Abstract:
The invention relates to murine monoclonal antibodies (MAbs), A, B, C and D, which are directed against tumor-associated antigens. The nearly complete nucleotide sequences of the V genes of these MAbs are described, so that the relevant variable domains can be put together to give chimeric MAbs, or “humanized” MAbs are obtained by inserting the hypervariable regions (complementarity determining regions=CDR) into a human MAb framework. Antibody constructs of this type can be employed in human therapy and in vivo diagnosis without the disadvantages observed with murine MAbs.

Description:
CROSS-REFERENCES TO RELATED APPLICATIONS 
     This application is a continuation of application Ser. No. 08/118,788, filed Sep. 9, 1993, now abandoned, which is a continuation of application Ser. No. 07/949,359, filed on Sep. 23, 1992, now abandoned, which is a continuation of application Ser. No. 07/497,201, filed on Mar. 22, 1990, now abandoned, which is based on German Application Ser. No. P3909799.4, filed on Mar. 24, 1989. 
    
    
     STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     Not applicable. 
     BACKGROUND OF THE INVENTION 
     The invention relates to murine monoclonal a tibodies (MAbs), A, B, C and D, which are directed against tumor-associated antigens. The nearly complete nucleotide sequences of the V genes of these MAbs are described, so that the relevant variable domains can be put together to give chimeric MAbs, or “humanized” MAbs are obtained by inserting the hypervariable regions (complementarity determining regions CDR) into a human MAb framework. Antibody constructs of this type can be employed in human therapy and in vivo diagnosis without the disadvantages observed with murine MAbs. 
     MAb A reacts with antigen 2, MAb B reacts with antigen 11 and MAb C reacts with antigen 7, all of which are described in EP-A2 0,141,079 and are membrane-associated antigens on permanent human tumor cells lines such as the CaLu-1, Chago, Oat 75, PaTuII and Bewo cell line. MAb D is directed against a Vibrio cholerae neuraminidase (VCN)-sensitive epitope on the ganglioside GD 2  which is exposed on human melanoma cell lines. 
     MAbs A to D were generated as described in EP-A2 0,141,079 and isolated by standard methods. 
     MAb A binds to cells of the granulocyte compartment and to carcinomas of the colon, pancreas and some of the lung and breast, as shown in Tab. 1. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Binding characteristics of MAb A 
               
             
          
           
               
                   
                 Malignant tissue samples 
                 Number of 
                 Total 
               
               
                   
                 investigated 
                 positives 
                 number 
               
               
                   
                   
               
             
          
           
               
                   
                 colorectal carcinomas: 
                   
                   
               
               
                   
                 primary carcinomas 
                 6 
                 6 
               
               
                   
                 liver metastases 
                 15 
                 16 
               
               
                   
                 carcinomas of the pancreas 
                 6 
                 8 
               
               
                   
                 carcinomas of the lung: 
               
               
                   
                 small-cell 
                 1 
                 2 
               
               
                   
                 adeno 
                 9 
                 10 
               
               
                   
                 squamous cell 
                 3 
                 4 
               
               
                   
                 large-cell 
                 1 
                 2 
               
               
                   
                 carcinoma of the breast 
                 1 
                 3 
               
               
                   
                   
               
             
          
         
       
     
     MAb B binds to virtually all carcinomas of the gastrointestinal tract and to some ovarian carcinomas and adenocarcinomas of the lung, whereas it does not react with most normal human tissues or reacts only with secretion-containing sites thereon. The binding characteristics are summarized in Tab. 2. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Binding characteristics of MAb B 
               
             
          
           
               
                   
                 Malignant tissue samples 
                 Number of 
                 Total 
               
               
                   
                 investigated 
                 positives 
                 number 
               
               
                   
                   
               
             
          
           
               
                   
                 colorectal carcinomas: 
                   
                   
               
               
                   
                 primary 
                 6 
                 6 
               
               
                   
                 liver metastases 
                 9 
                 10 
               
               
                   
                 carcinomas of the pancreas 
                 5 
                 6 
               
               
                   
                 carcinomas of the stomach 
                 4 
                 4 
               
               
                   
                 carcinomas of the lung: 
               
               
                   
                 small-cell 
                 2 
                 11 
               
               
                   
                 adeno 
                 9 
                 10 
               
               
                   
                 squamous cell 
                 2 
                 12 
               
               
                   
                 large-cell 
                  4* 
                 12 
               
               
                   
                 carcinoma of the breast 
                 2 
                 9 
               
               
                   
                 ovarian carcinomas 
               
               
                   
                 (secretion-containing sites) 
                 4 
                 6 
               
               
                   
                 carcinomas of the kidneys 
                 1 
                 12 
               
               
                   
                   
               
               
                   
                 *weak, heterogeneous reaction  
               
             
          
         
       
     
     MAb C shows a distinct reaction with 70-80% of stage I and II primary tumors of carcinoma of the pancreas (11/14). Like the primary tumors, most grade I and II metastases of carcinoma of the pancreas appear to have a positive reaction (3/4). The type of reaction on these positive tissues indicates that the epitope recognized by MAb C is located in the cytoplasm, on the membrane and in the intercellular space. 
     Grade III carcinomas of the pancreas do not express the epitope (primary tumor 0/2, metastases 0/5). However, since grade I and II carcinomas of the pancreas comprise up to 95% of carcinomas of the pancreas, MAb C ought to react with 60-70% of all carcinomas of the pancreas. 
     Another important property of MAb C is its reactivity with the duct system in the chronically inflamed pancreas (pancreatitis 10/13), whereas there is no detectable binding to healthy exocrine and endocrine pancreatic tissue (0/8). Only a minority of the investigated primary tumors of carcinoma of the colon was reactive (4/14), whereas most of the liver metastases of carcinoma of the colon showed distinct binding (7/10). 
     Cross-reactivities of MAb C are confined to the mucus-producing goblet cells of the colonic mucosa and of the stomach. All the other normal tissue tested, including peripheral blood leukocytes and bone marrow, do not express the mucus-associated epitope recognized by MAb C (see Tab. 3). MAb C recognizes an epitope located on a tumor associated antigen which can be detected at increased levels in patients with gastrointestinal tumors e.g. carcinomas of the pancreas and thus may be used as a tumor marker. 
     
       
         
               
             
               
               
               
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Summary of the formaldehyde-fixed, paraffin-embedded 
               
               
                 human tissue investigated (indirect immunoperoxidase) 
               
               
                 with MAb C. 
               
             
          
           
               
                   
                 Pancreatic tumors 
                 Grade I/II 
                 Grade III 
               
               
                   
                   
               
               
                   
                 primary carcinoma 
                 11/14 
                 0/2 
               
               
                   
                 liver metastases of carcinoma 
                 3/4 
                 0/5 
               
             
          
           
               
                   
                 papillary carcinoma 
                 1/2 
               
               
                   
                 cystadenoma 
                 0/1 
               
               
                   
                 Pancreatic tissue 
               
               
                   
                 ducts with changes due to 
               
               
                   
                 pancreatitis 
                 10/13 
               
               
                   
                 normal exo- and endocrine 
                 0/8 
               
               
                   
                 pancreas 
               
               
                   
                 Carcinomas of the colon 
               
               
                   
                 primary tumors 
                 4/14 
               
               
                   
                 liver metastases 
                 7/10 
               
               
                   
                 Normal colonic tissue 
               
               
                   
                 goblet cells of the mucosa, 
                 9/10 
               
               
                   
                 luminal part 
               
               
                   
                 part of the mucosa facing the muscularis 
                 3/10 
               
               
                   
                 muscularis mucosae 
                 0/7 
               
               
                   
                 tunica muscularis 
                 0/7 
               
               
                   
                 submucosa 
                 0/7 
               
               
                   
                 Carcinomas of the lung 
                 8/18 
               
               
                   
                   
                 (a few tumor cells) 
               
               
                   
                 Carcinomas of the breast 
                 0/3 
               
               
                   
                 Other normal tissue investigated 
               
               
                   
                 lung 
                 0/3 
               
               
                   
                 liver 
                 0/5 
               
               
                   
                 breast 
                 0/1 
               
               
                   
                 Stomach 
               
               
                   
                 muscle 
                 0/2 
               
               
                   
                 mucus-prod. cells 
                 3/3 
               
               
                   
                 kidney 
                 0/5 
               
               
                   
                 lymph nodes 
                 0/4 
               
               
                   
                 spleen 
                 0/2 
               
               
                   
                 bone marrow 
                 0/1 
               
               
                   
                 peripheral blood leukocytes 
                 0/2 
               
               
                   
                 connective tissue 
                 0/2 
               
               
                   
                 muscles 
                 0/10 
               
               
                   
                   
               
             
          
         
       
     
     MAb D recognizes a VCN-sensitive epitope on the ganglioside GD 2  which does not occur on other bovine cerebral gangliosides, including GD 3 , GM 3 , GM 1 , GT 1a , GD 1b , GD 1a  and GM 4 . Using MAb D it was possible to stain all gliomas, meningiomas and neurilemmomas in normal human cerebral tissue. Tab. 4 summarizes the binding properties of MAb D with respect to intracranial tumors. 
     
       
         
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 4 
               
             
             
               
                   
                   
               
               
                   
                 Number of tumors 
                   
               
             
          
           
               
                   
                 Type of tumor 
                 Positive 
                 Total number 
               
               
                   
                   
               
             
          
           
               
                   
                 well-differentiated gliomas, 
                 6 
                 6 
               
               
                   
                 grade I-II 
               
               
                   
                 malignant gliomas, 
                 10 
                 10 
               
               
                   
                 grade III-IV 
               
               
                   
                 meningiomas 
                 11 
                 11 
               
               
                   
                 neurilemmomas 
                 4 
                 4 
               
               
                   
                 pineal adenomas 
                 0 
                 5 
               
               
                   
                 metastases of carcinomas 
                 0 
                 1 
               
               
                   
                   
               
             
          
         
       
     
     The epitope defined by MAb D is also present on neuroblastomas, ganglioneuroblastomas and ganglioneuromas. Tab. 5 shows a summary. 
     
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Reactivity of MAb D with neuroblastomas and small round- 
               
               
                 cell tumors in children 
               
             
          
           
               
                   
                 Number of tumors 
                   
               
             
          
           
               
                   
                 Neuroblastomas 
                 Positive 
                 Total number 
               
               
                   
                   
               
               
                   
                 Grade I 
                 1 
                 1 
               
               
                   
                 Grade II 
                 6 
                 6 
               
               
                   
                 Grade III 
                 4 
                 4 
               
               
                   
                 ganglioneuroblastomas 
                  3 xx   
                 3 
               
               
                   
                 Ewing sarcomas 
                 0 
                 2 
               
               
                   
                 rhabdomyosarcomas 
                 0 
                 1 
               
               
                   
                 non-Hodgkin lymphomas 
                   2 xxx   
                 5 
               
               
                   
                   
               
               
                   
                   x Hughes classification  
               
               
                   
                   xx ganglion cells  
               
               
                   
                   xxx some positive tumor cells  
               
             
          
         
       
     
     Thus, MAb D is suitable for differential diagnosis of neuroblastomas and small round-cell tumors in children. 
     The specificity of MAb D for two other tumors derived from the neuroectoderm, namely melanomas and small-cell carcinomas of the lung (SCLC), and for unrelated tumors is shown in Tab. 6. 
     
       
         
               
               
               
             
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 6 
               
             
             
               
                   
                   
               
               
                   
                 Number of tumors 
                   
               
             
          
           
               
                   
                 Positive 
                 Total number 
               
               
                   
                   
               
             
          
           
               
                   
                 melanoma 
                  4* 
                 10 
               
               
                   
                 SCLC 
                  2* 
                 11 
               
               
                   
                 carcinomas of the colon 
                 0 
                 3 
               
               
                   
                 carcinomas of the breast 
                 0 
                 3 
               
               
                   
                 non-SCLC: 
               
               
                   
                 adenocarcinomas 
                 0 
                 3 
               
               
                   
                 squamous cell carcinomas of 
                 0 
                 3 
               
               
                   
                 the lung 
               
               
                   
                 large-cell carcinomas 
                 0 
                 3 
               
               
                   
                   
               
               
                   
                 *Weak and heterogeneous staining of a few tumor cells  
               
             
          
         
       
     
     The immunoglobulin V genes of MAb A to MAb D were isolated using the methods detailed in the Examples. 
     The nucleotide and protein sequences are shown in Tables 7 (V gene of MAb A), 8 (V gene of MAb B), 9 (V gene of MAb C) and 10 (V gene of MAb D). The CDRs can be identified therein as described by Kabat and Wu (loc. cit., see Examples). 
     BRIEF SUMMARY OF THE INVENTION 
     Accordingly, the invention relates to the epitopes specified by MAbs A, B, C and D, monoclonal antibodies against these epitopes, with MAbs A, B, C and D being particularly preferred, and monoclonal antibodies which contain the V genes specified above, or parts thereof (complementarity determining regions), with a human antibody framework or framework parts being preferred. The invention furthermore relates to constructs which contain these V genes or parts thereof together with enzymes or radioactive labels or toxins or catalytic antibodies or combinations of various V-gene specificities or MHC class I or class II antigens or cytolytic components. Finally the invention relates to pharmaceuticals containing the MAb&#39;s specified above and the use of said MAb&#39;s or in vivo or in vitro diagnosis. The invention is furthermore contained in the Examples and the patent claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 , Detection of different antigens by Mab C. NS signifies serum samples of healthy blood donors (n=141); AP means serum samples of patients with acute pancreatitis (n=56). CP means serum samples of patients with chronic pancreatitis (n=40) and Pan-Ca means patients with carcinoma of the pancreas (n=82). A means absorbance. 
         FIG. 2 , Competition of CA 19-9, C50 and Mab C. Ca 19-9 means MAb Ca 19-9. 
         FIG. 3 , Nucleotide sequences and amino acid sequences of the variable heavy Ig chain (V H ) and the variable light Ig chain (V K ) of MAb A. 
         FIG. 4 , Nucleotide sequences and amino acid sequences of the variable heavy Ig chain (V H ) and the variable light Ig chain (V K ) of MAb B. 
         FIG. 5 , Nucleotide sequences and amino acid sequences of the variable heavy Ig chain (V H ) and the variable light Ig chain (V K ) of MAb C. 
         FIG. 6 , Nucleotide sequences and amino acid sequences of the variable heavy Ig chain (V H ) and the variable light Ig chain (V K ) of MAb D. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     EXAMPLES 
     The polymerase chain reaction (PCR) described by Saiki et al., Science 230, 1350-1354 (1985) was employed hereinafter for the cloning and expression of the variable domain of murine immunoglobulins (Ig). 
     1. Identification of the Conserved Regions at the 5′ and 3′ End of the Murine Heavy Ig Chain (VH) and the Light Ig Chain (VK). 
     The mutually aligned sequences of the variable regions were taken from the data file of Kabat et al. Sequences of Proteins of Immunological Interest, US Dept. of Health and Human Services, US Government Printing Office (1987). The nucleotide sequences start there at the amino terminus of the mature protein and do not include the signal sequences. Computer screening (DBUTIL, R. Staden (1986) Nucleic Acids Res. 14, 217-231) was used to find suitable primers for cDNA synthesis and amplimers for use in the PCR: 
                     Oligonucleotide I:                      Bst EII              VH1FORWARD       5′ TGA GGA GAC  GGT GAC C GT GGT CCC TTG GCC CCA 3′       Oligonucleotide II:                CK1       5′ TGC AGC ATC AGCC 3′       Oligonucleotide III:       5′ AG GTC CAG  CTG CAG  GAG TCT GG 3′               G A A         C     A                     PstI                         VH1 BACKWARD       Oligonucleotide IV:       5′ GAC ATT  CAG CTG  ACC CAG TCT CCA 3′                  PvuII                            VK1 BACKWARD       Oligonucleotide V:           VK1FORWARD       5′ GTT  AGA TCT  CCA GCT TGG TCC C 3′              Bgl II            
2. cDNA Synthesis
 
     RNA was prepared from about 3×10 8  cells of the particular hybridomas which secrete MAb A, B, C or D, and poly A +  mRNA was enriched from the latter using oligo dT Sepharose. The poly A +  mRNA was used for the cDNA synthesis. The first strands of cDNA were synthesized using oligonucleotide primers which hybridize in the J region of the V H  nucleotide sequences (oligonucleotide I) and at the 5′ end of the kappa C gene nucleotide sequences (oligonucleotide II). 
     The RNA is then decomposed by NaOH treatment. The second strands of cDNA were synthesized using oligonucleotide primers which hybridize at the 5′ ends of the V H  (oligonucleotide III) and of the V kappa  (oligonucleotide IV) nucleotide sequences. 
     3. Amplification of the Synthesized cDNA and Sequencing of the Variable Domains 
     The DNA generated as described in 2. was amplified using oligonucleotides I, III, IV and V (oligonucleotide V hybridizes in the J region of the V kappa  nucleotide sequences) and the Taq DNA polymerase from  Thermophilus aquatius . A typical mixture contained in 50 ll total volume 5 ll of ds DNA (prepared in 2.), 25 pmol of amplimers, and was 250 lM in each of DATP, dTTP, dCTP and dGTP, 67 mM tris-HCl pH 8.8, 17 mM (NH 4 ) 2 SO 4 , 10 mM MgCl 2 , 200 lg/ml gelatin and 2 units of Taq polymerase. A layer of liquid paraffin was placed on the mixture and then 25 cycles each of 1 min at 95° C. (for denaturation), 1 min at 30° C. (hybridization) and 2 min at 72° C. (DNA synthesis) were carried out using a Techne PHC-1 programmable heating block. 
     The oligonucleotides used for the cDNA cloning and amplification contain restriction cleavage sites. The cDNA cloning and the amplification resulted in these restriction cleavage sites being introduced at the 5′ end and at the 3′ end of the V H  and V Kappa  nucleotide sequences (Pst I and BstEII in V H  and PvuII and BglII in V Kappa ). 
     These restriction cleavage sites were then used to clone V H  and V K  cDNA fragments in M13 vectors (Lys 19, Lys 17) (Verhoyen et al. Science 239, (1988), 1534-1536). 
     Finally, the nucleotide sequences of the particular V H  and V Kappa  cDNA fragments were determined using the method of Sanger (PNAS, USA, 74, 5463-5467, (1977)) from the Lys 19 and Lys 17 vectors (see Tab. 7, 8, 9, 10). 
     Examples 4 and 5 shall explain the use of the MAb C described using MAb C: 
     Example 4 
     Mab C was fixed to the wells of microtitration plates (Nunc) by adsorption. Into these wells 20 μl sample plus 100 μl buffer-solution (OSND, Behringwerke A G) each was pipetted and 2 respective 3 hours incubated. After threefold washing with diluted Enzygnost R  washing solution (OSEN, Behringwerke A G) 100 μl conjugate-solution was filled into each well. Used here were conjugates of peroxidase with lectin (e.g. wheat germ agglutinin WGA) or with other antibodies recognizing different epitopes of the tumor-associated antigens defined by MAb C. The following two or three hours incubation step at 37° C. was terminated by 3 wash cycles. For the third incubation step at room temperature 100 μl each of a buffer/substrate-chromogene solution (OUVG/OUSF Behringwerke A G) was filled into the wells and the enzyme reaction was terminated after 30 min. with 100 μlof stopping solution Enzygnost R  (OSFA, Behringwerke A G). Absorbance of the samples was measured at 450 mm. 
     Result: 
     The absorbance values determined as shown above correspond to the concentration of the antigen(s) in the samples. The concentration of the antigen defined by the specific binding of MAb C, in serum or plasma of tumor patients is significantly elevated as compared to said concentration of healthy control persons or patients with benign disease. This is especially true for patients with carcinoma of the pancreas (FIG.  1 ), significantly elevated concentrations were also determined in serum or plasma of patients with carcinoma of the stomach, colon or rectum. Equally good results were obtained irrespective of the conjugate system (antibody-peroxidase or WGA-peroxidase). With the use of MAb C as specific binding-component a sensitive test for tumor markers of especially gastrointestinal tumor disease can be made. 
     Example 5 
     5 μg each of MAb C or MAb C 50 as control (Pharmacia; Holmgren et al. (1984) British Med. J. 288, 1479) were pipeted in 50 μl phosphate-buffered-saline (PBS) into a CA 19-9 Test (CA 19-9 EIA “Roche”). The highest standard (100 U/ml) was added before start of the incubation. The tests were performed according to the instructions of the test-kit above and the absorbances of the respective samples were determined. 
     Result: 
     As shown in  FIG. 2 , the additional dilution of the standard-antigen after addition of the PBS-solution reduces the signal of the highest standard even without addition of MAb. No further reduction of the signal results by the presence of MAb C in the assay. In contrast hereto the signal formation is totally inhibited by MAb C 50, which as is known, binds among others specifically to the epitope “sialosyl Le αn  recognized also by MAb 19-9: From the above one may conclude that MAb C recognizes a different epitope as MAb 19-9.