AU660445B2 - Fusion proteins for prodrug activation, the preparation and use thereof - Google Patents

Abstract

The invention relates to fusion proteins for prodrug activation of the general formula huTuMAb-L- beta -gluc, where huTuMAb is a humanised or human tumour-specific monoclonal antibody or a fragment thereof, L is a linker and beta -gluc comprises human beta -glucuronidase. These fusion proteins are prepared by genetic manipulation. huTuMAb ensures specific location of tumours, L connects huTuMAb to beta -gluc in a way which does not hinder the specific properties of the two fusion partners, and beta -gluc activates a suitable prodrug compound by elimination of glucuronic acid, and owing to the humanised or human fusion partner a virtually autologous system is available for use in humans.

Description

Regulation 3.2(2)

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: FUSION PROTEINS FOR PRODRUG ACTIVATION, THTJ' PREPARATION AND USE THEREOF

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The following statement is a full description of this invention, including the best method of performing it known to us BEHRINGWERKE AKTIENGESELLSCHAFT HOE 91/B 004 Ma 876 Dr. Lp/Bi Description Fusion proteins for prodrug activation, the preparation and use thereof The rnvention relates to fusion proteins for prodrug activation of the general formula huTuMAb-L-p-Gluc, where huTuMAb is a humanized or human tumor-specific monoclonal antibody, a fragment or a derivative thereof, L is linker, and p-Gluc comprises human p-glucuronidase. These fusion proteins are prepared by genetic manipulation.

huTuMAb ensures the specific localization of tumors, L connects huTuMAb to p-Gluc in such a way that the specific properties of the two fusion partners are not impaired, and A-Gluc activates a suitable prodrug compound by elimination of glucuronic acid, where a virtually autologous system for use in humans is provided by the humanized or human fusion partners.

Combination of prodrug and tumor-specific antibody-enzyme conjugates for use as therapeutic agents is described in Sthe specialist literature. This has entailed injection of antibodies which are directed against particular tissue S 25 and to which a prodrug-cleaving enzyme is covalently bonded, into an animal which contains the transplanted tissue, and subsequently administering a prodrug compound which can be activated by the enzyme. The action of the antibody-enzyme conjugate which is anchored in the tissue converts the prodrug compound into thG cytotoxin which e sets a cytotoxic effect on the transplanted tissue.

WO 88/07378 describes a therapeutic system which contains two components and is composed of an antibody-enzyme component. Tha use of non-mammalian enzymes for the preparation of the antibody-enzyme conjugate is described in this case, and that of endogenous enzymes is ruled out 2 because of the non-specific release of active substance.

Since the exogenous enzymes are recognized as foreign antigens by the body, the use thereof is associated with the disadvantage of an immune response to these nonendogenous substances, on the basis of which the enzyme immobilized on the antibody is inactivated and, possibly, the entire conjugate is eliminated. Furthermore, in this case p-bis-N-(2-chloroethyl)-amino-benzylglutamic acid and derivatives thereof are used as prodrug, the chemical half-life thereof being only 5.3 to 16.5 hours. The chemical instability of a prodrug compound is a disadvantage because of the side effects to be expected.

EP A2-0 302 473 likewise describes a therapeutic system containing two components, in which the antibody-enzyme conjugate which is localized on the tumor tissue cleaves a prodrug compound to give a cytotoxic active substance.

The combined use, which is described herein inter alia, of etoposide 4'-phosphate and derivatives thereof as prodrug and antibody-immobilized alkaline phosphatases to •20 liberate the etoposides is a disadvantage because of the presence of large amounts of endogenous alkaline phosphatases in serum. DE Al-38 26 562 describes how etoposide 4'-phosophates have already been used alone as therapeutic antitumor agent, with the phosphatases present in serum liberating the etoposide from the prodrug.

It has been found that huTuMAbs coupled via L to p-Gluc and prepared by genetic manipulation represent a particularly advantageous, because virtually autologous, system. It has additionally been found that the catalytic activity of p-Gluc in the fusion protein at pH 7.4 (i.e.

physiological conditions) is significantly higher than that of the native enzyme when the fusion protein is bound to the antigen via the V region. Furthermore, a fusion protein with only one hinge region (see -Fg -P-and- -Example 0) can be generated by genetic manipulation in high yield because most of the product which is formed 3 results as one band (in this case with molecular weight 125,000) and can easily be purified by affinity chromatography with anti-,Ldiotype MAbs or anti-glucuronidase MA bs.

It has furthermore been foind that a chemical modification of the fusion proteins, in particular partial or complete oxidation of the carbohydrate structures, preferably with subsequent reductive amination, results in an increased half-life. Enzymatic treatment of the fusion proteins according to the invention with alkaline phosphatase from, for example, bovine intestine or E. coli has in general not resulted in a significant increase in the half-life.

Consequently, the invention relates to fusion proteins of 15 the formula huTbMAb-.L-p-Gluc

(I)

where huTuMAb is a humanized or human tumor-specific monoclonal antibody or a fragment or a derivative thereof, and preferably comprises the MAbs described in EP-Al-0 388 914. The fusion proteins according to the invention particularly preferably contain the humanized MAb fragment with the V, and VH genes shown in Table 3.

L is a linker and preferably contains a hinge 25 region of an immunoglobulin which is linked via a peptide sequence to the N-terminus of the mature enzyme.

B-Gluc is the complete amino-acid sequence of human p-glucuronidase or, in the relevant gene constructs, the complete cDNA (Oshima A. et al., Proc. Natl. Acad. Sci. USA 84, (1987) 685-689.

Furthermore preferred are constructs with a CH, exon and a hinge exon in the antibody part, and particularly preferred constructs are those in which these parts 4 derive from a human IgG3 C gene. Most preferred are construct6, as described in Example where the corresponding light chain of the humanized TuMAb is coexpressed in order, in this way, to obtain an huTuMAb portion which is as similar as possible to the original TuMAb in the binding properties. Finally, the invention relates to processes for the preparation by genetic manipulation of the abovementioned fusion proteins, to the purification thereof and to the use thereof as pharmaceuticals. Fusion proteins as described can be used for prodrug activation in oncoses.

In another embodiment, the fusion proteins according to the invention are chemically modified in order to achieve an increased half-life and thus an improved localization of tumors. The fusion proteins are preferably treated with an oxidizing agent, for example periodate, which ii9..: generally results in partial or complete cleavage of the carbohydrate rings and thus in an alteration in the carbohydrate structure. This alteration generally results 20 in an increased half-life, It is furthermore advantageous to derivatize, in a second reaction step, existing aldehyde groups, for example by reductive amination. The partial or complete destruction of the aldehyde groups generally results in a reduction in possible side reactions with, for example, plasma proteins. Accordingly, it is advantageous for the fusion proteins according to the invention to be oxidized in a first reaction step, for example with periodate, and to be reductively aminated in a second reaction step, for example with ethanolamine and 30 cyanoborohydride.

The following examples describe the synthesis by genetic manipulation of a particularly preferred fusion protein according to the invention, the derivatization thereof and the demonstration of the ability of the two fusion partners to function.

Example The starting material was the plasmid pGEM4-HUGP13 (Fig. pGEM4-HUGP13 contains a cDNA insert which contains the complete coding sequence for the human p-glucuronidnae enzyme (Oshima et al. loc. cit.). All the techniques used were taken from Maniatis et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor USA (1989).

Example The plasmid pGEM4-HUGP13 was cut with the restriction endonucleases PstI and Sall, and the 34.bp-long PstI/SalI fragment which harbors the NotI restriction cleavage site was isolated. The PstI/SalI fragment was cloned into the 15 PstI/SalI-cleaved vector pTZ (Fig. B.I) and the clone pTZpGlc350 was isolated (Fig. B.2).

.Example The plasmid clone pTZpGlc350 was cleaved with PstI, and the double-stranded DNA fragment pGlc linker, which is S 20 composed of the oligonucleotides pGlc linker 1 and fGlc linker 2 (Tab. 1) and has cohesive PstI ends, was ligated e into the opened PstI cleavage site.

-6- Tab. 1 (3Glc Linker 1: 3' GOG GCG GCG GCG GTG CA BG-~g Linker 2: 3' COG CCG CCG CCG CTG CA so,* The clone pTZgGlc37O in which the A-glucuronidase fragment is extended at its 5'1 end by oligonucleotide I and which has lost the previously present PstI cleavage site 5 but has acquired instead at its 5'1 end a new PstI cleavage site was isolated (Fig. C) Example The plasmid clone pTZpGlc37O was cleaved with PstI and p. ligated to the hinge-linker fragment which is composed of the hinge oligonuc leot ides 1 and 2b and which has two cohesive PstI ends (Tab. This results in the PstI cleavage site at the 5' end of the #Glc37O fragment being destroyed. The plasmid clone pTZfiGlc420 in which the pGlc insert is extended at the 5' end by the hinge linker H :is was isolated (Fig. D).

7- Tab 2 Hinge 1 Oligo: GAG CCC AAA TCT TGT GAC ACA OCT CCC CCG TGC CCA 3.

CGG TGC CCA GTT GCA Hinge 2b Oil=g ACT GGG CAC CGT GGG CAC GGG GGA GGT GTG TCA CAA 3' GAT TTG GGC TCT GCA 6 Exmle 60000 h lsi T*l42 a lae ih Pt Sall The plasi copltelywa cleaved with tl anadtSaill, clandd the Pt h ioae 420bp insert was isolated.Tepard o Ighis Fa) 2 P-A2- 352.)ca 76, igs3 iide, wn hc I:.:lo pami lewihcontains the CH, exon, anaw hinge exons fahmnIG Cgenei wasfomatoy cleavied with Sailean partially ave wt P-lcrns. The isolated 42bp insertH wa (Fig E) -8 Tab. 3 30 s0 110 accgtgtctggcttc-accatcagcagtggttatagctggcactgggtgagar-agcacc -n rlerl~ellee~xl~re~pLL~paAj3nr~ 130 150 170 1,9O 210 230 tcctcaaaa,,,tagagtgacatgctggtagacaccagcaacacagttagc-tjS SertLeuLysSerAxgVaThxb~tLeuVaAspThSeLysAsnGnPhe~jerLuA 250 270 290 ctcagcagcgt'acagccgccgacaccgcggtctattattggcaagagaagactatg Le~re~lhAal~phXaa~ry~sl~c~l~py.- 310 330 taccactggacttcgatgtctggggtcaagcagcctcgtc-aragtctcctca Ty~s~~rh~pa~plG~~ye~ua~ra~re 43/2 VI 1 30 -:130 350 170 ca4oggtgagtccactccgcatccgatgacccagaaccaacctgcagctgc )Gsy-oa13i~s~eAaProeG yst~eu~neulle' erySererAasner~la1qG: 790 210 110 lPrg~eAJTrIherhzy SereSererha1speTyr~thiTrp~erG 2510 250 270 cagcagccaggtaggctccacactgcctaccacacgatagactcctct VauIP oAGlupheerleahr~yerGy~iGh.nAs=ehr~erThyrIroehe 210 27029 ggccaagggaccaaggtggaaatcaaacgt GlyG~nGlyThrT-ysValGuleLysAxq 9- Example The piasmid pGE.X4-HUGPl3,lGlc was cleaved with Sail, and th~e 1750bp Sail fragment from the O-glucuronidase cDNkA waa isolated, The isolated 1750bp SlI fragment was ligated to the Sal-cleaved plasmid pUC CHI H PGlc42O. The plasmid clone ptJC CI H huflGlc which contains a fusion gene composed of a CHI exon, a hinge exon and a fq',on exon between a hinge exon and the human fi-glucuronidap-! cDNA was isolated (Fig. F).

Example The expression vector pABstop (Fig. 1. 1) was cleaved with Hindll and Sall. The plasmid pUC CHI H hupGlq- was cleaved completely with HindlII iirid partially with Sail, and the CHI H huPGlc insert was isolated. The CH, H huPGlc inser~t was ligated to the HindIIl/Sall- *.:cleaved pABsl and the clone pABstop CHI H hufiGlc **too: was isolated (Fig. G).

Example :9.:The pABstop vector pABstop BW 431/26 hum which contains the humanized version of the Va gene of the anti-CEA I4Ab BW 431/26 (Bossiet K. et al., Sur. J. Nuci.

14, (1988) 523-528) (see Tab. 3 for the sequences of the humanized Va and VK gene), was cleaved with HindIII and BainHI, and the insert which contain.i, the signal exon *:25 and the Vs exon was isolated. The plasmid el ne pABStop 6: CHI H huPGic was cleaved with Hindll and "i,gated to~ the HindIII/BaniHI 431/26 hum V 9 fragment. After ligation at room temperatulre for 2 h, the ligation was stoppftd by incubation at 70*C for 10', and the ends which weze still free were filled in with Kienow polymerase and dNTPs.

Further ligation was then carried out overnight. After transformation, the clone pABStop 431/26 hum Va hupGlclH which contains an immunoglobulin F(ab') 2 gene with a hinge exon, which is fused to the coding region of human 10 p-glucuronidase, was isolated with the aid of restriction mapping and nucleic acid sequence analysis (Fig. H).

Example The clone pABStop 431/26 hum V H hufGlclH was transfected together with a plasmid clone which carries the light chain of humanized BW 431/26 (Fig.~4) and two plasmids which carry a neomycin- (Fig. K) and a methotrexateresistance gene (Fig. L) into BHK cells. A fusion protein which has both the antigen-binding properties of MAb BW 431/26hum and the enzymatic activity of human p-glucuronidase was expressed.

Example Demonstration of the antigen-binding properties and of the enzymatic activity of the 431/26 hum V3hupGlc 1H 15 fusion protein The ability of the 431/26 hum VhupGlc 1H fusion protein to bind specifically to the epitope defined by 431/26 on CEA (carcinoembryonic antigen) and, at the same time, to exert the enzymatic activity of human p-glucuronidase was 20 determined in a specificity/enzyme activity assay. This assay is carried out as described below: C C Polystyrene (96-well) microtiter plates (U shape, Type B, supplied by Nunc, Order No. 4-60445) are incubated with purified CEA (1-5 pg of CEA/ml, 75 pl S 25 of this per well) or with GIT mucin (same amount as CEA) at R.T. overnight.

The non-adsorbed antigen is removed by aspiration and washed 3 x with 0.05 M tris/citrate buffer, pH 7.4.

The microtiter plates are left to stand at R.T. with the opening facing downwards on cellulose overnight.

The microtiter plates are incubated with 250 pl of 1% strength casein solution in PBS, pH 7.2, per well 11 (blocking solution) at 20°C for 30 minutes.

During the blocking, the substrate is made up. The amount of substrate depends on the number of supernatants to be assayed. The substrate is made up fresh for each assay.

Substrate: 4-methylumbelliferyl p-D-glucuronide (Order No.: M-9130 from S3igma), 2.5 mM in 200 mM sodium acetate buffer, pH 5.0, with 0.01% BSA.

The blocking solution is removed by aspiration, and in each case 50 pl of BHK cell supernatant which contains the fusion protein are loaded onto the microtiter plate coated with CEA or GIT mucin (that is to say the sample volume required is at least 120 pl).

Incubation at R.T. is then carried out for minutes.

The plates are washed 3 x with ELISA washing buffer (Behring, OSEW 96).

The substrate is loaded in (50 pl/well) and in- 20 cubated at 37°C for 2 hours. The plate is covered because of the possibility of evaporation.

After 2 hours, 150 pl of stop solution are pipetsed into each well (stop solution 0.2 M glycine 0.2% SDS, pH 11.7).

25 Evaluation can now be carried out under a UV lamp (excitation energy 380 nm) or in a fluorescence measuring instrument (Fluoroscan II, ICN Biomedicals, Cat. No.: 78-611-00).

It was possible to show using this specificity/enzyme 30 activity assay that fluorescent 4-methylumbelliferol was detectable in the wells coated with CEA when the enzyme activity was determined at pH 5, the catalytic optimum (Tab. 4).

12 Table 4: Dilution out of fusion protein cell culture supernatant (B73/2) on CEA and GIT mucin Substrate in various solutions Dilution 0.2 M sodium PBS, pH 7.2, PBS, pH 7.2 steps acetate buffer on CEA on GIT mucin 0.01% BSA, pH 5, on CEA Concentrated 9118 2725 115.7 1:2 7678 2141 93.37 1:4 4662 1195 73.39 1:8 2927 618.5 60.68 1:16 1657 332.1 53.69 1:32 853 168.2 40.44 1:64 425 99.26 48.21 1:128 192.5 57.89 47.48 Determination of the conversion rate at pH 7.2 showed that at this physiological pH that of the fusion protein was still 25% of the conversion rate at pH 5. No significant methylumbelliferol liberation was measurable on the negative control plates coated with GIT mucin and measured at pH 5. This finding shows that the humanized 25 V region of the 431/26hum VghupGlc 1H fusion protein has retained its epitope specificity, and the p-glucuronidase portion of the fusion protein is able, like the native human enzyme, to cleave the p-glucuronide of 4-methylumbelliferol.

Example Demonstration of the functional identity of the V region of the 431/26hum VghupGlc 1H fusion protein with that of the humanized MAb BW 431/26 and that of the murine MAb BW 431/26 13 It was shown in Example that the 431/26hum VehufGlc 1H fusion protein has a certain CEA-binding potential and p-glucuronidase activity. The antigen-specific competetive assay described hereinafter provides information on the identity of the CEA epitopes which are recognized by the competing molecules, and on the strength of the epitope/fusion protein and epitope/antibody interactions.

This assay is carried out as described below: Polystyrene 96-well microtiter plates (U shape, Type B, supplied by Nunc, Order No. 4-60445) are incubated with purified CEA (1-5 pg of CEA/ml, 75 pl of this per well) or with GIT mucin (same amount as CEA) at R.T. overnight.

S The non-adsorbed antigen is removed by aspiration and washed 3 x with 0.05 M tris/citrate buffer, pH 7.4.

The microtiter plates are left to stand at R.T. with the opening facing downwards on cellulose overnight.

o The microtiter plates are incubated with 250 pl of S: 20 1% strength casein solution in PBS, pH 7.2, per well (blocking solution) at R.T. for 30 minutes.

50 pl of the MAb BW 431/26 in a concentration of 5 ng/ml are mixed with 50 pl of 10-fold concentrated supernatant of the humanized MAb BW 431/26 or of the fusion protein, as well as serial 2 x dilutions.

50 pl aliquots of these mixtures are pipetted into the wells of microtiter plates coated with CEA or GIT mucin.

The microtiter plates are incubated at R.T. for 30 minutes.

The plates are then washed 3 x with ELISA washing buffer (supplied by Behringwerke AG, Order No.

OSEW 96, 250 pi).

Then 50 pl of a 1:250-diluted goat anti-mouse Ig antibody which is coupled to alkaline phosphatase (Southern Biotechnology Associates, Order No.: 1010-04) are added.

14 After incubation at R.T. for 30 minutes and washing 3 times, the substrate reaction is carried out as follows: Add 30 pl of 0.1 mM NADP per well (dissolve 7.65 mg in 100 ml of 20 mM tris; 0.1 mM MgSO 4 pH the solution can be stored at -20°C for several months.

Incubate at R.T. for 30 minutes.

Make up the enhancer system during the incubation with NADP: (5 ml per plate) 2 parts of INT (dissolve 2.5 mg/ml in 30% strength ethanol in an ultrasonic bath; always make up fresh) 1 part of PBS, pH 7.2 1 part of diaphorase (1 mg/ml PBS, pH 7.2) 1 part of ADH (0.5 mg/ml PBS, pH 7.2) add 50 pl of the enhancer system when the extent of reaction is as required, stop the reaction with 0.1 N H 2

SO

4 100 il per well measure at 492 nm in a TITERTEK' MULTISCAN (blank 50 pl of NADP 50 pl, of enhancer solution 100 pl of 0.1 N HSO0 4 NADP supplied by Sigma, Order No. N-0505 INT supplied by Sigma, Order No. 1-8377 ADH supplied by Sigma, Order No. A-3263 Diaphorase supplied by Sigma, Order No. D-2381 25 Reduction of the extinction in this antigen-specific competetive assay means that there is competition between the molecules competing with one another for epitopes which are the same or lying very close together spatially.

30 The inhibition data which are obtained show that both the fusion protein 431/26hum VshupGlc Hi and the humanized MAb 431/26 block binding of the murine MAb BW 431/26 to its CEA epitope. 50% inhibition ie reached at a 200 molar excess of the relevant competitors. The conclusion from this is that the avidity of the fusion protein for the CEA epitope is comparable with that of the humanized 15 MAb 431/26. Furthermore, the fusion protein and the humanized MAb bind to the same epitope or to an epitope which lies spatially very near to that defined by the murine MAb BW 431/26 on CEA.

Example Demonstration of the tissue specificity of the 431/26hum VghupGlc 1H fusion protein Example showed, inter alia, that the 431/26hum VEhupGlc 1H fusion protein is able to bind to purified

CEA.

Example showed that the V region of the fusion protein is able to compete with the V region of murine BW 431/26 for the same, or a very close, epitope. The indirect immunohistochemical assay which is specific for A-glucuronidase and is described hereinafter can be used to determine the microspecificity of the fusion protein on cryopreserved tissues.

The assay is described below: 6 pm-thick frozen sections are placed on slides and dried in air for at least 30 minutes.

The slides are subsequently fixed in acetone at for 10 seconds.

The slides are washed in tris/NaCl washing buffer, pH 7-4, with 0.1% BSA for 5 minutes.

25 20-100 pl of fusion protein-containing BHK cell supernatant (concentrated or diluted in tris/BSA, pH 7.4) is applied to each section and incubated in a humidity chamber at R.T. for 30 minutes.

The slides are washed in tris/NaCl washing buffer, pH 7.4, with 0.1% BSA for 5 minutes.

S 50 pl of hybridoma supernatant of the murine antip-glucuronidase MAb BW 2118/157 are added to each section, and the slides are incubated in a humidity 16 chamber at R.T. for 30 minutes.

The slides are then washed in tris/NaCl washing buffer, pH 7.4, with 0.1% BSA for 5 minutes.

20-100 pl of bridge Ab (rabbit-antimouse IgG diluted 1:100 in human serum, pH 7.4) are applied to each section and incubated in a humidity chamber at R.T.

for 30 minutes.

The slides are then washed in tris/NaCl washing buffer, pH 7.4, with 0.1% BSA for 5 minutes.

Subsequently 20-100 pl of APAAP complex (mouse anti- AP diluted 1:100 in tris/BSA, pH 7.4) are applied to each section and incubated in a humidity chamber at R.T. for 30 minutes.

The slides are then washed in tris-NaCl washing buffer, pH 7.4, with 0.1% BSA for 5 minutes.

The substrate for alkaline phosphatase is made up as follows (100 ml of substrate solution sufficient for one glass cuvette): Solution 1: 3.7 g of NaCl 20 2.7 g of tris base (dissol--e in 75 ml of distilled water) 26.8 ml of propanediol buffer, pH 9.75, adjust with HCI 42.9 mg of levamisole clear, colorless solution Solution 2: Dissolve 21.4 mg of sodium nitrite in 535 pl of distilled water Sclear, colorless solution Solution 3: Dissolve 53.5 mg of naphthol AS BI phosphate in 642 pl of dimethylformamide (DMF) clear, yellowish solution Add 368 pl of 5% strength new fuchsin solution to solution 2 (sodium nitrite) and leave to react for 1 minute (stopclock) to give a clear, brown solution Add solution 2 (sodium nitrite with new fuchsin) and solution 3 (naphthol AS BI phosphate) to solution 1 (tris/NaCl/propanediol buffer) clear, 17 yellowish solution adjust to pH 8.8 with HC1 cloudy, yellowish solution filter solution and place on the slide and leave to react on a shaker for 15 minutes solution becomes cloudy.

wash slide in tris/NaCl buffer, pH 7.4, for minutes wash slide in distilled water for 10 minutes after drying in air for 2 hours, the slides are sealed in Kaiser's glycerol/gelatin at 56 0

C.

Specific binding of the fusion protein was demonstrated under the light microscope by the red coloration of the epitope-positive tissue sections. Comparative investigations with the murine MAb BW 431/26, which was detected by the indirect APAAP technique (Cordell et al., J. Histochem. Cytochem. 32, 219, 1984), revealed that the tissue specificity of the fusion protein agreed exactly 2 with that of the murine MAb BW 431/26, i.e. that there is 20 identity both of the reaction type in the individual specimen and of the number of positive and negative findings from a large number of different carcinomas and normal tissue.

Example Purification of the 431/26hum VhupGlc IH fusion protein Murine and humanized MAbs can be purified by immunoaffinity chromatography methods which are selective for the Fc part of these molecules. Since there is no Fc part in the 431/26hum VghupGlc 1H fusion protein, it was necessary to develop an alternative highly selective immunoaffinity chromatography method. Besides the selectivity of this method to be developed, it is necessary for the isolation conditions to be very mild in order not to damage the p-glucuronidase, which is very labile in the acidic and in the alkaline range.

18 The principle of the method comprises purification of the fusion protein from supernatants from transfected BHK cells using an anti-idiotype MAb directed against the humanized V region. The preparation of such MAbs is known from the literature (Walter et al., Behring Inst. Mitt., 82, 182-192, 1988). This anti-idiotype MAb can be both murine and humanized. The MAb is preferably immobilized on a solid phase so that its V region has not been damaged. Examples of this are known from the literature (Fleminger et al., Applied Biochem. Biotechnol., 23, 123- 137, 1990; Horsfall et al., JIM 104, 43-49, 1987).

The anti-idiotype MAb thus immobilized on the solid phase by known methods binds very efficiently the fusion protein to be purified from transfected BHK cells, for example at pH 7, but has the surprising property that it no longer binds the fusion protein when the pH is lowered by only 1.5, to pH 5.5. This mild pH elution technique •has no adverse effect on the fusion protein, either in its ability to bind to CEA or in its enzymatic activity 20 (for methods, see Example Tab. 5 shows the OD values S• and fluorogenic units (FU) of the individual fractions S• from a purification using the solid phase-immobilized, anti-idiotype MAb BW 2064/34.

S* o.

19 Table lAnti-idiotype affinity chromiatography OD in %FU in %pH Chromatography procedure Fractions 6-142 143-162 1 0 7.2 Preliminary washing of the column with PBS, pH 7.2 7.2 Sample loading 7.*2 Washing of the column with PBS, pH 7.2 0 40 o too.

to.

163 164 165 166 167 168 169 170 171 20 172 173 174 175 176 177 178 179 180 0 0 0 0 10 20 40 80 100 80 60 40 30 25 20 10 5 0 7.2 7.2 7.2 G .8 6.1 5.7 5.6 5.4 5.3 5.2 5.2 5.1 5.1 5.1 Elution with PBS, pH 4.2 1 fraction 18 mi/h) 2 ml collection for 6.6 min (at pumping rate of The FU values are indicated as of the highest value (fraction 171).

20 The elution of the fusion protein was measured as protein by measurement of the OD at 280 nm. In addition, the isolated fractions were examined for specific binding to CEA and simultaneous enzyme activity in the specificity/ enzyme activity assay (Example The values show that all the specific binding and enzyme activity was concentrated in one peak (peak eluted from around pH 5.0 to pH The conclusion from this is that the described method of anti-idiotype affinity chromatography is a very efficient and selective purification technique for thie 431/26hum V1huGlc 1H fusion protein.

Example Gel chromatography of the fusion proteins The supernatants from the BHK cells secreting the 431/26hum V, hupGlc 1H fusion protein (B 70/6, B 74/2, B 72/72, B 73/2) were removed, sterilized by filtration and subjected to analytical gel filtration. For this, a TSK G3000 SW-XL column (7.8 x 300 mm) was equilibrated with 0.1 M sodium phosphate buffer, pH 6,7, 0.02% NaNa, 20 Jl of the supernatant were loaded on, and elution was carried out with a flow rate of 0.6 ml/min. Starting with an elution time of 9 min (exclusion volume 9.5 min), Sfractions (0.3 min each) were collected and assayed for Sp-glucuronidase activity.

For this 25 ul of the particular fraction were mixed with pl of substrate solution (2.5 mM 4-methylumbelliferyl p-glucuronide in 200 mM sodium acetate buffer, pH 5, 0.1 mg/ml BSA) and incubated at 37 0 C for 2 hours. The S" reaction was then stopped with 1.5 ml of 0.2 M glycine/0.2% SDS solution, pH 11.7, and the fluorescent label liberated by the glucuronidase was quantified in a Hitachi fluorometer (with excitation wavelength of 360 nm and emission wavelength of 450 nm).

21 Result: All 4 constructs show a single main activity peak between fractions 4 and 6 (Table This corresponds to retention times of about 10.2 10.8 min. The fusion proteins W,th glucuronidase activity in the supernatants thus hav6 retention times which are ol" the same order of magnitude as those of chen~ically prepared atibodr-p-glucuronidase co~nstructs (10.4 min). The retention time for the free enzyme is 11.9, and for the free antibody is 12.3 min.

22 Table 6: Ge). filtration of various fusion proteins Incubation: 25 p1, 370C, 120 min Substance concentration: 1.875 mM; 0.1 mg/mi BSA Each fraction 0.3 min; start at 9 min Liberated label/assay (FU) Fractions B70/6 B74/2 B72/72 B73/2 1 11 17 15 2 22 35 44 38 3 125 97 873 1014 4 1072 196 2959 3994 1588 165 2206 3141 6 1532 120 1133 1760 7 941 103 581 926 8 710 69 376 723 *9 500 108 302 626 71123 316 613 1320 107 263 472 12 254 91 210 456 *13 *224 57 146 357 14 190 65 134 332 171 52 83 294 167 44 99 243 17 100 38 73 217 18 129 34 55 179 19 75 45 48 155 66 41 36 129 21 118 30 22 93 23 78 24 61 24 26 51 23 Example Molecular characterization of the3 431/26 hum V, hupGlclH fusion prCtein The fusion proteins were puriQ.id by anti-idiotype affinity chromatography in Example o Aliguots from the peak eluted at pH 5.5 were subjected to 10% SDS PAGE electrophoresis under non-reducing and reducing conditions and immunostained in a Western blot using antiidiotype MAbs or with anti-p-glucuronidase MAbs (Towbin and Gordon (197P), Proc. Natl. Acad. Sci. USA 76: 4350- 4354).

Under non-reducing conditions with the 431/26 hum V, hupGlclH fusion protein, a Ihain band of a 125 kDa and a band of 250 kDa were detecte& and were detactable both by anti-idiotype MAb and by anti-p-glucuronidase MAb in the Western blot. Under reducing conditions there was no detectable immunostaining either by the anti-idiotype or by the anti-p-glucuronidase MAbs. A 100 kDa and a 25 kDa band were detected in the reducing SDS PAGE. However, these molecules analyzed under denaturing conditions are, according to TSK G 3000 SW-ZL gel filtration under native conditions in the form of a higher molecular weight product which has a molecular weight of 250 kDa (Example Diagrammatic representations of the 431/26 25 hum Va hu9GiblH fusion protein are shown in Fig. M. Figure NMb shows the monomer which has a s 25 kDa light chain and a 100 kDa heavy chain. This monomer and a dimer linked by inter-heavy chain disulfide bridges can be detected under denaturing conditions (Fig. Ma). Under native conditions, the fusion protein is in the form of a dimer of 250 kDa, with or without inter-heavy chain disulfide bridges (Fig. Mc).

24 EXAMPLE Chemical modification of the fusion protein The fusion protein purified as in Example (110 pg/ml) was adjusted to pH 4.5 and mixed with sodium periodate (final concentration 1 mM). After incubation at room temperature in the dark for 1 hour, the sodium periodate was removed by gel chromatography, and the fusion protein was then readjusted to pH 8. Addition of ethanolamine to a final concentration of 0.1 M was followed by incubation at 4"C for a further 3 hours, then sodium cyanoborohydride (final concentration 5 mM) was added and incubated for 30 min (reduction). This was followed by another gel filtration to remove the reducing agent and to change the buffer of the fusion protein. The chemical modification had no effect on the functional activity of the fusion protein. Tab. 7 shows the change in the plasma concentrations of unmodified and modified fusion protein in the nude mouse. The elimination of the fusion protein from the plasma is greatly slowed down by the modification.

Table 7: Plasma levels of p-glucuronidase activity in the nude mouse p-Glucuronidase fusion protein t Treated Untreated mm) [min] activity activity 0 100 100 54 30 76 22 240 40 4 480 1 1380 19 1440 1 5880 3 25 EXAMPLE

(Q)

Enzymatic treatment of the fusion protein 53 pg of fusion protein (Example N) in 0.01 M tris/HCl, 0.15 M NaCl were incubated with 1 unit of soluble alkaline phosphatase coli) or immobilized alkaline phosphatase (bovine intestine) at room temperature for h. Tab. 8 shows the change in the plasma concentration of untreated and treated fusion protein in the nude mouse. The elimination is not significantly affected by the enzyme treatment.

Table 8: Plasma levels of p-glucuronidase activity in the nude mouse f-Glucuronidase fusion protein *c 4 .Vee .4 4*4* as.

t [min] untreated (bovine intestine, immobil.) treated

AP

coli) treated 4 44 0 240 1440 100 78 44 35 22 4 100 76 57 36 27 5 100 53 22 AP alkaline phosphatase Example Pharmacokinetics and tumor retention of the 431/26 hum VH hupGlc 1H fusion protein By way of example, 5 x 4 pg of purified fusion protein 26 which was mixed with 100 pg of HSA/ml were injected in unmodified (Example N) and chemically modified form (Example P) i.v. at 24-hour intervals into CEA-expressing nude mice harboring human tumors. After defined time intervals, 3 animals in each case were sacrificed by cervical dislocation. The organs were removed, weighed and mixed with 2 ml of 1% strength BSA in PBS, pH 7.2.

The tissue and cells from these organs were then broken down in a Potter (10 strokes) and the amount of functionally active fusion protein was determined in the supernatant after centrifugation of the suspension at 3000 rpm and RT for 10' (Heraeus Labofuge GL, Type 2202) in the specificity/ enzyme activity assay (see Example The data from a representative experiment are shown in Tab. 9. It is clearly evident that the chemically modified fusion protein, which has a tl/2p of 4 h, specifically accumulates in the tumor from z 3 days after completion of the repetitive injection phase. The unmodified fusion protein, which has a tl/2p of 20 min, showed no significant accumulation 20 in the tumor under the same experimental conditions.

It may be concluded from these data that the hu 431 p-Gluc fusion protein is able to bind in vivo to CEA-positive tumors and to remain there as enzymatically active molecule over long time periods 9 days). The time the prodrug is administered in this system should be between day 3 and 9 after completion of the fusion protein injection.

SC t Table 9:- Tumor retention experiment with CSA-positive human stomach carcinoma xenograft (liz Sto 1) i.v. Injection of 5 x 4 pg of fusion protein per mouse at intervals of 24 hours Time after %Activity/g of -tumor or organ last i.v. injection Tumor Liver Lung Spleen Kidney Intestine Plasma 3 100 100 100 100 100 100 100 24 86.7 49.3 26.3 64 123 44.4 72 26.4 11 4.4 8.1 19 216 24.4 1.5 0.13 1.2 0.4 0.5 0.015 4 28 Example Isoelectric focusing of the 431/26 hum V, hupGlc 1H fusion protein The fusion protein purified by anti-idiotype affinity chromatography (Example N) was subjected to isoelectric focusing in the Pharmacia Fast System by the method of Righetti et al. (1979). It emerged from this that the isoelectric point of the molecule lies in a pH range from 7.35 to 8.15.

Example Demonstration that a cytostatic prodrug can be cleaved by the 431/26 hum V, hupGlc 1H fusion protein It was shown in Example that the p-glucuronidase portion S of the fusion protein is able, like the native human enzyme, to cleave the p-glucuronide of 4-methylumbelliferol. In S" the investigations which are described hereinafter, the substrate used for the enzymatic cleavage was a p-glucuronide, linked via a spacer group, of the cytostatic daunonycin. The specific procedure for these investigations was as follows: 4 mg of the compound N-(4-hydroxy-3-nitrobenzyloxy- :carbonyl)daunorubicin p-D-glucuronide (prodrug), which is described in French Patent Application (No. d'Enregistrement National: 9105326) were dissolved in 1 ml of 20 mM phosphate buffer, pH 7.2. 35 pl of the fusion protein (Example N) or of human p-glucuronidase (total concentration in each case 6.5 U/ml; 1 U cleavage of 1 pmol of 4-methylumbelliferol/min at 37 C) were pipetted into 5 pl portions of this substrate solution and incubated in the dark at 37 0 C. Samples (5 pl) of the incubation mixture were removed after various times and immediately analyzed by high pressure liquid chromatography under the following conditions: 29 Column: Nucleosil 100 RP 18, 5 /pm particle diameter, 125 x 4.6 mm Mobile phase: Gradient of solution A (100% acetonitrile) and solution B (20 mM phosphate buffer pH 0 min: 30% solution A min: 70% solution A min: 70% solution A Flow rate: 1 ml/min Detection: fluorescence, excitation 495 nm, emission 560 nm Data analysis: Beckman System Gold Software The retention time of the starting compound (prodrug) under these chromatography conditions was 11 min. The compound produced during the incubation (drug) had a retention time 15 of 8.9 min, identical to daunomycin (DNM, analysis of a oo standard under the same conditions). The kinetics of the cleavage of the starting compound by the fusion protein and human p-glucuronidase are shown in Tab. 11 and Tab. respectively.

20 The half-life of the cleavage of the prodrug by the fusion protein was 2.3 h. Cleavage by human p-glucuronidase took place with a half-life of 0.8 h. As already demonstrated in Example the results of the investigations show that the 6-glucuronidase portion of the fusion protein is functionally active and able to cleave p-glucuronides. The kinetics of the elimination of the glucuronide portion and the liberation of the drug (daunomycin) from the prodrug used show a rate comparable in magnitude to human p-glucuronidase, so that the substrate specifity of the fusion protein essentially agrees with that of human p-glucuronidase.

30 Table Kinetics of prodrug cleavage by p-glucuronidase (human, recombinant) t Prodrug DNM min area area 0 99.2 0.8 57 36.0 64.0 130 10.3 89.7 227 9.3 90.7 Table 11: Kinetics of prodrug cleavage by ,p-glucuronidase (fusion protein) to.

t Prodrug DNM 00 15 min area %area 0 98.9 1.1 81ll 18.9 135 51.7 48.3 190 33.0 67.0 *247 22.0 78.0 *S5317 12.4 87.6

Claims (14)

1. A fusion protein for prodrug activation of the formula huTuMAb-L-p-Gluc, where huTuMAb is a humanized tumor- specific monoclonal antibody or a tumor-binding fragment thereof, L is a linker, and f-Gluc is human p-glucuronidase.

2. A fusion protein as claimed in claim 1, in which the antibody fragment is composed of a V, exon, a CH, exon and a hinge exon and leads to expression of fusion proteins which are not linked together by disulfide bridges.

3. A fusion protein as claimed in claim 1, in which the antibody fragment is composed of a V, exon, a CH, exon and two hinge exons and leads to the expression of fusion proteins in which the heavy chains fragments are linked together by disulfide bridges. *o

4. A fusion protein as claimed in claim 1, in which the Santibody fragment is composed of a V, exon, a CH i exon Sand three hinge exons and leads to the expression of fusion proteins which are linked together by disulfide bridges. A fusion protein as claimed in claim 1, in which the antibody fragment is composed of a V, exon and a CH 3 exon and on expression can associate with a modified light chain composed of VL and CH 3 domain.

6. A fusion protein as claimed in any of claims 1 to where the huTuMAb portion derives from MAb BW 431/36. wher fuithn prohuin as lrtioed in anyf climone of the in which L contains a polypeptide spacer as shown in Tab. 1 and/or 2.

8. A fusion protein as claimed in any of claims 1 to 7, in which L contains 1, 2 or 3 hinge regions of a human IgG3 C gene.

9. A plasmid which contains the cDNA for peptides as claimed in claim 1 to claim 8. A transformed eukaryotic cell which is transformed with a plasmid as claimed in claim 9.

11. A process for the preparation of proteins as claimed in claim 1 to claim 8, which comprises expressing these fusion proteins in transformed cells by means of plasmids, and isolating said fusion proteins via anti-idiotype MAbs.

12. A fusion protein as claimed in any of claims 1 to 8, which fusion protein is treated with an oxidizing agent.

13. A fusion protein as claimed in claim 12, which fusion protein is reductively aminated in a second reaction step.

14. A process for the preparation of fusion proteins as claimed in claim 13, which comprises treating the fusion proteins as claimed in any of claims 1 to 8 with an oxidizing agent.

15. A process for the preparation of fusion proteins as claimed in claim 13, which comprises the fusion proteins as claimed in any of claims 1 to 9 being oxidized in a first reaction step and reductively aminated in a second reaction step.

16. A pharmaceutical composition comprising a fusion protein as claimed in any of claims 1 to 8 and claims 12 and 13 in adjunct with pharmaceutically acceptable carriers and excipients.

17. A diagnostic composition comprising a fusion protein as claimed in any of claims 1 to 8 and claims 12 and 13 in adjunct with diagnostically acceptable carriers and excipients. DATED this 19th day of April, 1995. BEHRINGWERKE AKTIENGESELLSCHAFT S S 5555 S S WATERMARK PATENT 8, TRADEMARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA DBM:KJS:JZ (Doc,3) AU1125192.WPC BEIRINGWERKE AKTIENGESELLSCHAFT HOE 91/B 004 Ma 876 Dr. Lp/Bi Abstract of the Disclosure Fusion proteins for prodrug activation-, thie prepatation and use thereof The inv,,ention relates to fusion proteins for prodrug activation of the general formula huTuMAb-L-p-Gluc, where huTuI4Ab is a-,hiumanized or human tumor-specific monoclonal antibody or a fragmunt thereof, _L is linker, and p-Gluc O S comprises human p-glucuronidase. Therse fusion proteins are prepared by genetic manipulation. huTuMAb ensures the S. *specific localization of tumors, L connects huTuMAb to pl-Gluc in such a way that the specific properties of the two fusion partners are Yiot imparied, and p-Gluc activates a suitable prodrug compound by elimination of crlucuronic acid, where a virtually autologous system for uso in humans is provided by the huma~nized or human fusion partners. too*