AU622446B2 - A monoclonal antibody against pseudomonas aeruginosa, the preparation and use thereof - Google Patents

Abstract

A monoclonal antibody (mAb) which cross-reacts with all 19 of the hitherto known Pseudomonas aeruginosa serotypes is described. The sequence of the variable regions is given. This mAb can be used as diagnostic aid, as active substance or as active substance carrier.

Description

T 1 -la BEHRINGWERKE AKTIENGESELLSCHAFT 88/B 010 Ma 675 Dr. Lp/rd A monoclonal antibody against Pseudomonas aeruginosa, the preparation and use thereof The invention relates to a monoclonal antibody (mAb) which cross-reacts with all the 19 Pseudomonas aeruginosa serotypes hitherto disclosed. This antibody can be used as a diagnostic aid, as an active substance or as an active substance carrier.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogenic bacterium. It may cause life-threatening illnesses, for example pneumonia in patients with cystic Sfibrosis or septicemia in patients with burn injuries or 15 infections of bone or the urinary tract. The therapy of diseases caused by Pseudomonas aeruginosa using antibiotics is difficult and often unsuccessful owing to its resistance pattern. This is why attention has been directed at immunoprophylaxis and immunotherapy for controlling infection. Protection against all 19 sero- S types of Pseudomonas aeruginosa was provided by a mixture L of purified outer membrane proteins (OMP), F, H2 and I S'(von Specht et al. (1987) Infection 15, 408 412). In c the abovementioned investigation, lymphocytes were t 25 isolated from immunized mice and fused with NS-1 myeloma cells in a known manner (Kearny et al. (1979), J.

SImmunology, 123, 4, 1548 1550). From about 500 hybrid- S c omas obtained, one mAb called mAb 6A4 hereinafter was Sselected and has the advantageous properties listed hereinafter: strong and uniform reaction with all 19 Pseudomonas aeruginosa serotypes positive Clq fixation test protective on exposing mice to infection with Pseudomonas aeruginosa.

Tab. 1 and Tab. A human mAb can now be obtained by replacing the constant regions of the mouse antibody gene by the corresponding human sequences. Expression of this "humanized" antibody sequence in eukaryotic cells, for example CHO cells or yeast, provides the corresponding human mAb which can be used for diagnostic purposes, for prophylaxis and therapy.

Consequently, the invention relates to a) purified and isolated DNA sequences which code for mAb 6A4 or parts thereof, including the transcription products thereof, as well as the corresponding .ts combination of "humanized" sequences, 4 oitt I, b) DNA structures and vectors containing these sequen- '15 ces whole or in part, c) pro- or eukaryotic cells transformed with such DNA, g o d) polypeptides expressed by these cells by reason of the transformation, or parts thereof, e) the amino acid sequences thereof, C4' C f) diagnostic aids, prophylactics or therapeutics which contain peptides or amino acid sequences from (e) alone or in combination, g) a process for the preparation, by genetic manipulation, of the polypeptides mentioned under or parts thereof, h) and the epitope to which antibodies encoded by sequences under bind.

Further embodiments of the invention are detailed in the following examples, tables and the patent claims.

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3 Example 1: Preparation of mAb 6A4 la) Purification of the outer membrane proteins OMP F, H2 and I of Pseudomonas aeruginosa serotype 12, which are to be used for the immunization OMP F, H2 and I were purified from Pseudomonas aeruginosa serotype 12 as described by T. Mizuno and M. Kageyama in J. Biochem. 84, 179-258 (1978). According to this, the cells were harvested in the late log phase and disrupted by ultrasound treatment, then the cell membranes were collected by centrifugation at 100,000 x g for one hour.

The cell membranes were extracted stepwise with 2% SDS, glycerol and 2% SDS and finally 0.1 M NaCl. The remaining insoluble fraction contains mainly OMP F, OMP ,cc H2 and some OMP I.

'l5 lb) Immunization of Balb/c mice with OMP F, H2 and I r C C C C For the immunization, each mouse received 50 pg of purified OMPs (80 pl) in a suspension with 20 pg of Al(OH) 3 i.p. on day 1, which was repeated with the same dose on day 14 and day 21. After a further 4 weeks, another c 20 booster (same dose) was given, and after the subsequent 3 days the spleens were removed.

Ic) Cell fusion The procedure for the cell fusion essentially followed the method of G. K6hler and C. Milstein, Eur. J. Immunol.

6, 511 519 (1976). About 1 x 108 spleen cells prepared from the removed spleens from (Ib) were fused with 5 x 107 NS-1 myeloma cells (Kearny et al., loc. cit., commercially available from the American Type Culture Collection under ATCC No. TIB 18) in 2 ml of 50%. polyethylene glycol 1500 (Serva, Heidelberg). After the fusion, the cells were plated out with 1 x 10 6 /ml normal spleen cells in Dulbecco's modified minimal medium (MEM) which contains hypoxanthine, aminopterin and thymidine (HAT). Antibody 4 production was tested by the enzyme-linked immunosorbent assay (ELISA) described hereinafter, and clones of interest were obtained by diluting out.

Id) ELISA to detect antibody-secreting clones A direct ELISA was used to determine whether and to what extent the various mAbs bind to the antigen.

Microtiter plates from Dynatech, Plochingen, made of polyvinyl chloride with 96 wells, were coated with 100 pl of Pseudomonas aeruginosa sonicate (see below, diluted 1 200 with PBS 4.5 pg/ml) of serotype 12 and incubated at 4 0 C overnight. The plates were then washed three times and saturated at 37 0 C for 1 hour with bovine serum albumin diluted in PBS. After renewed washing, the wells were covered with film, and the plates were stored at 115 minus 20 0 C until used.

pl of ascites (as duplicates) were pipetted in for each clone to be tested (from serial logarithmic dilutions 1 2, 1 4, 1 8, 1 16, The positive control was polyclonal mouse serum from mice t t 20 actively immunized and given 4 boosters with the membrane C proteins (1 100, 1 1000, 1 10,000). The serum from non-immunized mice was used as negative control serum.

PBS (phosphate buffered saline) was used as diluting medium. After the plate had been incubated at 37 0 C for one hour and washed three times, 50 pl of peroxidase-conjugated anti-mouse F(ab) 2 were added in the dilution 1 500.

After renewed incubation at 37 0 C for one hour and three washes, 50 pl of the substrate o-phenylenediamine (0.2% OPD, 0.015% H 2 0 2 were added. All the ELISA reagents were derived from the NEI 501 kit from NEN New England Nuclear, Dreieich.

Ij F After a further incubation for 30 minutes, the reaction was stopped with 50 pl of 4.5 M H 2

SO

4 and extinction at 450 nm was determined using an ELISA evaluator MR 580 from Dynatech. Positive clones showed an extinction 0.2 OD.

From 7 clones on the shortlist, the clone which secretes mAb 6A4 was selected for the subsequent investigations, because mAb 6A4 has the advantages described on page 1 and, in addition, is secreted in large quantities (about 1% of the gamma-globulin fraction in ascites fluid comprises mAb 6A4). mAb 6A4 belongs to the IgG2a class and reacts with OMP I.

Preparation of sonicate: ct t The bacteria of serotype 0-12 were cultured in trypr c;t15 ticase-soya broth. They were then washed 3 x and resuspended in sodium chloride buffer. The suspension was sonicated 3 x 5 minutes (with intervals of 2 minutes each time).

1 1 After renewed centrifugation (1 x at 5000 rpm/10 minutes) C1 20 the supernatant was divided into portions which were C t t stored at minus 20 0 C until used. The protein concentra- 1 tion on the coated plates was 4.5 pg/well.

Example 2: Isolation and sequencing of the cDNA of mAb 6A4 coding for the variable regions 2a) cDNA isolation 6A4 hybridoma cells were removed from ascites fluid by centrifugation (10 min at 1500 x g and The poly (A) RNA was obtained therefrom by the method of H. Domdey et al., Cell 39, 611-621 (1984). cDNA was synthesized with a commercially available cDNA synthesis kit (from Amersham) starting from 15 pg of poly (A) RNA. The doublestranded, blunt-ended cDNA was provided with single-

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B b i: -6 stranded oligo-dC ends using the cloning kit obtainable from NEN New England Nuclear, and annealed with the plasmid pBR322 which has corresponding oligo-dG ends.

Transformation (Hanahan, D. (1983) J. Mol. Biol. 166, 557-580) of competent E. coli K12 strain JM 109 cells (Yanisch Perron C. et al., (1985) Gene 33, 103-119) resulted in about 12,000 transformants, which were hybridized with the 32 P end-labeled oligonucleotide CAGGCATCCTAGAGTCAC-3' to detect clones which contain cDNA of the heavy chain of subgroup II B. Likewise used for hybridization was the Hind III-Bam HI fragment (II) of the plasmid Cl Weischert et al., (1982) Nucleic Acids Res. 10, 3627-3645) which is 2850 base-pairs (bp) in size and has been nick-translated with deoxynucleotide "P-triphosphates. originates from the region of the i IgG2a" allele in the mouse (heavy chain). (II) codes for the kappa constant region (light chain). The hybridizations were carried out as described by D. Woods, Focus 6, S"1-2 (1984). About 4.2% and 1.6% of the colonies reacted positively with the kappa- and gamma-specific probes respectively.

I 2b) cDNA sequencing Plasmid-containing colonies which presumably contained complete cDNAs of the light or heavy chain were sought from those obtained above. The insert DNA of the plasmids (containing the cDNA of the light chain) and pBH7 (containing the cDNA of the heavy chain) were sequenced I c, by the standard method Sanger et al. (1977) Proc.

Natl. Acad. Sci USA 74, 5463-5467) modified as described by E.Y. Chen and P. Seeburg, DNA 4, 165-170 (1985). The nucleotide sequences of the relevant variable regions are shown in Tab. 1 (light chain) and Tab. 2 (heavy chain).

The nucleotide sequences which are not included in the tables for the constant regions are in each case identical to those known from the literature (gamma chain: Schreier et al., (1981) Proc. Natl. Acad. Sci USA 78, 4495-4499; kappa chain: P.H. Hamlyn et al. (1981) i -7- Nucleic Acids Res. 9, 4485-4494). The total length of the coding regions is 717 bp for the light chain and 1407 bp for the heavy chain, with the first 20 amino acids of the light chain representing the leader peptide, so that the mature light chain is 259 amino acids long. The heavy chain has a leader peptide of 19 amino acids and, accordingly, the mature molecule is 450 amino acids long.

Ct t C I t I 1 i Tab. 1 -36 AM ATTGCTGCTGCTATGGGT.TCTGGTCGTGTGGGCATTGTGhVPCTCAC-AGTCTCCA -12 lIeLeuLeuLeuLeuTrpValSer~lyThirCysGlyAspleVal~eLSerGl nSerPrd 9 Se rSe rLeuAl aVa IS e rhlaG IyGI l~y sVa fh r~e tSe rCy sLy sSe rSe rG IfnSe r I156 CTCCAATTACGAG ATCTGCTGACACGACGGA 2 9 LeuLeu~snSerIeThrArLyAsflPheLeuAaTrpTyIGflLysProG1yGln 216 TCTCCT)XCTGCTGATCTACTGGGCATCCACI'GGGA)TCTGGGGTCCCTGKTCGCTTC 49 Se rP roLy sLeuLeu 11eTy rTrphl aSe rTh rArgG luSe rGyVal roA spArg P le 216ACA GG CA GT GGATCTG G GAC-AG.T T T CACT CT CAC CA TCA GCAG TG T GCA GG CT G "GA C 336 CT G GCAG T T TATTAC T GA-A GCAAT CT TA TAATCT TC GG AC GT TC GG TG G AG GCA CCAA G 0 9 LeuPLIaVa ITy rTyrC sLy sGIn 5e rTy rAs nLeuArqTh rP)eG IyG IyG IyTh rLy s 39r, CTGGAAATCA" CGGCTGATCT GCA CCAA CTGTA TCCATC'rTCCCA CC ATCA LTCGG i~ 109 eu~lu~l lys h 9 lahspAlaAlaPrOThrValSerIePheProProSerSerGlu t t 4 9- Tab 2 -120 ctgcagggggggggAAATl\CGTCGCA'rCC1C'fCCACAGACACI'GAAAJC-C'rCTCcc AC A1TGGAAGG CACTG GAT CTTT CTCT TCC'fG'TTTCAGTT ACT GCAG G TG T C CACTCC *1-19 He GluArlIisTrpIleL'heT..euPIheLeu~theSerVaiThirAlaG1yVal1~iisSer I ChGGTCQ-AGCTTCAGC-lAGTCTGCGGGCTG1ACTGGCAAAJ'ACCTGGGGCC'rCAlGTGAAGPLrG I GlnValGlPLeuGlnGl nSe rG) yhl aGluLeuA1 aLy sProGlyAl aSe rVa 61 TCCTGCAAGGCTTCTGGCTACACCTTTACTGCCTACTGGAJCC-CTGGGTAAAC-AGAGG 21 S erCy sLy sAla Se rG IyTy rTh rP heTh rA aTy rT rpMeLI I is T rpVa I Ly 5G InA rc 121 CCTGchCAGGGTCTGG kATGG1kT.IGG kTACATTAMkTCCTAA C-ACT GGTTATA CTG JvrAC 41 11 l ATC.AGAACTTCAGGAC.A GGCCA CATTA CTCAGA C.A-A-ATCCTCCA GCA CA GCCT A C 61 AsnGlnAspPheLysAspLysPhlaThrLeu'FhirAlaAspLysSerSerSer'fhrAlaTyr 241 ATGCAACTGAGCAGCCTGACATCTGA GACT CTGCA TCTATTATT GT CAAGAA GCT A C 61 MetGlnLeuSerSerLeuTbrSerGlu~spSerAlaVa lTyrTyrCysThir)rgSerry r 301 TA"CAG GGCAGACACG GT.AGA CCGCACTTCC 101 361 GCC-ACAACCCCJAGCCGGTCTTCC-ACTGGCCCCTGT~jGTGG~A'UTCAACTGGC 121 AIa Ly s Th rThr A Ia Pr o 5erV aIT y r:Pr oLeu A Ia Pr oV a ICys G 1y Asp Th rT hr GIy

Claims (10)

1. DNA sequences coding for the variable antibody regions or parts thereof shown in Table 1 and Table 2.

2. Monoclonal antibodies (mAbs), wherein the variable regions contain the amino acid sequences of Tab. 1 and Tab. 2 or parts thereof, including the alleles and mutants as long as these alleles and mutants react with OMP I of Pseudomonas aeruginosa.

3. mAbs as claimed in Claim 2, wherein the constant regions or parts thereof are of murine origin.

4. mAbs as claimed in Claims 2 or 3 wherein the constant regions or parts thereof correspond to IgG2a.

5. mAbs as claimed in Claim 2, wherein the constant regions or parts thereof are of human origin.

6. Hybridomas which secrete mAbs as claimed in Claim 2, 3 or 4.

7. Expression systems which express mAbs as claimed in Claim

8. An epitope to which monoclonal antibodies as claimed in Claim 2, 3, 4 or 5 bind.

9. A process for the preparation of hybridomas as claimed in Claim 6, which comprises a mammal being immunized with outer membrane proteins (OMP) of Pseudomonas aeruginosa, spleen cells being taken from such an animal and fused with myeloma cells, preferably NS-1 cells, and the resulting hybridomas being selected for secretion of mAbs against OMP of Pseudomonas aeruginosa. A process for the preparation of mAbs as claimed in Claims 2, 3 or 4, which comprises the DNA sequences, or parts thereof, coding for the variable regions being attached to the antibody sequences, or parts thereof, coding for the constant regions, and being expressed in an expression system. i ,I- I JI.

31- 1 11 11. A process for the preparation of mAbs as claimed in Claim 5, which comprises the DNA sequences, or parts thereof, coding for the variable regions as claimed in Claim 1 being attached to the antibody sequences, or parts thereof, coding for the constant human regions, and being expressed in an expression system. 12. A pharmaceutical composition comprising monoclonal antibodies as claimed in Claim 2, 3, 4 or 5 in adjunct with pharmaceutically acceptable carriers of excipients. 13. The use of mAbs as claimed in Claim 2, 3 or 4 in a diagnostic aid. 14. The use of mAbs as claimed in Claim 2, 3, 4 or 5 as carriers for a pharmaceutical active substance. DATED this 17th day of January, 1992 BEHRINGWERKE AKTIENGESELLSCHAFT 0 r a e o r cI t I B t t .Ma t 1tP astt^ WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRAUA 1; i r B I; r: i x: f :-r 1 r I i:i DBM/KJS:JJC AU3310189.WPC (DOC.07) I- 1- ~rr i