuPont Zorbax gf-250 column run at lml/min in 0.2M phosphate buffer pH7.0 and SDS-PAGE was carried out as described by La email (1970). Typically 30-50% of the CB72.3 Fab* δ Cys was cross-linked to tri-Fab by this method. In a second method of cross-linking with CT557, freshly reduced, desalted cB72.3 Fab' δ Cys was prepared as described above and CT557 added as a solution in dry DMF such that a molar ration of 5:1, Fab’CTSS? was achieved. The mixture was incubated at 37*C for 1 hour or longer and the extent of cross-linking - him.no tuii 11-no Π71 XOC 41RC p an assessed by HPLC gel filtration and SDfi-PAGE as described above. The extent of cross-linking to tri-Fab with this method of preparation was typically 40-60%. Typical HPLC analyses of cross-linking mixtures as shown in figure l.

Tri-Fab was purified by gel filtration chromatography either using preparative HPLC on a DuPont Zorbax GF-250XL column at 3ml/mln in 0.2M phosphate buffer pH7.0 or on a 2.6cm di ana tar 183 cm long column of Sephacryl S-200HR run in o.lM acetata buffer containing 0.21( potassium chloride and 2mM EDTA at pH 6.0.

The antigen binding ability of tri-Fab was compared to IgG di-Fab and monomeric Fab* using a mucin binding ELISA.

Titrations of monomeric, dimeric and trimeric chimeric Fab and chimeric B72.3 IgG were allowed to bind to solid-phase mucin in wells of a microtitre plate for 1 hour. Unbound antibody was washed off before addition of a goat antihuman Fab-HRPO conjugate followed by development with tetra20 methyl benz idine (TMB). The signal obtained was plotted against concentrations of antibody expressed as nH binding sites. This allows a direct comparison of the efficiency of the binding site in each multimerio state. Results of the antigen binding ELISA are shown in figure 2. The sonomerio Fab* is poor in avidity as expected whereas the di-Fab and IgG titrate in a very similar fashion also as expected. The cross-linking to tri-Fab appears to result in a 2-3 fold advantage in the ability of the binding site to bind antigen. If an increase in avidity of the molecule was achieved by cross-linking, a significant change in the off rate of the molecule would be seen, off-rates of OB72.3 IgG and tri-Fab were compared by allowing tri-Fab and CB72.3 IgG each at 70nM (expressed in binding sites) to bind to solidphase mucin in wells of a microtitre plate for 16 hours.

Duplicate wells were then subjected to continuous washing for 23,8,4,2 and 1 hours. Residual antibody was revealed with a goat anti-human Fab-HRPO conjugate followed by development with TMB. The signal obtained was read off standard curves of tri-Fab or IgG, and the data plotted ae nM binding sites remaining in either tri-Fab of IgG format against time, figure 3 shows the results of this analysis, λ significant improvement in off rate is seen for the tri5 Fab, indicating a greatly improved ability for the molecule to remain bound to the antigen over a long time period.

CB72.3 IgG and tri-Fab (0.5mg of each at lmg/ml in 0.2M phosphate buffer pH 7.0} where labelled with 1251 using Bolton Hunter reagent with standard methodology. The quality of the labelled tri-Fab and IgG were assessed by SDS-PAGE/autoradiography. There was no apparent breakdown of thetrL+Fab &t Jgfr byJZtbe labelling procedure- Groups of four female nude mice bearing subcutaneous 2-3 week old LS174T human tumour xenografts on the flank were injected i.v. in the tail vein with approximately 17pg/9pCi of triFab and 19/*Ci of IgG. Groups of animals were killed at 3h, 24h, 48h and I68h for collection of tissues which were weighed, dissolved in 7M potassium hydroxide and counted in an LKB model 1270 gamma counter. Results were expressed as mean percentage of the injected dose per gram of tissue +/standard deviation (n=4).

The biodistribution results for the iodinated IgG were consistent with previous experiments (King et al., 1992) iodinated tri-Fab wae found to clear significantly faster from the animals than the IgG despite the two molecules being approximately the same molecular weight (figure 4). The tri-Fab was able to localise well to the tumour with no significant accumulation in any other tissue (figure 5}. consequently the tumour;blood ratios for the tri-Fab were significantly better than those seen for IgG (figure 6}. The tumour: blood ratio is important as this means that less toxicity from blood radiation is expected for a given radiation dose to the tumour.

The biodistribution of CB72.3 tri-Fab in nude mice bearing sub-cutaneous LS174T xenograft tumours was also assessed r> i r\ ββ when labelled with Y. The 12H4 macrocycle for labelling with ’°Y was attached to purified tri-Fab using a 12N4maieimide derivative (CT77, prepared from the compound of Example lb in International Patent Specification W089/01476 and the N-hydroxysucc in imide eater of N-(2carboxyethyl)malei»ide). λ sample of purified tri-Fab was buffer exchanged into 0.1M sodium bicarbonate buffer pH8 containing 2mN EOT A. Thiol groups were then introduced into the tri-Fab by reaction with a 10 fold molar excess of 210 iminothiolane over tri-Fab for 30 minutes at room temperature. The thiolated tri-Fab was then desalted into 0.1M sodium bicarbonate buffer pB8 containing 2aM EDTA using a column at - Sephadex G-25 (Pharmacia PEh-10) to remove the unreacted 2-iminothiolane. The number of thiol groups present were determined by titration with di thiodipyridine. 12H4 macrocyle was then conjugated to the thiolated triFab by addition of CT77 at a ten fold molar excess over the number of thiol groups present followed by incubation at 37*C for 2 hours. The conjugate was then purified by desalting on a Sephadex G-25 column (Pharmaoia PD10) into 0.1M potassium acetate pH6. Radiolabelling was achieved by the addition of 90¥C13 to the conjugate, ensuring that the buffer in the conjugate solution was sufficient to buffer the acidic 90YC13. After incubation at 37 *C for 15 minutes the radiolabelling was quenched by the addition of 10AK DTPA and the labelled tri-Fab purified by gel filtration HPLC on a DuPont Zorbax GF-250 column in 0.2M phosphate pH7. cB72.3 tri-Fab labelled with 90Y was assessed by SDS30 PAGE/autoradiography. There was no apparent breakdown of the tri-Fab by the labelling procedure. Groups of four females nude mice bearing subcutaneous 2-3 week old LS174T human tumour zenogrefts on the flank were Injected i.v. in the tail vein with approximately 3/ig/3^ci of tri-Fab.

Groups of animals were killed at 2.5h, 24h, 48h and 120h for collection of tissues which were weighed, dissolved in 7M potassium hydroxide and counted in an LKB model 1270 gamma counter. Results were expressed as mean percentage of the injected do*· per gram of tissue +/- standard deviation (n=4).

Results of this biodistribution experiment (figure 7) revealed similar fast clearance of the 90Y labelled triFab to that seen when labelled with 125-iodine. Good tumour localisation was seen with low levels detected in all other tissues. Iaportantly, low level* of activity vers detected in the kidney. Retention of 90Y in the kidney when administered on other antibody fragments such as Fab and F(ab')2 bas limited their usefulness, thus low kidney levels for tri-Fab represent a considerable advantage over other antibody- fragment».1S Example 7 CB72.3 tetra-Fab was prepared from reduced desalted Fab' with the tetra-maleimide linker CT55B was then carried out. CT558 was dissolved in dry DMF and added to the freshly reduced, desalted Fab' in a five times molar excess of CT558 over Fab’.

After incubation at 37 *C for 1 hour an excess of Nethylmalemide was added, a further ten minutes incubation at 37 *C was carried out and the mixture was then desalted on a column of Sephadex 0-25 into 0.1X acetate/citrate pH 6.0 containing 2mM EDTa. This procedure generated cB72.3 Fab' Cys with CT558 attached. Meanwhile a further batch of freshly reduced and desalted CB72.3 Fab* ί Cys was prepared as described above and added to the Fab'-CT558 in a ratio of . 3:l (Fab· :Fab'-CT558). The reaction mix was maintained overnight at 37 eC and then the extent of cross-linking assessed by HPLC gel filtration and SDS-PAGB as described in exanple 6. Typically 20-408 of th* CB72.3 Fab* 4 Cys was cross-linked to tetra-Fab by this method. Typical HPLC analyses of cross-linking mixtures are shown in figure 8. The tetra-Fab was purified by HPLC gel filtration as described for the tri-Fab in Example 6.

The antigen binding ability of tetra-Fab was measured in activity assays as deecribed for tri-Fab in Example 6. A similar improvement in avidity over IgG vas observed for the purified tetra-Fab (figure 2).

Purified tetra-Fab vae buffer exchanged into o.lM sodium bicarbonate buffer pH8 containing 2wK KOTA and thiolated by incubation with a 15 fold excess of 2-iminothiolane over tetra-Fab incubated at room temperature for 30 minutes. 2-iminothiolane vas then removed by desalting the thiolated tetra-Fab on a Sephadex G-25 (Pharmacia, PD-10, column in phosphate , /buffered, fl^lipe. A'"Sn 3 macrocycle was then conjugated to the tetra-Fab by the addition of a 10-fold excess of CT82 (prepared from the compound of example 2b in International Patent Specification WO89/01475 and the Nhydroxysuccinimide ester of N-(2-carboxyethyl)maleimide) over the number of thiol groups present (number of thiol groups determined by titration with dithiodipyridine as described above}. After incubation et 37*C overnight, an excess of N-ethylmalaimide was added and incubation continued for a further 10 minutes. The conjugated tetraFab was then purified by desalting into O.lM sodium acetate pH 5.0 using a Sephadex G-25 column (Pharmacia, PD-10). The tetra-Fab conjugate vas radiolabelled with Ill-indium by adding lll-InC13 directly to the purified tetra-Fab 9N3 conjugate and incubation for 30 minutes at 37*C. The labelling was quenched by the addition of DTPA to 5mK, and the radiolabelled tetra-Fab purified by HPLC gel filtration as described above for tri-Fab. cB72.3 tetra-Fab labelled with ill-In was assessed by SDS-PAGE/autoradiography. There was no apparent breakdown of the tetra-Fab by the labelling procedure. Groups of four female nude mice bearing subcutaneous 2-3 week old LS174T human tumour xenografts on the flank were injected i.v. in the tail vein with approximately 3.5ug/ll#Ci of tetra-Fab. Groups of animals were killed at 2 Results of this biodistribution experiment (figure 9) revealed fast blood clearance of the tetra-Pab and good tumour localisation, similar to that seen for tri-Pab (example 6). Again low kidney levels were observed suggesting e significant advantage for tetra-Pab, similar to that observed for tri-Pab (exaaple 6). . -.- i SKUBlS-l CB72.3 tri-Pab was prepared with the cross-linker CT998 by the same method described for tri-Fab with CT557 (example 6). This cross-linker contains a 12N4 macrocycle for labelling with 90Y or X1ln. Tri-Pab prepared with CT998 had equivalent activity in antigen binding assays to tri-Pab prepared with CT557. The biodistribution of CB72.3 triPab(998) in mice was assessed when labelled with 90Y. *°Y labelling was achieved as described for CB72.3 tri-Fab CT77 conjugate in exaaple 6. Por CB72.3 tri-Pab (998) it was not necessary to conjugate a macrocyle as the macrocycle is already present in the cross-linker and provides a site for labelling. A biodistribution experiment was then carried out by injecting groups of 4 mice with approximately 4gg/8pCi of 90Y labelled OB72.3 tri-Pab (988). Groups of animals were killed at 3h, 24h, 48h, 72h and 168h for collection of tissues which were weighed, dissolved in 7M potassium hydroxide and counted in an LKB model 1270 gamma counter. Results were expressed as mean percentage of the injected dose per gram of tissue +/- standard deviation (n35 5). Results of this biodistribution experiment demonstrated that tri-Pab (988) behaved in a similar manner to CB72.3 tri-Pab made with the linker CT557. The tri-Pab (988) cleared rapidly fro· tha circulation and was able to P. 4R localise well to the tumour with no significant accumulation In any other (figure 10).

Kaaapi* a The murine monoclonal antibody A5B7 has been studied and shown to recognise the tumour associated antigen known as careinoembryonic antigen (CSA) (Harwood et al., 1986). A mouse:human chimeric version of this antibody was produced, and the genes for e suitable cA5B7* δ eye were constructed in an expression vector for use in HSO cells as described in Patent application W092/01059. An HSO cell line producing oA5B7 Fab1 was prepared 15y liheaftsiftg 50pg of* plasmid DMA (PHMC30) with the enzyme Fspl, electroporating into HSO cells and selecting producing cell lines as described for chimeric B72.3 (Bebbington et al. 1992).

CA5B7 Fab* δ cys was purified from HSO oell aulture supernatant by firstly adjusting the pB of the supernatant fluid to 5 with HCI and applying to a column of protein G sepharose (Hi-trap, Pharmacia) which had been preequilibrated in lOOmM phosphate buffer pH 5.0 containing 150mM sodium chloride. After loading the supernatant the column vas washed with equilibration buffer and the Fab* eluted with 0.1H citric acid. Fractions containing the purified Fab* were collected directly into sufficient IK tri to adjust the pH to between 6 and 7. The fractions containing the Fab* were pooled and concentrated by ultrafiltration, cross-linking to CA5B7 tri-Fab with CT557 and purification of the tri-Fab was achieved by substantially the same method as described for cB72.3 Fab* δ Cys in example 6. Antigen binding ability of the cA5B7 tri-Fab was compared to A5B7 IgG using a CHA binding ELISA. This was carried out substantially as described for the mucin binding ELISA in example 6, except that CEA coated plates were substituted for mucin coated plates. Results (figure 11) demonstrate similar improved avidity of the CA5B7 tri-Fab over IgG. The biodistribution of cA5B7 D 47 tri-Fab ove lgG. The biodistribution of cA5B7 tri-Fab was also examined in a nude mouse xenograft experiment.

Approximately 3/tg/2.4MCi of CA5B7 tri-Fab labelled with USI (by Bolton Hunter reagent as described for ΟΒ72.3 above) wae injected into groups of 4 nude mice bearing subcutaneous LS174T xenografts and the biodistribution measured at 24h' and 72h. Results showed rapid clearance of the oA5B7 triFab with localisation to the tumour (figure 12), demonstrating similar favourable properties ae seen for io CB72.3 tri-Pab.

A CDR grafted version of A5S7 Fab* 6 Cys (gA5B7 Fab*) was also produced as described in Patent application W092/01059. Plasmid pHHC53 was constructed from pAL54 (described in W092/01059) by removing the ampR gene and GS minigene on a BamHl-Clal fragment and replacing it with a BamH2-Clal fragment consisting of ampR gene and GS cDNA. This produces a vector suitable for expression in NSO cells (Bebbington et al., 1992). An NSO cell line secreting CDR grafted A5B7 Fab* S Cys was produced using pHKC53 as described for the chimeric Fab* above. gA5B7 Fab* was purified from NSO cell culture supernatant by firstly adjusting the pH of the supernatant fluid to 8 with IK tris and applying to a column of protein A sepharoee (Pharmacia) which had been pre-equilibrated in lOOnH boric acid buffer pH 8.0 containing 150mM sodium chloride. After loading the supernatant the column was washed with equilibration buffer and the Fab* with 0.1M citric acid. Fractions containing the purified Fab’ were collected directly into sufficient 1M tris to adjust the pH to between 6 and 7. The fractions containing the Fab* were pooled and concentrated by ultrafiltration. Cross-linking to gA5B7 tri-Fab with CT557 and purification of the tri-Fab was achieved by substantially the method as described for CB72.3 Fab* t Cys in example 8. Again antigen binding analysis Ρ ΛΒ demonstrated that the gA5B7 tri-Fab was of high avidity and a biodistribution experiment of 1251 labelled material carried out in a similar manner to those described above, showed similar fast blood clearance and good tumour localisation (figure 13).

Claims (24)

1. A tri- or tetra-valent monospecific antigenbinding protein comprising three or four Feb fragments bound 5 to each other by a connecting structure, which protein ie not a natural immunoglobulin.

2. An anigen-binding protein according to claim 1 wherein the Fab fragments are bound together covalently by io a single linker molecule.

3. An antigen-binding protein according to claim 2 where Lev the.. Fab /fragments included hinge region having at least one cysteine residue and tbe linker molecule is a tri15 or tetra-valent maleimide cross-linker which links via at least one cysteine residue in each fragment.

4. An antigen-binding protein according to claim 2 or claim 3 wherein the hinge region has a reduced number of 20 eye residues compared to the natural hinge region of tbe Fab'-like fragments.

5. An antigen-binding protein according to any preceding claim which demonstates increased antigen avidity, 25 improved blood clearance performance and superior localisation to antigen-containing tissues when administered to an animal.

6. An antigen-binding protein according to any one 30 of claims 3 to 5 wherein the linker molecule is a maleimide cross-linker.

7. An antigen-binding protein according to any preceding claim which is speoific for a tumour-associated 35 antigen.

8. An antigen-binding protein according to claim 7 which is specific for CEA. d cn

9. An antigen-binding protein according to claim 7 which is specific for TAG72. 5 10. A cross-linker compound of the general formula (1):

10.R 1 CB(R a )MHCOR 3 wherein R 1 is a carboxyl (-co 2 H) or esterified carboxyl (10 C0 2 R) group or a group -COA where A is an effector or reporter molecule attached to the -co group either directly or via a spacer group to form a carbon-carbon, or carbonhet aroatemlinkagerdR 2 - and R 3 » which may be the same or different, is each an optionally substituted straight or 15 branched alkylene, alkenylene or alkynylene chain [optionally interrupted by one or more -O- or -S- atoms, or -M(R«) (where R* is a hydrogen atom or a c x _ 6 alkyl group), —N(R*)CO—, -CON (R*)-, C 5 _ 8 cycloalkylene, C 6 _ 12 arylene or c $ _ XQ heteroarylene groups] containing one or more reactive 20 functional groups such that the total number of reactive functional groups in R 2 and R 3 together is three or more.

11. A cross-linker compound of the general formula (10): R 9 CH(R 2 )CONHCH(R 2 )CONHCB(R 2 )CONH 2 (10) where R 9 is -NH 2 or a substituted amino group, e.g. a group -NHCOA, and A and R 3 are as defined for compounds of formula 30 (l).

12. A cross-linker according to claim 10 or claim 11 further comprising a macrocycle. n π

13. A cross linker according to claim 12 wherein the macrocycle is of the following general formula: wherein L is a substituent containing a reactive group, B is a C 2-i4 elkylene chain interrupted by one or two optionally substituted nitrogen atoms; W 1 and hi 2 , vhich may be the same or different, is each an optionally substituted nitrogen atom; p ie zero or an integer 1 and q is zero or an integer-! or. 2. with-the ,proviso that when~p is zero, q is an integer l or 2

14. λ cross-linker according to any one of claims 10 to 113 wherein the macrocycle chelates a radioisotope.

15. A cross-linker according to claim 14 wherein the radioisotope is m In, 90 Y or l2S I.

16. A cross-linker of the formula CT998:

17.A cross-linker of the formula CT557: 2-C.H co

18.P (0

19.A cross-linker of the formula CT558i 2 γ Κ u X 18. Λ vudk 10. 19. A Fab-like protein according to any one of claims 1 to 9 comprising a cross-linker according to any one of claims 10 to 18.

20. A method far the therapy or diagnosis of cancer 15 comprising the administration of an effective amount of a Fab-like protein according to any one of claims 1 to 9 attached to a suitable therapeutic or diagnostic agent to a human or animal subject. 20 20. The method of claim 19 where the therapeutic agent is a radioisotope.

21. The use of a Fab-like protein according to any one of claims 1 to 9 for the treatment or diagnosis of 25 cancer.

22. The use of a Fab-like protein according to any one of claims 1 to 9 in the manufacture of a composition for the treatment or diagnosis of cancer. η (-λ

23.-5123. A tri- or tetravalent monospecific antigen-binding protein as claimed in claim 1, substantially as herein described.

24. A cross-linker compound as claimed in claim 10, substantially as herein described.