CHYMOPAPAIN FRAGMENTATION OF MONOCLONAL ANTIBODIES

Abstract: Certain selected (Kallestad W0214 and W4507, DAS5, B72.3, GAC 39) monoclonal antibodies are fragmented by digestion with chymopapain. F(ab)2 fragments can be further reduced to Fab-fragments with dithiotreitol (DTT).

Description: CHYMOPAPAIN FRAOMENTATTON OF MONOCLONAL ANTIBODIES Field of the Inven ion This invention relates to a method of fragmenting monoclonal antibodies and to Fab' monoclonal antibody fragments. BACKGROUND OF TEE INVENTION A number of monoclonal antibodies have found use in targeting tumor cells. B72.3 is a mouse monoclonal antibody prepared from immunizations using a membrane enriched fraction of breast tumor metastases to the liver (ATCC HB8108). It reacts with 50% of breast carcinomas and 807o of colon carcinomas. However, it has no significant reactivity with normal human tissues in vitro. Therefore, it is clinically useful for the detection of colon carcinoma lesions in situ. Monoclonal anti-tumor antibodies have great promise for radioimmunodetection and localization of tumors. B72.3 detects a high molecular weight human tumor associated glycoprotein TAG-72, which has been purified and characterized. This antigen is obtained from LS174T cells. LS174T is a cell line originally derived from a human colon carcinoma which expresses TAG—72 on its surface and in its cytoplasm. Expression of this antigen is increased significantly in xenografts of LS174T cells in nude mice compared to LS174T cells maintained in tissue cell culture. In humans, TAG-72 is a shed antigen so that B72.3 has been used as the basis of an assay for serum levels of this molecule. A correlation is claimed between serum levels and the presence of certain cancer types. In radioimaging studies using a murine monoclonal antibody to carcinoembryonic antigen in a human colon carcinoma xenografted into hamsters, F(ab') 7 fragments were shown superior to Fab fragments and the intact antibody for scintiscanning. In double-label experiments with anti-CEA antibody and control monoclonal IgG, F(ab') 2 fragments were found to give better and more rapid specific tumor localization than intact antibody or Fab fragments. F(ab') 2 fragments also offer significant promise for tumor imaging and possibly therapy (J . Nucl. Med. 24: 316-326, 1983). For a number of reasons it is better to use fragments of antibodies [F(ab') 2 and Fab] rather than intact antibodies for im unoscintograph. Because of their smaller size, it is easier for antibody fragments to penetrate tumors. Fragments are also cleared from the circulatory system faster than intact antibodies thereby lowering the background signal in tissues perfused with blood. More rapid clearing of fragments would allow the use of radionuclides with shorter half lives and thus reduce the radioactive burden a patient receives. Fragments lacking the Fc region of the intact antibody would not interact with the tissues that have Fc receptors as part of their normal structure. This would reduce non-specific binding (background). PROBLEM OF TEE INVENTION Certain monoclonal antibodies such as B72.3 are difficult to fragment with pepsin and papain. These are the proteolytic enzymes conventionally used for fragmentation. See On The Fragmentation of Monoclonal IgGl. IgG2a and IgG2b BALB/c Mice. Parham et al, Journal of Immunology, 131, December 1963 and Preparation of FC b'^ Fragments From Mouse IgG of Various Subclasses. Lamoyi et al, Journal of Immunological Method 56 (1983) 235-243. Bromelain is also ineffective in that the yield of F(ab') 2 fragments is low. Also, bromelain fragmentation produces additional fragments having molecular weights and structure similar to B72.3 F(ab') 2 fragments thereby increasing the difficulty of purifying B72.3 F(ab') 2 fragments. SUMMARY OF TEE INVENTION A convenient method for making F(ab') 2 fragments from certain monoclonal antibodies has been found. The method comprises the steps of: a) selecting a monoclonal antibody from the group consisting of Kallestad W0214, Kallestad W4507, DAS5, Phe 1.9, B72.3 and GAC 39; and b) subjecting the monoclonal antibody to chymopapain digestion, thereby fragmenting the antibody to form F(ab') 2 fragments. The fragments can be purified with anion exchange chromatography. Cleavage of B72.3 with chymopapain produced F(ab') 2 fragments in good yields. The fragments were easy to purify using anion exchange chromatography. Moreover, Fab' fragments can be prepared from B72.3 F(ab') 2 fragments by selective reduction of the inter-heavy chain disulfide bonds of the F(ab') 2 fragments with dithiothreitol (DTT). The F(ab') 2 and Fab' fragments retained the ability to bind to TAG-72, the tumor associated glycoprotein recognized by the intact antibody. DETAILS OF TEE INVENTION Chymopapain is a proteolytic enzyme from papaya latex. Experiments were performed to study the effect of various concentrations of chymopapain and various times of proteolysis on the fragmentation of B72.3. In general at least 47. up to about 107, by weight (compared to weight of B72.3) of chymopapain is required at temperatures as low as 25°C for several hours. The preferred range of conditions for giving primarily F(ab') 2 fragments with very few side products are 4 to 87. chymopapain (percent by weight compared to weight of B72.3) for 1 to 3 hours at about 37 β C. Procedures used in carrying out the following examples to confirm that the method of this invention is operative were as follows. ELISA ELISA determination of binding activity of B72.3 and its fragments was carried out as follows. The antibody or its fragments were diluted to 3.16 x 10~ 7 M in 0.57. BSA-PBS. BSA-PBS is 0.010 M sodium phosphate, pH 7.9, containing 0.15 M sodium chloride and 5 mg/ l bovine serum albumin. Further dilutions (square root of 10) were made in the same buffer. One hundred μL of each dilution was added to the appropriate wells of microtiter plates that contained antigen (and had been blocked by the addition of 200 μl of 0.57. BSA-PBS for 1 hour at 37 β C) to allow the antibody-antigen complex to form. The wells were then washed 3 times with PBS solution and 100 μL of a 1:5000 dilution of an anti-mouse F(ab' ) 2 ~peroxi- dase (ERP) conjugate was added to each well. The plates were incubated for 1 hour at room temperature with shaking. The plates were then washed six times with PBS solution. Color was developed in each well by adding 100 μl of peroxidase (ERP) substrate buffer (9.0 mL nanopure water; 1.0 mL of 170 mM citric acid, 650 mM sodium phosphate, 0.17, merthiolate, pE 6.3; 5 μL 307. E 2 0 2 and 2 tablets of orthophenylene diamine tablets). Color development was stopped by the addition of 100 μL of 4.5 N sulfuric acid to each well, after the desired color density was reached. The plates were read colorimetrically using a 492 nm filter. Slab Gel Electrophoresis - Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was carried out using either a Eoefer Protean Apparatus (Eoefer Scientific Instruments, San Francisco, CA) or a Pharmacia Phastgel System (Pharmacia Biotechnology, Inc., Piscataway, NJ). Ten percent polyacrylamide gels (1.5 mM thickness) were prepared for use as the resolving gel with the Eoefer apparatus. Pharmacia 10-157. gradient gels were used with the Phastgel System. Manufacturer's instructions were followed for the respective systems. Western Blots Electrophoretic separation (SDS-PAGE) was done using the Eoefer apparatus under both reducing and non-reducing conditions. The gels were run at 50 volts (constant) overnight with cooling. Fifteen μg of B72.3 fragments in 100 μL were placed in the sample wells . The electrophoresed samples were transferred from the acrylamide gel to nitrocellulose paper (BioRad Trans-Blot cell). A transfer "sandwich" was formed according to conventional procedures. The sandwich was immersed in transfer buffer (240 mL "10X" electrode buffer, 60 g Tris, 280 g glycine in 2 L distilled water), 1600 mL chilled methanol, 24 mL 107. SDS, and 2136 mL chilled distilled water in the transfer unit and transferred for 1.5 hours, 70 volts, with cooling. One group of the samples was stained for protein by immersion in a solution of 0.17. Naphthol Blue Black in 457. methanol, 107. acetic acid and 457. water for about 10 minutes or until protein bands were visible. The rest of the nitrocellulose paper was blocked with 17. BSA-PBS for 12 hours before being scored. For F(ab') 2 scoring, either a rabbit anti—mouse F(ab' ) 2 -peroxidase conjugate (Jackson Labs, 315-3547, 1:1250 dilution in PBS containing 0.057. Tween 20 and 0.017. merthiolate) , or a goat anti-mouse F(ab' ) 2 —peroxidase conjugate (Bohringer Mannheim, 60530, diluted according to manufacturer's instructions in PBS containing 0.057. Tween 20 and 0.017. merthiolate) was used. The blocked nitrocellulose paper was a) incubated with the selected diluted conjugate for 1 hour with gentle shaking; b) washed three times with PBS containing 0.057. Tween 20 and 0.017. merthiolate; c) washed once with substrate buffer (17 mM citric acid, 65 mM phosphate, 0.017. merthiolate, pE 6.3); and d) incubated with substrate solution (60 mg of 4-chloro-l-naphthol in 20 mL methanol at -20 β C plus 80 mL of 17 mM citric acid, 65 mM phosphate, pE 6.3; 60 μL of 307. H 2 0 2 ) with shaking until the desired amount of color formed (about 5-20 minutes). The paper was washed with distilled water to stop the color development. For Fc scoring, the same procedure was used except the scoring reagent was a rabbit anti-mouse Fc—peroxidase conjugate (Jackson Labs, 315-3546, 1:1000 dilution with PBS containing 0.057. Tween 20 and 0.017. merthiolate) . Example 1 Chymopapain Fragmentation Ten mg of chymopapain (Sigma) was dissolved in 2.5 mL of a phosphate buffer solution (PBS) comprising 0.01 M sodium phosphate and 0.15 M NaCl adjusted to pH 7.4. The chymopapain solution was equlibrated with PBS by passing it through a size exclusion column (PD-10 from BioRad) which had been pre-equilibrated with PBS. The enzyme was removed by elution with 3.5 mL of PBS. For these experiments, 10 mg of B72.3 was equilibrated with PBS using the size exclusion column. For a typical fragmentation with chymo¬ papain, chymopapain was added to the antibody at a concentration of 47. of the antibody weight (400 μg of papain to 10 mg antibody). The proteolysis was allowed to proceed for the desired time (usually 3 hours) at 37 ° C after which it was stopped by the addition of iodoacetamide to a final concentration of 30 mM. Purification of F(ab' Fragments The purification was achieved by anion exchange chromatography. A diethylaminoethyl (DEAE) cellulose anion exchange resin (10 mL of DEAE- Sephacel obtained from Pharmacia LKB Biotechnology, Piscataway, NJ) was washed with 5 mM Tris-ECl, pH 7.5, in a 15 mL scintered glass funnel. Tris is tris(hydroxymethyl)aminoethane. Two mL of the washed suspension were packed into a column (1 cm x 1 cm) and washed with a further 15 ml of 5 mM Tris-ECl, pE 7.5. Chymopapain fragmented B72.3 (0.5 mL, 1.8 mg/mL) was equilibrated with 5 mM Tris-ECl, pE 7.5 on the size exclusion column (PD-10). The 3.5 mL volume in which the equilibrated B72.3 fragments were collected was passed through the DEAE column. The F(ab') 2 fragments were not bound to the column. Fractions containing the F(ab') 2 fragments were collected until the initial protein peak was eluted. The fractions containing the F(ab") 2 fragments were pooled and concentrated. Analysis of F(ab')^ Fragments The eluted F(ab') 2 fragments were analyzed by electrophoresis described supra and found to be free of the cleaved Fc. After anion exchange purification, the yield of F(ab') 2 fragments was calculated to be 75-807. using a conventional protein assay procedure with bovine IgG for calibration. The F(ab » ) 2 fragments retained the ability to bind antigen using the ELISA described supra. Weεtern blot analysis indicated that the fragments had the correct structural characteristics of F(ab') 2 fragments. More specifically, the fragments were scored by anti—F(ab') 2 antigens but not by an anti-Fc antigen. Reduced samples showed both the light chain and a slightly larger band (presumably the cleaved heavy chain) when stained for protein. To analyze the reduced samples, two anti-F(ab') 2 scoring reagents were used. One seemed to be specific for light chains only, while the second reagent seemed to cross react with the Fc region of the heavy chain. Using either probe, the cleaved heavy chain in the reduced sample was not detected indicating that it probably originated from an F(ab') or F(ab') 2 -like fragment. The anti-Fc reagent in this test was not as sensitive as the anti-F(ab') 2 reagents. Example 2 Fab' Fragments from Ffab 1 ^ Fragments Purified, F(ab') 2 fragments from example 1 served as the starting point for the preparation of Fab' fragments. The approach was to selectively cleave the inter-heavy chain disulfide bonds while leaving the heavy chain-light chain sulfide bonds intact. By adding excess DTT to a sample of F(ab') 2 fragments for an incubation period of 90 minutes at 37 β C, and subsequently removing the excess DTT using the size exclusion column, Fab' fragments were produced. Example 3 The Reaction of Chymopapain with Other Monoclonal Antibodies Other monoclonal antibodies (Table I) were digested with chymopapain (47., 4 hours, 37 β C). Preεence or absence of F(ab') 2 fragments was confirmed using sodium dodecysulfate electrohporesiε which separated the fragments according to size. The results show that chymopapain is not a general fragmentation reagent. It only fragments selective antibodies . Table_I The Effect of Chymopapain on Other Monoclonal Ant i-b.ojdJLfis Antibodv*** Antigen ISOt pe. Fragmented S_ource Kallestad, #W0729 Theophylline IgGl.k No Kallestad Laboratories Kallestad, 0WO214 Phenytoin IgGl.k Yes Kallestad Laboratories Kallestad, #W4507 Phenobarbital IgGl.k Yes Kallestad Laboratories Cambridge, C07 hCG ND* No Cambridge Medical Technolgies Damon, Theo 1.7 Theophylline ND ** Damon Corp. Cambridge, C06 hCG ND No Cambridge Medical Technolgies DAS4 Digitoxin IgGl.k No Beckman Instruments, Inc. DAS5 Digitoxin IgGl.k Partial Beckman Instruments, Inc. GAC 39 Strep A Carbohydrate IgG3 Fab' Mallinckrodt Inc. B72.3 TAG—72 IgGl Yes American Type Culture Collection * Not determined **0riginal antibody was fragmented Comparative Example Fragmentation of B72.3 with Bromelain Bromelain is a proteolytic enzyme from pineapple stem. Initial experiments using the preactivated bromelain indicated F(ab') 2 fragments were being produced. Western blot analysis confirmed that the major polypeptide fragment reacted with the proper detection antigens. For example, the non-reduced sample of F(ab') 2 migrating slightly slower than the 116,000 molecular weight marker reacted with the anti-F(ab') 2 antigen and not with the anti-Fc antigen. When electrophoresed under reducing conditions, a light chain and partial heavy chain were observed. Eowever, overall yield of F(ab') 2 was not good as indicated by the intensity of the F(ab') 2 band in the electrophoretic analysis which was considerably weaker than the corresponding amount of B72.3. Production of other fragments by bromelain close in size to the desired F(ab') 2 fragments was also observed. It was felt that these contaminants would be hard to remove during purifica¬ tion of F(ab') 2 because of their similarity in size and, presumably, in structure to the desired fragments . Fab' Fragments from Bromelain-Produced F(ab' Fragments Attempts were made to prepare Fab' fragments from the bromelain-produced F(ab') 2> The methods centered on ways to selectively reduce the inter- heavy chain disulfide bonds while leaving the light chain-heavy chain disulfide bonds intact. Conditions included cleaving F(ab') 2 with dithiothreitol (DTT), β-mecrcaptoethanol, or cysteine (Cyε) followed by treatment with H 2 0 2 (to reoxidize the appropriate disulfide bonds), or alkylation with iodoacetamide (to prevent reformation of the inter—heavy chain disulfide bonds), or removing excess reducing reagent. No Fab' fragments were detected. The invention has been described in detail with particular reference to preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.

Claims: I claim : 1. A method of making F(ab') 2 fragments from certain monoclonal antibodies comprising the steps of: a) selecting a monoclonal antibody from the group consiεting of Kallestad W0214, Kallestad W4507, DAS5, B72.3 and GAC 39; and b) subjecting the selected monoclonal antibody to chymopapain digestion thereby fragmenting the antibody. 2. The method of claim 1 wherein the digestion is carried out using at least 47. up to about 107. by weight of chymopapain based on the weight of the antibody at a temperature of at least 25 C C. 3. The method of claim 1 wherein the digestion a) is carried out from 1 to 4 hours at b) a temperature of 37°C with c) 4 to 87. chymopapain based on the weight of the selected antibody. 4. The method of claim 1, 2 or 3 wherein the monoclonal antibody is B72.3. 5. B72.3 F(ab') fragments. 6. The method of claim 2, 3 or 4 wherein the digestion is stopped after 1 to 4 hours by the addition of iodoacetamide to a final concentration of 20 to 30 mM. 7. A method of making Fab' fragments from B72.3 monoclonal antibody F(ab') 2 fragments comprising the step of reducing the F(ab') 2 fragments with dithiothreitol (DTT). 8. The method of claim 3 further compris¬ ing the step of separating the B72.3 F(ab') 2 fragments from the digestion mixture by carrying out anion exchange chromatography on the digestion mixture.

Inventors: HILBORN DAVID ALAN, HILBORN, DAVID, ALAN
Applicants: EASTMAN KODAK CO, EASTMAN KODAK COMPANY
Application year: 1990
Main classification: A61K39
IPC codes: A61K39, A61K47, C07K16, C12P21, G01N33