Ovine derived human TNFα polyclonal antibody composition for oral administration

Abstract

The present invention is directed to an antibody composition for oral administration comprising intact blood-derived polyclonal antibodies that bind to a human tumour necrosis factor α (TNFα), and means for protecting the antibodies during gastrointestinal transit, as well as methods for manufacturing, kits, and therapeutic uses of the same.

Claims

1. An antibody composition for oral administration comprising: a. intact blood-derived polyclonal antibodies that bind to a human tumour necrosis factor a (TNFα); and b. a protease inhibitor and/or an antacid, wherein the intact blood-derived polyclonal antibodies are derived from serum or plasma of an ovine mammal administered TNFα or a fragment thereof.

2. The antibody composition of claim 1, wherein the antibody composition and the intact blood-derived polyclonal antibodies therein have not been affinity purified.

3. The antibody composition of claim 1, wherein the intact blood-derived polyclonal antibodies are purified using sodium sulphate precipitation or caprylic acid precipitation, and optionally, filtration.

4. The antibody composition of claim 1, wherein the protease inhibitor is selected from the Bowman-Birk inhibitor family of proteins.

5. The antibody composition of claim 1, wherein the protease inhibitor comprises one or more of: a. a polypeptide which binds specifically to and suppresses or inactivates the proteolytic activity of trypsin and/or chymotrypsin; and/or b. an antibody that binds to trypsin and/or chymotrypsin and suppresses or inactivates the protease activity of said trypsin and/or chymotrypsin.

6. The antibody composition of claim 1, wherein the protease inhibitor comprises egg whites.

7. The antibody composition of claim 6, wherein the egg whites comprise powdered egg whites.

8. The antibody composition of claim 1, wherein the protease inhibitor comprises ovomucoid, ovostatin, ovomacroglobulin, or combinations thereof.

9. The antibody composition of claim 1, formulated as a liquid.

10. The antibody composition of claim 1, wherein at least 5% of the total antibodies present in the composition bind to TNFα.

11. The antibody composition of claim 1, wherein at least 10% of the total antibodies present in the composition bind to TNFα.

12. A method for manufacturing an antibody composition for oral administration, said method comprising: a. obtaining a blood sample from an ovine mammal that has been administered an immunogen comprising human tumour necrosis factor a (TNFα) or a fragment thereof, thereby providing a sample comprising intact blood-derived polyclonal antibodies that bind to TNFα; b. admixing the intact blood-derived polyclonal antibodies that bind to human TNFα with a protease inhibitor and/or an antacid.

13. The method of claim 12, wherein the obtaining further comprises obtaining serum from the blood sample.

Description

FIGURES

(1) Embodiments of the invention will now be described, by way of example only, with reference to the accompanying Figures, in which:

(2) FIG. 1 shows inhibition of bovine trypsin by Intact Anti-TNFα purified with either caprylic acid (.box-tangle-solidup.) or sodium sulphate (.square-solid.). TNFα antisera (.circle-solid.) was used as a control.

(3) FIG. 2 shows results of a direct ELISA for detection of anti-TNFα IgG in antisera (.circle-solid.) and IgG purified by way of caprylic acid precipitation (.box-tangle-solidup.).

(4) FIG. 3 shows results of an immunocytotoxicity assay (IOTA) testing the neutralising activity of Anti-TNFα Fragment (.square-solid.), Intact Anti-TNFα (.box-tangle-solidup.) and TNFα antisera (.circle-solid.) using L929 cells. Starting concentration: Anti-TNFα Fragment −50 mg/ml; Intact Anti-TNFα −210 mg/ml; TNFα antisera protein concentration −86 mg/ml.

(5) FIG. 4 shows the results of an ELISA comparing binding to murine TNFα by ovine blood-derived intact PcAbs (anti-human TNFα IgG) and monoclonal antibody Infliximab. Anti-human TNFα IgG (.circle-solid.), Infliximab (.square-solid.), and negative control (PCB) (.box-tangle-solidup.).

EXAMPLES

Example 1

(6) Preparation of Ovine Antisera to Human TNFα

(7) Mature human TNFα (hTNFα) (UniProtKB Accession No.: P01375) was obtained from R&D Systems, Boehringer. The amino acid sequence is shown as SEQ ID No. 1.

(8) The immunogen for the primary immunisation of merino wether sheep comprised Freund's complete adjuvant and 100 μg of hTNFα per sheep. The protein:adjuvant mixture was injected subcutaneously and equally into 6 injection sites chosen for their proximity to the axillary, inguinal, and prescapular drainage lymph glands. Each sheep was reimmunized at 28-day intervals with 100 μg of hTNFα and Freund's incomplete adjuvant, and blood samples were collected 14 days later at approximately 4 weekly intervals at processing facilities at Turretfield Research Centre (Rosedale, South Australia, Australia) according to strict state and national ethical guidelines for animal welfare. The animals were not terminally bled. A total of 10 mL of blood per kg of body weight can be collected from the external jugular vein without detriment to the animal.

(9) Ovine antisera was subsequently stored at −20° C.

Example 2

(10) Purification of Polyclonal Ovine Antibodies to Human TNFα (Intact Anti-TNFα)

(11) Two different methods for purification of ovine PcAb were used in order to determine which method would co-isolate to a larger extent the efficient inhibitor of human trypsin, α1-anti-trypsin. Either caprylic acid precipitation, which precipitates albumin and keeps the IgG in solution, or the sodium sulphate precipitation that precipitates IgG were used. The purified IgG was filtered and stored at −20° C. ready for inclusion in the proposed formulation for oral administration or for further characterisation.

(12) The presence of protease inhibitors in antisera and the two IgG fractions purified using either caprylic acid or sodium sulphate was assayed against trypsin and chymotrypsin by way of a colorimetric assay. The assay was based on the methods of Kakade M L et al. Determination of trypsin inhibitor activity of soy products: a collaborative analysis of an improved procedure. Cereal Chem 51: 376-381, 1974 (which is incorporated herein by reference) and measured cleavage of Na-Benzoyl-DL-Arginine-p-Nitroanilide Solution (L-BAPNA) (colourless) to p-nitroanaline (a yellow substrate) by trypsin.

(13) Two-fold dilutions of inhibitor sample were diluted in 50 mM Tris-buffer, pH 8.2, containing 20 mM CaCl.sub.2 (100 μl final volume) across a 96-well plate (Grenier UV-Star). To each well, 100 μl of trypsin (0.2 mg/ml diluted in 1 mM HCL) or chymotrypsin (1 mg/ml in WFI) solution was added followed by 100 μl of either L-BAPNA or NSLPN (2 mM in DMSO) for trypsin or chymotrypsin, respectively. Reactions were terminated after 5 minutes for the trypsin assay and after 60 minutes for the chymotrypsin assay with the addition of 50 μl stop solution (30% v/v acetic acid) and the absorbance measured spectrophotometrically at 410 nm using Omega PolarStar. Blank samples were prepared by adding the stop solution prior to the substrate solution. The average absorbance value was then plotted against the dilution, resulting in a dose response curve. Representative curves are shown in FIG. 1, which demonstrate that the sodium sulphate purification retains the al-anti-trypsin inhibitor much better that the caprylic acid precipitation procedure.

(14) These two methods can also be used to assess the survival of protease inhibitors in the oral formulation after incubation in simulated gastric and intestinal fluids.

Example 3

(15) Enzyme-Linked Immunosorbent Assay for Characterising Antibody Binding

(16) The specific antibodies produced bound to multiple epitopes on the surface of recombinant human TNFα (rhTNFα) but not to recombinant rodent TNFα. The avidity of binding was extremely high.

(17) A direct ELISA assay was developed for detection of anti-TNFα IgG in the ovine antisera and in the purified fraction of IgG (purified by way of caprylic acid precipitation) from this antisera (Intact Anti-TNFα). Immulon 4HBx microtiter plates were coated with 1 μg/mL hTNFα. Plates were washed with 3 changes of phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST) and blocked for 1 hour at 37° C. with blocking buffer (2.5% fetal calf serum diluted in PBS). Plates were washed and incubated for 1 hour at 37° C. with antisera at initial dilutions of 1:1000, followed by 1:2 serial dilutions; washed with PBST; and incubated with a donkey anti-ovine IgG horseradish peroxidase conjugate for 1 hour at 37° C. After further washing, 3,3′,5,5′-Tetramethylbenzidine (TMB) liquid substrate solution was added, and the reaction was stopped after approximately 10 minutes by the addition of 1.0 M HCL before reading the optical density at 450 nm.

(18) The developed assay showed very low background which was tested using pre-immune ovine serum (FIG. 2).

Example 4

(19) Immunocytotoxicity Assay for Characterising Antibody Neutralisation

(20) The L929 mouse fibrosarcoma cell line (commercially available from Sigma-Aldrich, The Old Brickyard, New Road, Gillingham, Dorset, SP8 4XT, UK) was used to test the cytotoxic effects of TNFα as well as the neutralising ability of antibodies to TNFα. An assay was therefore developed to test neutralisation of the cytotoxic effect of rhTNFα by the ovine PcAb in the antisera, and by purified IgG from the antisera (Intact Anti-TNFα), and by fragments thereof (Anti-TNFα Fragment).

(21) Anti-TNFα Fragment was prepared by subjecting a portion of the stock of ovine antisera of Example 1 to papain digestion. The Fab were present at a concentration of 10 g/L and about 10% of the total Fab were specific for TNFα. An affinity chromatography step was not included in its manufacture. In the presence of excess of Fab, about 12 molecules of Fab become attached to each TNFα trimer.

(22) As a challenging dose we used the 1090 hTNFα concentration of 13 ng/ml determined from a cytotoxicity assay (data not shown). Briefly, L929 cells containing twice the necessary challenging dose in DMEM were co-incubated with an equal volume of various dilutions of hTNFα antisera or Anti-TNFα Fragment or Intact Anti-TNFα for 24 h. As a positive control (maximum killing), 2.5 μg/ml hTNFα was used. Antibody toxin neutralisation titres were estimated by colorimetric assays based on cell staining with neutral red.

(23) Antibody toxin neutralisation titres were estimated by colorimetric assays based on cell staining with neutral red (representative curves shown in FIG. 3).

(24) The specific antibody concentration was calculated as follow:
Specific Ab conc [g/L]=[CCD(μg/L)−LC50(μg/L)]×[MW Ab/(MW Ag×BS)]×EC50×10.sup.−6 CCD (μg/L)−challenging dose=13 μg/L (LC90 determined from the TNFα cytotoxicity on L929 cells) LC50 (μg/L)=0.3 μg/L (determined from the TNFα cytotoxicity on L929 cells) BS−binding sites=2 for whole IgG MW Ab=160 000 Da MW Ag (TNFα)=51 000 Da

(25) Taking the above into account, the specific PcAb concentration in the antisera was calculated at 2.9 g/L.

Example 5

(26) Formulation of Intact Anti-TNFα

(27) The choice of protease inhibitors to protect the PcAb of the invention (Intact Anti-TNFα) from digestion as well as the inhibitor's survival in gastric and intestinal fluids was assessed against trypsin and chymotrypsin using colourimetric assay, based on the method of Kakade M L et al. Determination of trypsin inhibitor activity of soy products: a collaborative analysis of an improved procedure. Cereal Chem 51: 376-381, 1974. The trypsin assay is described above. The chymotrypsin assay utilised a colourless NSLPN substrate producing a yellow colour.

(28) The two antacids in Table 1, magnesium hydroxide and aluminium hydroxide gel, were added to neutralise the low gastric pH, and thereby prevent pepsin in the stomach from degrading the active components of Intact Anti-TNFα—the ovine IgG against TNFα and the egg white trypsin and chymotrypsin inhibitors. Other constituents were antimicrobials methylparaben and propylparaben which are believed to maintain a low bioburden. The antifoaming agent simethicone was added to prevent protein denaturation when the suspension is mixed prior to use. Sweeteners in the form of sodium saccharin and mannitol reduce the bitterness of the two antacids. A flavouring agent, peppermint oil, was used to improve the taste. A suspending agent, xanthan gum, was added to keep all the above components in suspension.

(29) It was experimentally determined that adding 200 mM Glycine to the formulation significantly improved stability of the suspension.

(30) TABLE-US-00008 TABLE 1 Composition of Intact Anti-TNFα oral formulation. Category Material name Concentration Active IgG Intact Anti-TNFα  50 g/l components Protease Egg white dried (EWD)  60 g/l Excipients inhibitor Antacid Magnesium hydroxide 23.4 g/l  Dried aluminium 26.4 g/l  hydroxide gel Antimicrobial Methylparaben (E218)   2 g/l Propylparaben (E216) 0.6 g/l Suspension Glycine (200 mM)  15 g/l stability Antifoaming Simethicone 16.89 g/l  agent Sweeteners Sodium saccharin 0.4 g/l Mannitol  21 g/l Flavour Peppermint oil 0.2 g/l Suspending Xanthan gum   4 g/l agent

Example 6

(31) Physicochemical Characterisation of the Composition Formulated for Oral Delivery

(32) The physical appearance of the suspension and its purity were ensured by carefully performing visual inspections. Sedimentation volume of the suspension was determined by pouring 50 ml of the formulation into a 100 ml measuring cylinder and the sedimentation volume was monitored and recorded at different time intervals.

(33) Duplicate results were obtained and the sedimentation volume was calculated according to the equation:
F=Vu/Vo

(34) Where F is sedimentation volume, Vu—ultimate height of sediment and Vo—initial height of total suspension.

(35) Within 48 hours of the beginning of the sedimentation experiment, no sedimentation was observed (Vu=0). Therefore, the pharmaceutical suspension remained very stable within 48 hours without any separation. Furthermore, after 48 hours the cylinder with the suspension was turned upside-down and none of the layers were observed to be disposed at the bottom of the cylinder. This demonstrates that there were no layers forming within the suspension. In other word, there was no flocculation observed within the prepared suspension during the 48-hour period of the experiment.

Example 7

(36) Case Study

(37) A 27 year old white male on an emergency admission to hospital is diagnosed as suffering from acute, severe ulcerative colitis which is confirmed by colonoscopy and biopsy. The subject is immediately administered an intravenous course of hydrocortisone but fails to respond over the next six days. There is concern that he might require a total colectomy and, consequently, he is orally administered a formulation of the invention (e.g. as per Example 5). For the first two weeks he receives 50 ml twice daily and, thereafter, 50 ml daily for a further 12 weeks.

(38) The patient makes a quick and excellent recovery based on his answers to the UK bowel disease questionnaire (UK-IBDQ) and various clinical parameters. In addition measured parameters such as C-reactive protein return to normal values, and he experiences no serious adverse effects. At the end of the study there is significant mucosal healing as assessed by a second colonoscopy and biopsy.

Example 8

(39) Synergistic Effects of the Compositions Formulated for Oral Delivery

(40) The following formulations are provided: Formulation A: Intact Anti-TNFα; Formulation B: Egg White Dried Protease Inhibitor; Formulation C: Bowman-Birk Inhibitor (soybean); Formulation D: Intact Anti-TNFα+Egg White Dried Protease Inhibitor; and Formulation E: Intact Anti-TNFα+Bowman-Birk Inhibitor (soybean).

(41) 25 patients with ulcerative colitis and 25 patients with Crohn's Disease provide consent to be involved in a study to test the efficacy of Formulations A-E. The patients are split into groups of 5 and administered one of Formulations A-E (i.e. 5 patients with ulcerative colitis are administered Formulation A and 5 patients with Crohn's Disease are administered Formulation A, 5 patients with ulcerative colitis are administered Formulation B and 5 patients with Crohn's Disease are administered Formulation B, etc.). The dosage regime is 20 ml (equivalent to 1 g of Intact Anti-TNFα) twice daily for 4 weeks, and once daily thereafter.

(42) Physicians determine that patients in both disease groups administered Formulations D and E show a greater improvement and reduced symptoms when compared with patients administered Formulations A-C. Colonoscopies reveal an improvement in the surface layers of the intestinal tract post-treatment. The improvement in patients administered Formulations D and E is much greater than the improvement in patients administered Formulations A-C, and much greater than the expected combined improvement of Formulations A+B and Formulations A+C (as determined by way of colonoscopy). Thus, the combination of PcAbs and protease inhibitors (e.g. EWD protease inhibitors and/or Bowman-Birk Inhibitors) yields unexpected synergistic effects.

Comparative Example 9

(43) Comparative Analysis of Specific Antibody Titres from Sheep Serum (Ovine) & Hen Eggs

(44) A study was undertaken to assess the concentration and avidities of specific IgY obtained from hen eggs in comparison with specific antibody concentrations from ovine antisera.

(45) A group of 10 chickens and 5 sheep were immunised with human interleukin-6 (hIL-6, a pro-inflammatory cytokine like TNFα) and the titres and avidities of the resultant specific PcAb was compared. The average avidity constants were 1.3×10.sup.10 L/mol for chicken IgY vs 3.1×10.sup.10 L/mol for the ovine antibodies. However, the levels of specific PcAb attained in the sheep (with an average titre of ≥1:200,000) were more than ten times the titres found in egg yolk (≤1:20,000). This tenfold or more difference in the concentration of specific PcAb was also apparent when sheep and hens were immunised with a number of other immunogens.

(46) The above experiment shows the advantages of antibodies derived from blood, and from an ovine source in particular.

Example 10

(47) Comparative Analysis of Specific Antibody Titres Sourced from Sheep Serum (Ovine) & Cow's Milk (Bovine)

(48) A study was undertaken to assess the potential of colostrum and milk from suitably immunised cows as a source of PcAb.

(49) Cows were immunised with human TNFα and the titres of the resulting specific PcAb determined first in the colostrum and then in serial samples of milk. The maximum titre obtained in colostrum was 1:275,000 (as compared with 1:800,000 in ovine antisera) and, after the first milking, levels rapidly fell to approximately 1:27,500.

(50) Thus, blood-derived sources were shown to yield higher concentrations of antibodies that bind to human TNFα when compared to milk/colostrum-derived sources.

Example 11

(51) Stability of Oral Antibody Formulations

(52) The oral formulation of Example 5 was tested for antibody binding and neutralising activity following storage for approximately 12 months. It was shown that there was no deterioration in protease inhibitor activity, and no change in the physical stability of the formulation, which retained antibody binding and neutralising activity.

Example 12

(53) Efficacy of Antimicrobials of the Oral Antibody Formulations

(54) The antimicrobial agents in the oral formulation of Example 5—Methylparaben (E218) (concentration 2 g/L) and Propylparaben (E216) (0.6 g/L)—were subjected to external antimicrobial testing to European Pharmacopeia standards for such organisms as S. aureus, P. aeruginosa, E. coli, C. albicans and A. brasiliensis. Complete sterility was shown.

Example 13

(55) Comparison of Blood-Derived Polyclonal Antibodies with Monoclonal Antibodies

(56) A comparative antigen-binding assay was performed using blood-derived (ovine) polyclonal antibodies that bind to human TNFα and Infliximab (Schering-Plough Ltd), a monoclonal antibody that binds to human TNFα.

(57) FIG. 4 shows that the monoclonal antibody, Infliximab, does not bind to murine TNFα, whereas the ovine derived polyclonal antibodies raised towards human TNFα do.

(58) The blood-derived polyclonal antibodies of the invention were shown to bind to and neutralise murine TNFα albeit at a concentration approximately 100-fold higher than that needed to neutralise human TNFα. No neutralisation of murine TNFα was observed by Infliximab, indicative of an overall reduced neutralisation capability when compared to antibodies of the invention.

(59) TABLE-US-00009 SEQUENCES SEQ ID No. 1 VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQ LVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLL SAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDY LDFAESGQVYFGIIAL SEQ ID No. 2 MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTL FCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVA NPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFK GQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAK PWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQ ID No. 3 MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTL FCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVA NPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFK GQGCP SEQ ID No. 4 VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQ LVVPSEGLYLIYSQVLFKGQGCP SEQ ID No. 5 VRSSSRTP SEQ ID No. 6 HVVANPQAEGQLQWLNRR SEQ ID No. 7 NGVELR SEQ ID No. 8 VPSEG SEQ ID No. 9 CPSTHVL SEQ ID No. 10 ISRIAVSYQTK SEQ ID No. 11 PCQRETPEGAEAK SEQ ID No. 12 DRLSAEINRPDYLDFA SEQ ID No. 13 (Variant Human TNFα P84L) MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTL FCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTLSDKPVAHVVA NPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFK GQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAK PWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGI IAL

(60) All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.