WO1996014339A1

WO1996014339A1 - Antibodies

Info

Publication number: WO1996014339A1
Application number: PCT/GB1995/002585
Authority: WO
Inventors: Frederick Charles Kull, Jr.; Mary Elizabeth Fling; Julie Beth Stimmel
Original assignee: The Wellcome Foundation Limited
Priority date: 1994-11-05
Filing date: 1995-11-03
Publication date: 1996-05-17
Also published as: GB9422383D0; ZA959336B; JPH10508482A; EP0789713A1; AU3811495A

Abstract

The present invention relates to antibodies which are capable of being conjugated at specific sites, to processes for the site-directed conjugation of such antibodies, to antibodies that have been conjugated at a specific site and to the use of such antibodies in therapy and diagnosis.

Description

ANTIBODIES

Antibodies are globular proteins which represent a vital component of the mammalian immune response to foreign disease inducing agents. Antibodies may be manufactured ex vivo by any of a number of methods, and such antibodies, particularly monoclonal antibodies and/or fragments thereof, have proved valuable as both diagnostic and therapeutic agents. The utility of antibodies stems from their unique antigen specificity, i.e.. their ability to chemically recognise and remain bound to discrete chemical moieties such as pathogen antigens or tumour-associated antigens. Another aspect of their utility is their diversity, i.e., the ability of mammals (and now other processes such as phage display) to create a very large variety of discrete, genetically defined antibodies (monoclonal antibodies). A final aspect of their utility is their capacity to interact via their "constant" regions. This latter aspect determines other sets of properties, for example, those properties common to isotypes such as interaction with effector cells, complement or other binding moieties like protein A.

The usefulness of an antibody or fragment may be enhanced by chemically coupling one or more further molecular moieties (referred to herein as substances) either directly or indirectly that convey properties to the conjugate that are not naturally present in the constituents alone. Such properties may include a reporter function such as a dye or a radionuclide, an enzymatic function, a second binding function (such as with biotin- avidin), a drug (such as adriamycin), a cytotoxic function (such as with ricin), a chelator, or a chemical linkage moiety that may, in turn render the antibody capable of subsequent reaction with any of a variety of molecular moieties. Thus an antibody conjugate comprises an antibody and an active substance (substance) which is either directly or indirectly conjugated to the antibody. For example, when the active substance is a radionuclide, the radionuclide may be directly conjugated to the antibody or alternatively it may be indirectly conjugated to the antibody via a chelator such as for example TMT or even a chelator which is in turn linked to the antibody by a further protein reactive group (cross-linker) such as. for example, bromoacetyl. Similarly, the active substance could be a molecular chimaera for use in enzyme-prodrug therapy, such a chimaera comprises a transcriptional regulatory DNA sequence capable of being activated in a mammalian cell such as a cancer cell and a DNA sequence operatively linked to the transcriptional regulatory DNA sequence and encoding a heterologous enzyme capable of catalysing the conversion of a prodrug which is administered subsequently,into an agent toxic to the cancer cell. This molecular chimera may be directly conjugated to the antibody or alternatively, it may be contained within a viral vector or liposome with the viral vector or liposome then being attached to the antibody (see European application No.90309430.8).

Many means have been described for the chemical addition or crosslinking of molecular entities to antibodies and their fragments, and these reactions are commonly referred to as conjugation reactions. Conjugation reactions exploit the chemical functional groups that occur naturally in the antibody. For example, a common approach is to target primary amines (mainly the ε amino groups of lysine residues). Another common example of this approach includes the iodination (such as l-^I) of tyrosine residues. A disadvantage of this approach is that it is random. That is, lysine or tyrosine residues may occur throughout the structure and therefore the natural properties that these residues help convey ~ such as, for example, antigen recognition, complement reactivity or effector cell interaction — may be compromised. The behaviour of the conjugate is the average of the behaviour of all the individual unique components, some of which may be entirely useless or detrimental. The random approach is also known to affect antigen recognition.

Site-directed conjugation, i.e.. conjugation to a specific amino acid residue within the antibody structure, would convey the advantage that the resulting conjugate is not a mixture of different products. The properties conveyed by the antibody to the conjugate, such as antigen reactivity or pharmacokinetic stability, can thus be ascribed to a defined chemical structure. Furthermore, the site may be selected so as to be spatially removed from areas known to convey antigen-binding properties. For example, the variable region of an antibody is known to contain the antigen binding site and the CH2 domain of the heavy chain is known to contain effector (FcR) and complement (Clq) interactive residues. Thus, additions to the CH3 domain of the heavy chain may avoid compromising these functions. - j -

Two examples of site-directed conjugation procedures to antibodies have been described. The conjugation of antibodies to the carbohydrate portion of antibodies has been detailed (O'Shannessy and Quarles. J. Immunol. Met 99: 153-161, 1987). Human IgG antibodies have one common glycosylation site at asn297 in the CH2 domain. (Note that all antibody amino acid residues described herein are numbered according to the EU index. Kabat, et al.. Sequences of proteins of immunological interest, 5th ed.. NIH publication No. 91-3242, 1991. The common isotypes are referred to as Gl, G2 G4 etc.). The glycosylation conjugation procedure presents the risk of altering carbohydrate that might be required for antigen or effector interaction (Lund, et al., Molec. Immunmol 27: 1 145, 1990; Isaacs, et al. J. Immunol. 148: 3062, 1992).

Another example of site-directed conjugation to antibodies involves the creation, by site- directed mutagenesis, of a free thiol on the antibody. Antibodies naturally contain cvstine residues whose thiol groups are joined by disulfide bridges. The position of the naturally occurring cvstine residues is highly conserved among species indicating that these residues are essential for the structure and function of antibodies. Antibodies do not naturally contain free sulfhydryi groups. It is hypothetically attractive to engineer an antibody to possess a cysteine the thiol group of which is neither oxidised nor compromises the fidelity of the natural sulfhydryi bridges. Sulfhydryi groups of cysteine residues may of course be exploited for conjugation by numerous conjugation chemistries that are rather specific for sulfhydryls such as maleimides. alkyl and aryl halides, α-haloacyls and pyridyl disulfides. However, it has been determined that variant monoclonal antibodies that have been designed with unnatural cysteine residues do not de facto possess free thiol groups available for conjugation. (Since antibodies consist of two identical heavy and two identical light chains and depending on how the gene has been engineered and expressed, genetic replacement of a residue on one chain can result in two new residues in the complete H2-L2 structure; furthermore, the two residues need not be chemically identical due to their spatial arrangement and neighbourhood.) In one known example of an engineered thiol (Bodmer. et al. US # 5,219,996 and Lyons, et al, Protein Eng. 3: 703, 1990), an available free thiol was observed only when a cysteine residue was introduced into a discreet "concave" molecular pocket within the antibody structure — a pocket only accessible to small molecules (0.13 - 0.5 mm diameter) and inaccessible for forming disulfide bridges within the same or other antibody chains. Cysteine residues introduced on "convex" or "flat" surfaces of the monoclonal antibodv were found not to contain free thiols. One s illed in the an would therefore conclude that surface cysteine residues would form disulfide bridges within the same and/or another antibody chain and thus distort the macromolecular structure and functions of the antibody.

The published literature indicates that engineered surface thiols alter antibody structure and function. For example, both monomers and dimers (IgG-IgG) were observed in the crude antibody-producing cell supernatant of a ser444→cys variant (Shopes, J. Immunol. 148: 2918. 1992). Relatedly, a procedure was described for the production of dimeric antibodies also using a ser444→cys variant (Caron, et al, J. Exp. Med. 176: 1 191-1 195, 1992). The dimeric IgGs from both of these examples were found to have enhanced effector functions in vitro. In these examples and in the earlier example of Bodmer, et al, no physicochemical evidence was provided to indicate whether dimers formed specifically at the engineered site or to what extent normal disulfide bridges may have been altered. Finally, a "tethered" antibody has been described that was produced by creating a se 19— »cys variant (Shopes, Mol. Immunol 30: 603, 1993). The variant allegedly generated dimers and an interchain structural variant or "tethered" antibody. Mixtures of structural aberrations compromise the goal of producing a defined chemical entity.

It was therefore with some surprise and contrary to expectation that we found that monoclonal antibodies containing a cysteine residue exposed on the surface of the antibody, are, in fact, amenable to site-directed conjugation, especially those containing a ser442→cys heavy chain variant.

Accordingly, the present invention provides a monoclonal antibody comprising a cysteine residue exposed on the surface of the antibody such that the residue is capable of being conjugated to a substance and wherein the antibody is immunochemically functional, the term immunochemically functional primarily referring to the antibody^'s ability to bind but also encompassing effector functions if these are present. Bearing in mind that the variable region of the antibody contains the antigen binding site the preferred sites for conjugation are the surface residues on d e surface of the variable region which are not involved in antigen binding such as, for example, the sSv heavy chain - light chain linker peptide as well as the surface residues of the constant region of the antibody which encompass the constant region of the light chain, the CH1.CH2, and CH3 domains of the heavy chain and also includes the hinge region. Thus all residues on the surface of the antibody which are not involved in antigen binding, in particular those which are not part of the CDRs, are suitable for conjugation to a substance. If the antibody has effector functions, the preferred sites for conjugation are the same but excluding the CH2 domain which is known to contain the effector functions. The cysteine residue is in a substantially reduced form. More preferably, die reduced cysteine residue is in the CH3 domain of the heavy chain and more preferably at position 442 within the CH3 domain. Another preferred position for the reduced cysteine residue is the heavy chain - light chain linker peptide. Novel cys442 antibodies are capable of being expressed by their producer cells in a manner indicating both monomeric IgG and aggregated forms. Although the presence of aggregate suggested that the CVS442 variants were surface variants as had been observed in the works by Bodmer. et al. and by Shopes. surprisingly, most of the antibody was not in an aggregated form. Monomeric IgG was readily purified, for example, by gel filtration chromatography, and the monomeric form was stable upon long term storage. The monomeric IgG was found to possess no free thiol (Table 1 ). Whilst not wishing to be bound by theory, we believe that the thiol may be initially blocked (i.e. protected) by naturally occcurring adducts such as for example, glutathione.

We have discovered that the engineered thiol is reduced under controlled conditions that do not reduce the natural disulfide bonds. For example, milder conditions such as lower concentrations of the reductant which are not capable of reducing the natural disulfide bonds are found to be suitable for reducing the engineered thiol . The reduced antibody sustains a monomeric form even when stored for prolonged periods of time at pH 8. (Thiols are known to be reactive by judicious manipulation of pH and oxygen). Thus, by controlled reduction, the engineered antibody is rendered capable of site-directed chemical addition specifically at the engineered thiol.

Thus the present invention is also directed to a monoclonal antibody comprising a cysteine residue exposed on the surface of d e antibody wherein, by controlled reduction, the antibody is rendered capable of site-directed chemical conjugation to a substance, said cysteine residue being introduced at a site which does not interfere with the immunochemical function of the antibody.

The antibodies according to the present invention are preferably monoclonal antibodies, or fragments thereof, the term antibody encompassing both antibodies and antibody fragments. Antibodies according to the present invention can be from any species. The antibodies may be chimaeric antibodies that have variable regions from one antibody and constant regions from another, such as a human antibody. Thus, chimaeric antibodies may be species/species chimaeras or class/class chimaeras. Such chimaeric antibodies may have one or more further modifications to improve antigen binding ability or to alter effector functioning. Another form of altered antibody is a humanised antibody including a composite antibody, wherein the constant regions and die hypervariable regions other than the CDRs are transferred to the human framework. Additional amino acids in the framework or constant regions of such antibodies may be altered if required to restore binding. Thus the antibodies of use in the present invention include any altered antibodies in which the amino acid sequence is not one which exists in nature. However, CDR-grafted antibodies are most preferred. Antibodies of the present invention include different isotypes such, for example, as Gl. G2. G4. Examples of antibodies are the 40KD antibody (CO/ 17.1. A) as disclosed in J. Cell Biol 125(2) 437-446. April 1994 and in Proc.Natl. Acad. Sci. 87, 3542-3546. May 1990, preferably the humanised anti-40 D antibody and in paπicular humanised anti-40KD of the G4 isotype. A specific example of an anti-40KD antibody is 323/A3. preferably humanised 323/A3 and in particular humanised 323/A3 IgG4.

Another example of an antibody is an anti-folate recepter antibody as disclosed in A.Tomasetti et al. Federation of European Biochemical Societies Vol 317. 143-146, Feb 1993. preferably humanised anti-folate and in particular humanised anti-folate of the Gl isotype. A specific example of an anti-folate antibody is MOV 18, preferably humanised MOV 18 IgGl. Further examples of antibodies include anti-CEA. anti mucin. anti-20/200KD, anti-ganglioside, anti-digoxin. anti-CD4, anti-CD23, anti- CDw52 and more specifically Campath-IH which is a humanised anti-CDw52 antibody. The antibody chain DNA sequences including die CDRs of Campati -1H™ are set out in EPO328404, die disclosure of which is hereby incorporated by reference. (Page, M.J., and Sydenham, M.A., High level expression of the humanised monoclonal antibody Campath-IH in Chinese Hamster Ovary cells. Biotech. 9: 64-68, 1991.).

Antibody fragments of use in the present invention include Fab, F(ab)2, Fv and fragments comprising synthetic peptide sequences eg. as generated by recombinant DNA technology. Monoclonal antibodies of use in the invention may be prepared by any metJ od well known in the art or more particularly as described in GB 9022547.5. Purification may be carried out as described in EP-A-91917891.

Fragments may be prepared by any of the means known in the literature, for example Antibodies, a laboratory manual, eds. E. Harlow and D. Lane, Cold Spring Harbor Laboratory, 1988 or by molecular genetic means.

The invention also provides an antibody wherein the cysteine is conjugated eiti er directly or indirectly to a substance. When the substance is conjugated indirectly to the antibody it may be connected to the antibody via one or more linkage moieties such as for example chelators. Such substances which are connected to the antibody via one or more linkage moieties are commonly known as "bifunctional substances". Such linkage moieties may, for example, be a functional chemical moiety such as maleimide or bromoacetyl. that is capable of covalent attachment to thiol functional groups within proteins such as antibodies. The linkage moiety may also utilise a chemical spacer (e.g. a p-benzyl group) tiiat functions as a bridge between the substance(s) and the antibody attachment moiety.

Examples of the conjugated substance are dyes, radionuclides. enzymes, drugs, cytotoxins. and biotin/avidin. Specific examples of a drug and cytotoxin being adriamvcin and ricin respectively. Specific examples of chelators include d e following.

DOTA ( 1.4,7, 10-tetraazacyclododecane-NN' N",N" -tetraacetic acid):

PA-DOT A(α-[2-(/7-nitrophenyl)ethyl]- 1 ,4,7, 10- 1 -acetic-4.7.10-tris(methylacetic)acid);

TMT (6,6"-bis[NN",N'"-tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4- methoxyphenyl)-2.2':6'.2"-terpyridine);

IB4M-DTPA (NN,N',N"_!N"-pentakis(carboxymethyl)-2-[(4-aminophenyl)methyl]-6- memyldied ylenetriamine);

CHX-A-DTPA (-^-[2----mino-3-( -aminobenzyl)propyl]-trαrø-cyclohexane-1.2-diamine-

NN',N',N",N"-pentaacetic acid);

TRITA (l,4,7,10-tetraazacyclotridecane-N,N',N",N'"-tetraacetic acid); and

TETA 1,4.8.1 l-tetraazacyclotetradecane-NN',N",N" -tetraacetic acid When the substance is a radionuclide, it may be attached to d e free thiol of d e antibody by eitiier direct or indirect methods. For example, 99_mχ_c may be directly attached to the antibody by a modification of procedures similar to the Schwartz method (Schwartz. A., and Steinstrasser. A., J. Nucl. Med. 18:721 , 1987), wherein reduction of the natural disulfides would not be necessary. Metallic radionuclides such as 9 γ_t 186R_C 177L_U> H I In and ^^Cu may be attached by eidier indirect prelabelling methods, wherein radionuclide is first added to bifunctional chelator then conjugated to antibody, or by indirect postlabelling methods, wherein radionuclide is added to preformed chelator- antibody conjugate. Chelators of the present invention may be linked to antibodies by any hetero- or homo-bifunctional cross linker (i.e. chemical spacer) capable of linking a chelator to a thiol group in the antibody (M.McCall et al.. Bioconjugate Chem. 1. 222- 226, 1990) or by use of cross linkers described in Pierce "Immuno Technology Catalog and Handbook" 1992, pages E8 to E39 and in Parker. Chem.Soc.Rev. [1990], __, 271- 291 and the metiiods referred to therein.

Examples of chelators in combination with cross-linkers include:

bromoacetyl-DOTA (2-[p-(bromoacetamido)benzyl- 1.4,7, 10-tetraazacyclo-dodecane-

NN',N",N'"-tetraacetic acid); bromacetyl-TRITA (2-[p-(bromoacetamido)benzyl]-l .4,7.10-tetraazacyclotridecane-

N.N, N", N'"-tetraacetic acid); bromoacetyl-TMT (2-(^-(bromoacetamido)benzyl]-6.6''-bis[N.N"N''- tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4'-methoxyphenyl)-2.2' : 6'.2 "- terpyridine);

The bifunctional chelator cross-linker combination. bromacetyl-DOTA, also called BAD, is described in M.J. McCall, H. Diril. and C.F. Meares, Bioconjugate Chem. 1: 222-226. 1990

Most particularly the chelator of use in the present invention is eidier DOTA or TMT and die protein reactive group (cross-linker) is either bromoacetyl or a maleimide. most preferably bromoacetyl.

Chelators of use in the present invention may be prepared by any known technique (for example M.McCall et al.. ibid) Radionuclides which may be used in accordance witii die present invention include those appropriate for obtaining in vivo radio immunotherapy and/or imaging of a target cell or tissue. For radioimmunotherapy, a high dose of energy must be delivered to die target site in order that cellular DNA is damaged; both α and β emitting radionuclides produce emissions in a suitable energy range. However α-emitters are eidier shorter lived or decay to hazardous daughter products. Hence die radionuclide of choice for radioimmunotherapy will usually be a β-emitter. For imaging die radiation must interact as little as possible witii the body tissue yet produce a strong signal for external detection. Hence a gamma emitting radionuclide is most suitable for imaging. For botii imaging and radioimmunodierapy the radionuclide must possess a half-life suitable to permit activity or detection after the elapsed time between administration and binding to the target site. The radiolabelled antibody must travel from the bloodstream to d e extracellular fluids of the target via the endothelial pores. Large antibodies or antibody/chelator complexes may diffuse slowly and a radionuclide half-life of between several hours and several days is desirable. In a particular aspect of die present invention the radionuclide is selected from the group comprising of 195^^, 57^1, 5?Co. ¹⁰⁵Ag, ⁶⁸Cu, 52M . ⁵²Fe, ^{ι π}In. ^u3m_Inι 99m_Tc? 67_Ga. 1 ^ 166_Tm? l67_Tm. 146_Gd, 157_Dy₅ 95_mNb, 103_Ru, 97_Ru, 99_Ru, 101_mRh, 201_T1. 203_Hg, 197_Hg, 203p_b. 99^ 48_Cr. 57_Co, 125τ, 131τ, 35_S, 153_Sm, 88γ 90γ 186_Re, 188_Re, 211_AL 212_Bi. 212_{Pb and} 177_Lu.

In a more particular embodiment of the present invention d e radionuclide is selected fr , om th . e group composing I l l, In, 67^ C,u, 186_D Re. 188_ϋ Re. 177- Lu, 99 m _τTc. 131, I. 88_v Y,

90γ. 21 1 _At 212_Bi 212_Pb 57_Co, 153_Sm._, 88γ 90γ _md 177_Lu.

In a more particular embodiment of the present invention the radionuclide is l^Lu 153 s_m 90γ and U lin. The invention also provides a radiolabelled antibody comprising an antibody of the present invention conjugated eidier directly or indirectly to a radionuclide, in particular chelator-antibody conjugate that may be labelled witii ^υY or 17?Lu _{v a} DOTA. or TMT. The invention also provides metiiods for producing antibodies capable of being conjugated at specific sites and for site-directed conjugation of antibodies according to the invention.

According to another aspect of the present invention there is provided the use of a conjugated antibody of die invention in therapy and diagnosis. In particular there is prύvided the use of antibodies according to the invention for the diagnosis and/or tiierapy of conditions which are detectable or amenable to dierapy with dyes, radionuclides enzymes, drugs and cytotoxins. These antibody complexes are useful in treating cancers such as lymphomas and leukaemias and in particular small cell and non small cell lung cancer, prostatic cancer as well as ovarian cancer.. In a most particular aspect of the present invention there is provided an antibody complex according to the invention for use in the imaging and/or treatment of cancers and associated metastases. They may also be used for example as immunosuppressives and more particularly for the treatment of T-cell mediated disorders including severe vasculitis. rheumatoid ardiritis, systemic lupis, also autoimmune disorders such as multiple sclerosis, graft vs host disease, psoriasis, juvenile onset diabetes, Sjogrens' disease, thyroid disease, myasthenia gravis, transplant rejection and astiima.

The invention also provides the use of a conjugated antibody described above in the manufacture of a medicament for the treatment or imaging of any of the aformentioned disorders.

According to anoti er aspect of the present invention there is provided a method of treatment of conditions amenable to therapy and diagnosis witii a conjugated antibody complex according to d e invention comprising administering a tiierapeutically efficacious amount of antibody complex to a mammal requiring such treatment. In particular there are provided metiiods of treatment of cancers such as lymphomas and leukaemias and in particular small cell and non-small cell lung cancer, prostatic cancer as well as ovarian cancer and most particularly metiiods of treatment of cancers and associated metastases. They may also be used in a metiiod of treatment of T-cell mediated disorders including severe vasculitis, rheumatoid arthritis, systemic lupis and also autoimmune disorders such as multiple sclerosis, graft vs host disease, psoriasis, juvenile onset diabetes, Sjogren's disease, thyroid disease, myasd enia gravis, transplant rejection and astiima.

There is also provided in the present invention a pharmaceutically acceptable composition containing conjugated antibodies according to the present invention which comprise a conjugated monoclonal antibody or fragment tiiereof and one or more pharmaceutically acceptable excipients.

Such compositions include, in addition to conjugated antibodies a physiologically acceptable diluent or carrier possibly in admixture with odier agents such as other antibodies or an antibiotic. Suitable carriers include but are not limited to physiological saline, phosphate buffered saline, phosphate buffered saline glucose and buffered saline. Routes of administration are routinely parenteral including intravenous, intramuscular, subcutaneous and intraperitoneal injection or delivery.

In respect of radioimmunod erapy die dosages of compositions containing antibody conjugated to radionuclides according to die invention will vary witii the condition being treated and the recipient of the treatment, but will be in the range of to about 1- lOOmg for an adult patient, preferably 1-lOmg, most preferably 5mg, usually administered as an infusion. A repeat dosing regime may be preferable wherein 10 mg are administered for 1 day followed after weeks or months by a second treatment.

In respect of imaging the dosages of such compositions will vary witii the condition being imaged and the recipient of die treatment, but will be in the range 1 to about lOOmg, preferably 1-10 and most preferably 5mg for an adult patient.

Kits can also be supplied for use with the subject conjugated antibodies in d e protection against or detection of a cellular activity or for the presence of a selected antigen. Thus, a monoclonal antibody conjugated of the present invention may be provided, usually in a lyophilized form in a container, either alone or in conjunction with additional antibodies specific for the desired cell type. The conjugated antibodies, which may be conjugated to a dye, radionuclide. enzyme, drug, cytotoxin, chelator or biotin/avidin, are included in die kits witii buffers, such as Tris, phosphate, carbonate, etc., stabilisers, biocides, inert proteins, e.g., serum albumin, or the like, and a set of instructions for use. Generally, these materials will be present in less tiian about 5% wt. based on the amount of active antibody, and usually present in total amount of at least about 0.001% wt. based again on the antibody concentration. Frequently, it will be desirable to include an inert extender or excipient to dilute die active ingredients, where the excipient may be present in from about 1 to 99% wt. of the total composition. Where a second antibody capable of binding to the chimeric antibody is employed in an assay, tiiis will usually be present in a separate vial. The second antibody is typically conjugated to a label and formulated in an analogous manner with die antibody formulations described above. Generally the kit will also contain a set of instructions for use.

Description of Figures

Figure 1 . Immunoreactivitv of variant antibodies.

a) Five distinct antibody preparations bearing the Campath-IH antigen specificity — 2 natural isotypes (Gl and G4) and 3 variants (G4m. G4c and G4mc), the G4c and G4mc containing ser 442^→ cys substitutions — were constructed and expressed in NSO cells as described in example 1. The antibodies were labelled in situ witii ^^S and purified in the following manner. Two x 10 ' washed producer cells were incubated for 48-72hrs at 37° in 5ml methionine cystine-free DMEM (ICN Biomedicals. Costa Mesa. CA) that contained 6μg cystine/ml. 3μg methionine/ml. and 8-10mCi of 3 s methionine eg. Tran ^S-label (ICN). The supernatant was harvested by centrifugation. dialized and concentrated (Centricon, Amicon. Beverly, MA). The antibody was purified by protein A chromatography (HPLC Dynamax Hydropore-protein A mini column [Rainin, Woburn. MA]), eluted witii 1 M acetic acid, concentrated by ultrafiltration eg. verus a PM 30 membrane (Amicon) and further fractionated by HPLC gel filtration eg. S-5 200A diol, YMC, Willmington. NC. The protein A eluants resolved on gel filtration commensurate with unlabelled antibodies and had specific activites ~ 2μCi/μg. Immunoreactivitv was determined on Fixed Wein 133 Cl cells using the method of Lindmo et al. (J. Immunol Meth. 72: 77-84, 1984). Note that the v intercept, die inverse of which is a measure of immunoreactivity, was equivalent for all the preparations. (The slopes are a function of the specific activity and not d e immunoreactivity.) b) Equilibrium specific binding competition for Campath-IH Gl and G4mc on fixed Wein 133 Cl cells. The two antibodies were biosyndietically labelled in situ with 3 § to the same specific activity (0.3 mCi/nmole) and purified (35S-ligand) as described above. Equilibrium competition was carried out in parallel where each ligand was competed for by its respective unlabelled form. Binding was performed in a total volume of 0.2ml containing 5 x 10^ fixed Wein 133 cells, -0.1 nM of the indicated radiolabeled form of C1H and the indicated concentration of competitor. The binding buffer contained phosphate-buffered saline, 2% bovine calf serum. 0.01% triton XI 00, and 0.02% sodium azide (binding buffer) overnight at 4°. The cells were centrifuged, washed 3 times with cold binding buffer and radioactivity determined. Points depict die average of triplicate measurements ± SEM (bars). The two antibodies were biosyntiietically labelled in situ witii 35$ o the same specific activity (0.3 mCi/nmole) and purified (35s-ligand) as described above. Equilibrium competition was carried out in parallel where each ligand was competed for by its respective unlabelled form. The two profiles indicate that the antibodies have identical antigen binding potencies.

More definitive evidence for site-directed conjugation was obtained by radiolabeling a monoclonal antibody chelator conjugate prepared as described in Example 3. Peptide mapping (enzymatic digestion, fractionation of peptides and peptide amino acid sequence analysis) determined that all radioactive peaks were composed of heavy chain. C-teπninal peptide fragments that contained cys442 -chelator adduct.

Figure 2. Site specific conjugation as indicated bv SDS PAGE.

A ser442→ cys variant, Campath-IH G4mc, was specifically reduced as described in Example 2. By way of comparison, diiol groups were introduced onto nonreduced antibody using the procedure of McCall, et al, Bioconjugate Chem. 1: 222-226, 1990. (The latter procedure is a random conjugation process tiiat employs 2-iminodιiolane to introduce thiol groups onto the ε amino groups of lysine residues.) Both tiiiol- containing antibody preparations were then conjugated as described in Example 2. Equal amounts of d e labelled conjugates were subjected to reducing SDS PAGE, wherein the antibody heavy and li ht chain subunits were separated by virtue of their size. The gel was stained for protein (left side) and for radioactivity by autoradiography (right side). Lanes, left to right: Random, protein stain; specific, protein stain; random, autorad; specific, autorad. The figure shows that the addition of the conjugate was localised to the heavy chain for the specific labelling procedure, whereas for the random process, botii subunits were labelled.

Figure 3.

Biodistribution of ⁹⁰Y-TMT-Campath-1H G4mc conjugate. Reduced Campath-IH G4mc was conjugated to bromoacetyl-TMT, radiolabeled witii 90γcι-, and biodistribution carried out in tumor-bearing mice as described above for bromoacetyl- DOTA. bars show die average % injected dose/g tissue (% ID/g) conected for decay for 5 mice.

The following Examples are illustrative of the present invention and not intended to constitute any limitation thereof:

Example 1 production of variant monoclonal antibodies with a cvs substitution.

(a) Campath-IH and anti-digoxin variants. CVS442 was introduced into the heavy chain of various isotypes by conventional molecular genetic means. For example, genetic constructs of human IgG2. IgG4, Campath-IH and anti-digoxin (botii IgGl) were obtained from within Wellcome Laboratories. The terminal portion of the CH3 region was excised with the restriction enzymes Nsil(5') and EcoRl(3') and replaced with an annealed double stranded oligonucleotide ligated at die respective restriction sites. Antigen specificity was introduced onto constant regions by respective replacement of the variable and CHI regions. A ser228~ P^{r0 was} introduced into IgG4 in order to match die G4 sequence initially reported by Pink, et al, Biochem. J. 117: 33-47, 1970. An IgG4 cys442 variant entitled G4mc was further modified by changing tiiree residues in the CH2 region: leu235→ala. gly237~ *ala and glu3 \ g— ►ala. these latter changes were introduced based on rationale supplied by Winter, et al. tiiat such changes might reduce antibody interaction with Fc gamma receptors and complement Clq (Duncan, et al, Nature 332: 563-564, 1988: Duncan and Winter, Nature 332: 738- 740. 1988).

(b) 323/A3 variant. The murine antibody 323/A3 reacts witii an epitope on human epithelial tissues that may be useful in the identification of treatment of adenocarcinoma (Edwards, et al. Cancer Res. 46: 1306-1317, 1986). The complimentarity determining regions within the variable region of 323/A3 were first "humanized" and grafted onto a human IgGI isotype. To prepare a cys variant, the cDΝA expression construct was ligated in frame with d e cDΝA encoding die constant region of Campath-IH IgG4 cys442 variant. The humanized 323/A3 IgG4 cys442 variant was expressed in ΝSO cells and purified by conventional means.

(c) 323/A3 sFv fragment. A single chain sFv fragment of humanized 323/A3 was constructed by conventional PCR and cloning techniques. Cys variant constructs were produced by introducing a cys residue substitutions into the linker region. For example, the conventional linker region (gly4ser three repeat) was altered to contain gly4serglv2cys2sergly4ser by site-directed mutagenesis. The variants were expressed in E. coli and purified by affinity chromatography.

(d) Mov- 18 variant. Mov- 18 reacts with a folate binding protein that is prominently expressed on ovarian cancer tissue (Miotti. et al., Intl. J. Cancer 39: 297-303. 1987). A human IgGl isotype cDNA was cloned from a public source mRNA library by using reverse transcriptase. The variable region of Mov- 18 was humanized and ligated to die human Gl constant region. Cys442 was introduced into the heavy chain cDNA by site- directed mutagenesis. The humanized Mov- 18 IgGl cys442 variant was expressed in NSO cells and purified by conventional means.

Example 2. Reduction of variant monoclonal antibodies.

Antibody solutions were prepared in degassed buffer, such as 1 OOmM sodium phosphate or trimethylammonium phosphate at pH> 8.0, preferably pH 8.0 - 8.5. at a convenient concentration, for example 100 μM (15 mg/ml). An amount of reductant was mixed witii the antibody solution to achieve the desired extent of reduction. For example, fixed volumes of a 50% gel slurry of a solid phase reductant such as Reduce-Iπu-J (Pierce) were stined into the antibody solution. The reductant capacity of Reduce- ImmTM g_ej _as assumed from the manufacturer to be 30 μmole/ml packed gel. A 250 μl volume of 50% slurry was added to 1 ml of 100 μM antibody solution to generate a mixture with a 30-fold excess reductant capacity (mole reductant/mole antibody). The antibody-reductant mixture was shaken for 1 hr at room temperature, centrifuged and the solution subjected to additional procedures such as free tiiiol determination. pH reduction, purification or conjugation as described in Example 3.

Alternatively, antibody protein was reduced with soluble reductant such as mercaptoediylamine. For example, protein was concentrated to 200-300 μM in 0.1 M sodium phosphate, pH 6.0, 5 mM DTPA. Mercaptoediylamine was added to a final concentration 10-fold in excess of the protein concentration and mixed gently for 1 hour at room temperature. The reduced protein was then separated from reductant and prepared for conjugation by conventional means such as gel filtration. Commonly, protein solution was gel filtered through Bio Spin 30 columns (Bio-Rad Laboratories) that had been pre-equilibrated in 0.1 M tetrametiiylammonium phosphate pH 8.2. 25 uM DTPA for 2 min at 150 x g.

Example 3. Site directed corrugation of reduced variant antibody. A reduced ser442→ cys variant, Campath-IH G4mc, was conjugated to 2-[p- (bromoacetamido)benzyl]- 1 ,4,7, 10-tetraazacyclododecane-N,N'N",N" -tetraacetic acid (bromoacetyi-DOTA). Bromoacetyi-DOTA was a bifunctional moiety wherein one aspect was a chelator that had been prelabelled with 5?Co and the second aspect was a reactive group capable of covalent attachment to a free sulfhydryi. The bifunctional chelator and a metiiod for labelling it with ^Co has been described previously (Mears, et al, Analytical Biochem. 142: 68-78, 1984: ). Briefly, a solution of bromoacetyl DOTA was trace labelled with ^Co. and then a 10-fold molar excess was added to antibody solution. The conjugation reaction was carried out for 2 hrs at 37° and stopped by separation of the reactants by gel filtration using a Bio Spin 30 column (Bio Rad) as recommended by the manufacturer. Bifunctional chelators, such as bromoacetylTMT, were also employed prior to radiolabeling. The termperature and duration of d e conjugation reaction was varied for maximal site-specific conjugation. For example, reduced protein at a concentration of 150 - 200 uM in tetrametiiylammonium phosphate, pH 8.2. 25 μM DTPA, was added to a 10-fold excess of bromoacetylTMT in a metal- free reaction vial. The reaction was carried out for 24 hours at room termperature. and die conjugate was isolated by gel filtration as described above.

Example 4, Muring biodis-i- -tion o conjugates,

A reduced ser442→ cys variant, Campatii-IH G4mc, was conjugated to bromoacetyl DOTA by the site directed procedure described above without prelabelling. By way of comparison, random conjugate was prepared by introducing thiol groups into nonreduced Campatii- 1 H g4mc as has been described previously for Lym- 1. (The latter process follows the procedure of McCall, et al, Bioconjugate Chem. I: 222-226, 1990 and employs 2-iminodιiolane to introduce thiol groups randomly onto die ε amino groups of lysine residues.) Iminothiolated Campath-IH G4mc was conjugated to bromoacetyi-DOTA to the same extent (chelators per antibody) as site directed conjugate. The conjugates were rendered radioactive by mixing with ⁹^YC 13 in plastic ware by addition of the following reagents: an 8X volume of monoclonal antibody conjugate (> 25 mg/mL. 0.1 M ammonium acetate pH 6.7), a 2X volume of 90N. and cold yttrium up to a final concentration of 10 μM. Cold yttrium was added first. followed by 90γ and conjugate. Chelation was allowed to occur at room temperature for 90 minutes. Ten μg (2 μCi) were injected intravenously per mouse into mice bearing a subcutaneous tumour (CHO-10/D4) that expressed die Campath-IH antigen. Mice were sacrificed at d e indicated times, tissues excised, weighed and radioactivity determined. The results are shown in Table II below and are expressed as the % injected dose per gram tissue (average of 5 mice per group +/- sem). Note the diminution of normal tissue deposition and increase in tumour deposition in die site- directed (direct) conjugate groups relative to the random groups. Figure 3 illustrates the biodistribution of 90 Y-TMT-Campath- 1 HG4mc.

Example 5

A reduced ser442"^cys variant. Campath-IH was conjugated to TMT, tiiat is covalently attached via a thioether linkage to the cys442 residues in die heavy chains as illustrated in Figure 4. The reaction may be regulated to produce conjugates that contain an average of 1 - 2 chelators per antibody. Conjugate is purified free of unreacted bifunctional chelator by gel filtration in metal-free conditions and is stable in a buffered, metal-free environment.

Immunoconjugate chelation. Conjugates were radiolabeled in metal-free plasticware using the best metal-free reagents available. Carrier-free 90 YC 13 was purchased from Dupont/New England Nuclear, Amersham and odier sources. The specific activity was typically 5 mCi in 10-30 μL 0.05 N HC1, specific activity 5.6 X 10⁵ Ci/g. Prior to use, the 90γci was buffered witii 0.1 volume of 6 M ammonium acetate to ~ pH 5.8. Chelation was performed by adding the following reagents in sequence and incubating for up to 90 min at room temperature: a IX volume of cold yttrium (lOOμM yttrium in 0.1 M ammonium acetate, pH 6.8.), a 2X volume of 9 γ acetate, and an 8X volume of monoclonal antibody conjugate (25 mg/ml 0.1 M ammonium acetate, pH 6.5). Non- chelated radiometal was "scavenged" by d e addition of DTPA to a final concentration of 500μM and a 10X volume of 0.1 M ammonium citrate, pH 6.5. The mixture was incubated at room temperature for 30 minutes and fractionated by "spin column gel filtration," i.e., applied to a 1 mL Bio-Spin 30 (Bio-Rad Laboratories) tiiat had been pre-equilibrated in phosphate-buffered saline and centrifuged at 150 x g for 2 minutes. Spin column gel filtration was repeated for a total of two centrifugations. The efficiency of chelation (ability to chelate all the radiometal) and scavenging (ability to remove non-chelated radioactivity from radiolabeled conjugate) was monitored by thin layer chromatography as described by Meares et al, Anal. Biochem. 142, 68-78. 1984. More than 90% of the radiometal was routinely chelated. (The extent of chelation was dependent on die acid and metal content of die supplied radiometal.) After scavenging, die fraction of " γ that was tightly bound to conjugate was typically >98% when analyzed by HPLC gel filtration, thin layer chromatography or SDS PAGE.

90γ.postlabeled conjugates have been prepared with specific activities of up to 10 mCi/mg which is 30-fold below the theoretical capacity. Although every chelator is available to accept radiometal. higher specific activity causes radiolysis (a function of time and concentration) that can be reduced by inclusion of an anti-oxidant such as ascorbic acid. In principle, chelation can be optimized for complete efficiency given a consistent and high quality supply of radiometal and thus eliminate the need for scavenging.

Example 6. Large scale reduction of variant monclonal antibodies

Dilute ultrafiltered IgG product to 50mg/ml (+/- lmg/ml) and measure a volume V. Add V/10 of freshly prepared stock solution (13mg/ml ME A in 50mM phosphate and 5mM EDTA at pH7.0), mixing well during addition to ensure even distribution of reductant. Leave at ambient temperature for 60 minutes. The sample is applied to a gel filtration column such as, for example, Sephadex G25 or Superdex 75 or 30. The IgG peak is monitored at absorbance of 280nm and die peak collected and subjected to additional procedures such as free thiol determination, pH adjustment, purification or conjugation as described in Example 3.

Example 7. Large scale site directed coηiugation of reduced variant antibody.

Add 10 fold molar excess of bromoacetyl-TMT as a lOmM stock made up in 0.1 M Hepes + 25μM DTPA at pH8.4 over the IgG from example 6. Mix well and leave at ambient temperature for a minimum of 21 hours, maximum 37 hours. The conjugated antibody is then loaded onto a Superdex 200 column. The IgG peak is monitored at absorbance of 280nm and the monomer peak collected and subjected to standard analytical procedures such as estimation of binding, protein content etc. ________

Free thiol content following exposure to solid-phase reductant

A G4 ser442→ cys variant labelled "G4mc" and a natural G4 control were exposed to solid phase reductant as described in Example 2. The thiol content was determined by Ellman's reagent and is expressed relative to moles antibody. (It is assumed tiiat 2 moles of tiiiol were reduced per antibody ti iol.) The sem for triplicate measurements was ± 0.1 SH/antibody.

Molar Ex Gel Moles -SH/Antibody

______ __4

Expt.1

100 4.6 .6

30 3.0 10 1.2 .1

J .6 0 0 .1 0

Expt.2

50 J.J .J 30 2.3 20 1.7 10 1.0 - .5 0 .1

Iab JI

Murine biodistribution of ^--Y-coηjugates

% ID/g ⁽avg -/- se ^

24 hour 72 hour 168 hour direct

BLOOD 13.47 1.75 8.80 1.67 5.93 0.80

SPLEEN 3.51 0.63 2.46 0.50 2.83 0.39

TUMOUR 17.01 3.16 24.78 8.41 31.36 4.96

LIVER 5.24 0.62 3.32 0.69 2.56 0.28

LUNG 5.60 0.49 5.00 0.70 3.99 1.32

KIDNEY 5.09 0.30 5.21 0.65 4.16 0.54

BONE 1.56 0.19 1.09 0.18 0.95 0.15

random

BLOOD 11.97 3.96 9.48 2.00 6.66 0.76

SPLEEN 5.79 1.42 4.61 0.90 3.55 2.07

TUMOUR 15.42 5.52 22.37 9.29 29.91 4.70

LIVER 10.07 2.99 6.75 1.31 4.34 0.82

LUNG 6.19 0.95 4.56 1.22 3.67 1.08

KIDNEY 5.38 1.41 6.84 1.41 5.18 0.50

BONE 1.76 0.46 1.39 0.35 1.25 0.14

Claims

_________

1. An immunochemically functional monoclonal antibody comprising a cysteine residue exposed on the surface of the antibody such that die residue is capable of being conjugated to a substance.

2. An antibody according to claim 1, wherein the cysteine residue is in the variable region of the antibody but not part of the CDRs.

3. An antibody according to claim 1, wherein the cysteine residue is in die constant region .

4. An antibody according to claim 3, wherein the cysteine residue is in the CH3 domain of the heavy chain.

5. An antibody according to claim 4. wherein the cysteine residue is at position 442 within the CH3 domain.

6. An antibody according to any of the preceding claims, wherein the antibody binds to a 40KD antigen or folate receptor antigen.

7. An antibody according to any of d e preceding claims, wherein the antibody is humanised.

8. An antibody according to any of the preceding claims, wherein the antibody is a Gl or G4 isotype.

9. An antibody conjugate comprising an antibody according to any of the preceding claims and a substance, which is directly or indirectly conjugated to the cysteine- residue of the antibody.

10. An antibody conjugate according to claim 9, wherein the substance is indirectly conjugated via a chelator.

1 1. An antibody conjugate according to claim 9, wherein die substance is indirectly conjugated via a linker and a chelator.

12. An antibody conjugate according to claim 10 or 1 1. wherein the chelator is TMT or DOTA.

13. An antibody conjugate according to claim 1 1 or 12. wherein the linker is bromoacetyl.

14. An antibody conjugate according to any of claims 10 to 13, wherein die substance is Y or Lu.

15. An antibody conjugate according to claim 9, wherein the substance is a molecular chimaera for use in enzyme-prodrug therapy.

16. An antibody conjugate according to claim 15. wherein die chimaera comprises a transcriptional regulatory DNA sequence capable of being activated in a mammalian cell and a DNA sequence operatively linked to die transcriptional regulatory DNA sequence and encoding a heterologous enzyme capable of catalysing the conversion of the prodrug into an agent toxic to die cancer cell.

17. An antibody conjugate according to claim 16. wherein the transcriptional regulatory DNA sequence is a tissue- or cancer- specific transcriptional regulatory DNA sequence.

18. An antibody conjugate according to claim 15 or 17, wherein the chimera is contained witiiin a viral vector or liposome.

19. Use of an antibody conjugate according to any of claims 9 to 18, for the treatment and diagnosis of cancers and associated metastasis.

20. Use of an antibody conjugate according to claims 9 to 14, for the treatment of small cell and non-small cell lung cancer, prostatic cancer and associated metastasis.

21. Use of an antibody conjugate according to claims 15 to 18, for the treatment of ovarian cancer.

22. Use of an antibody conjugate according to claims 10 to 14 for radioimmuno¬ therapy.

23. A pharmaceutically acceptable composition comprising conjugated antibodies according to any of claims 9 to 18 togetiier with a physiologically acceptable diluent or carrier.

24. A pharmaceutically acceptable composition according to claim 23 for radioimmunodierapy, wherein the dosage is 1 to 10 mg.