Therapy for treating proctitis with polyclonal anti-TNF alpha antibodies
阅读说明：本技术 用多克隆抗TNFα抗体治疗直肠炎的疗法 (Therapy for treating proctitis with polyclonal anti-TNF alpha antibodies ) 是由 约翰·兰登 露丝·伊丽莎白·科克森 于 2019-07-29 设计创作，主要内容包括：本发明涉及一种用于治疗直肠炎的抗体组合物,其中所述抗体组合物包含与人肿瘤坏死因子α(TNFα)结合的完整血源性多克隆抗体。本发明还涉及使用/包括其的相关用途、方法、固体组合物和液体组合物。(The present invention relates to an antibody composition for use in the treatment of proctitis, wherein said antibody composition comprises an intact blood-derived polyclonal antibody that binds to human tumor necrosis factor alpha (TNF α). The invention also relates to related uses, methods, solid compositions and liquid compositions using/including the same.)
1. An antibody composition for use in the treatment of proctitis, wherein said antibody composition comprises an intact blood-derived polyclonal antibody that binds to human tumor necrosis factor alpha (TNF α).
2. Use of an antibody in the manufacture of a medicament for the treatment of proctitis, wherein the antibody is an intact blood-borne polyclonal antibody that binds to human tumor necrosis factor alpha (TNF α).
3. A method of treating proctitis comprising administering to a subject an antibody composition, wherein the antibody composition comprises an intact blood-derived polyclonal antibody that binds to human tumor necrosis factor alpha (TNF α).
4. The antibody composition for use according to claim 1, the use of claim 2 or the method of claim 3, wherein said blood-borne polyclonal antibody is a sheep blood-borne polyclonal antibody.
5. The antibody composition for use according to claim 1, the use of claim 2 or the method of claim 3, wherein said blood-borne polyclonal antibody is a horse blood-borne polyclonal antibody.
6. The antibody composition for use, the use or the method according to any one of the preceding claims, wherein at least 5% of the total antibodies present in the composition bind to TNF α.
7. The antibody composition for use, the use or the method according to any one of the preceding claims, wherein at least 10% of the total antibodies present in the composition bind to TNF α.
8. The antibody composition for use, the use or the method according to any one of the preceding claims, wherein the antibody composition or medicament is formulated as a solid, preferably as a suppository.
9. The antibody composition for use, the use or the method according to claim 8, wherein the concentration of said antibody is 0.2-20% w/w, preferably wherein the concentration of said antibody is 0.2-10% w/w (more preferably about 1-2% w/w or 2-6% w/w).
10. The antibody composition for use, the use or the method according to any one of the preceding claims, wherein a single dose comprises 1000mg or less of antibody (e.g. 500mg or less).
11. The antibody composition for use, the use or the method according to any one of the preceding claims, wherein a single dose comprises 250mg or less of antibody.
12. The antibody composition for use, the use or the method according to any one of the preceding claims, wherein the antibody composition is administered to a subject at least once daily.
13. The antibody composition for use, the use or the method according to any one of the preceding claims, wherein the antibody composition is administered to a subject at least twice a day, preferably three times a day.
14. The antibody composition for use, the use or the method according to any one of the preceding claims, wherein the antibody composition is administered rectally.
15. The antibody composition for use, the use or the method according to any one of the preceding claims, wherein the proctitis is idiopathic proctitis.
16. The antibody composition for use, the use or the method according to any one of the preceding claims, wherein the subject further suffers from a sexually transmitted disease, a celiac disease or a combination thereof.
17. The antibody composition for use, the use or the method according to any one of the preceding claims, wherein the subject for treating proctitis is receiving chemotherapy, radiation therapy or a combination thereof.
18. A solid composition for treating proctitis comprising:
a. intact blood-derived polyclonal antibodies that bind to human tumor necrosis factor alpha (TNF α); and
b. one or more of gelatin, cocoa butter, polyethylene glycol and suppository wax.
19. The solid composition of claim 18, wherein the solid composition is a suppository.
20. The solid composition according to claim 18 or 19, wherein the concentration of the antibody is 0.2-20% w/w, preferably wherein the concentration of the antibody is 0.2-10% w/w (more preferably about 1-2% w/w or 2-6% w/w).
21. The solid composition of any one of claims 18-20, wherein the solid composition comprises:
a. intact blood-derived polyclonal antibodies that bind to human tumor necrosis factor alpha (TNF α); and
b. gelatin.
22. A liquid composition (preferably an enema composition) for use in the treatment of proctitis comprising:
a. intact blood-derived polyclonal antibodies that bind to human tumor necrosis factor alpha (TNF α); and
b. one or more (preferably all): biocides, suspension stabilizers, defoamers and suspending agents;
wherein the composition does not comprise a device that protects the antibody during gastrointestinal transit.
23. The solid or liquid composition of any one of claims 18-22, wherein the blood-borne polyclonal antibody is a sheep blood-borne polyclonal antibody.
24. The solid or liquid composition of any one of claims 18-22, wherein the blood-borne polyclonal antibody is a horse blood-borne polyclonal antibody.
25. The solid or liquid composition of any one of claims 18-24, wherein at least 5% of the total antibodies present in the composition bind to TNF α, preferably at least 10% of the total antibodies present in the composition bind to TNF α.
26. The antibody composition for use, the method, the solid or liquid composition according to any one of the preceding claims, wherein said antibody or composition is not affinity purified.
27. Antibody composition for use, the method, the solid or liquid composition according to any one of the preceding claims, wherein only the antibody or composition is subjected to the precipitation (and optionally filtration) step.
28. The antibody composition for use, the method, the solid or liquid composition according to any one of the preceding claims, wherein said composition is a serum composition that is subjected only to a precipitation (and optionally filtration) step.
29. The antibody composition for use, the method, the solid or liquid composition according to any one of the preceding claims, wherein said composition is a sheep serum composition comprising intact blood-borne sheep polyclonal antibodies that bind to human TNF α and which is subjected only to a precipitation (and optionally filtration) step.
30. The antibody composition for use, the method, the solid or liquid composition according to any one of the preceding claims, wherein said composition is a plasma composition which is subjected only to a precipitation (and optionally filtration) step.
31. The antibody composition for use, the method, the solid or liquid composition according to any one of the preceding claims, wherein said composition is a horse plasma composition comprising intact blood-borne madoconclons antibodies that bind to human TNF α and only subjected to a precipitation (and optional filtration) step.
32. The antibody composition for use, the method, the solid or liquid composition of any one of claims 27-31, wherein the precipitation is caprylic acid precipitation.
33. The antibody composition for use, the method, the solid or liquid composition according to any one of claims 27-32, wherein said filtering comprises the use of a 0.2 μ ι η filter and optionally a 0.45 μ ι η filter (preferably consisting of the use of glass microfibers (0.45 μ ι η) and a 0.2 μ ι η filter).
Drawings
Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:
figure 1 shows the results of a direct ELISA for the detection of anti-TNF α IgG in antisera (●) and IgG purified by caprylic acid precipitation (a).
Figure 2 shows the results of an immunocytotoxicity assay (ICTA) using L929 cells to test the neutralizing activity of anti-TNF α fragment (■), whole anti-TNF α (a-solidup) and TNF α antiserum (●). Initial concentration: anti-TNF alpha fragment-50 mg/ml; intact anti-TNF alpha 210 mg/ml; TNF α antiserum protein concentration-86 mg/ml.
FIG. 3 shows the results of ELISA comparing the binding of sheep blood-derived intact Pcabs (anti-human TNF α IgG) and the monoclonal antibody infliximab to murine TNF α. Anti-human TNF α IgG (●), infliximab (■) and negative control (PCB) (. tangle-solidup).
Figure 4 shows the results of a second ELISA comparing the binding of sheep blood-derived intact PcAbs (anti-human TNF α IgG) and the monoclonal antibody infliximab to murine TNF α. Anti-human TNF α IgG (●), infliximab (■) and negative control (PCB).
Figure 5 shows the results of an immunocytotoxicity assay comparing cell viability in the presence of murine TNF α at an challenge dose of 5ng/ml when administered sheep blood-borne intact PcAbs (anti-human TNF α IgG) or the monoclonal antibody infliximab. Results were obtained from 2x plates of sheep blood-derived intact PcAbs ("sheep IgG" ═ ● +. diamond-solid.) 2x plates of infliximab ("infliximab" ═ ■ + open circles).
Examples
Example 1
Preparation of anti-human TNF alpha sheep antiserum
Mature human TNF α (hTNF α) (UniProtKB accession No.: P01375) was obtained from R & D Systems of Boehringer. The amino acid sequence is shown as SEQ ID No. 1.
Immunogens for primary immunization in merino sheep included freund's complete adjuvant and 100 μ g of hTNF α per sheep. Mixing the protein: the adjuvant mixture was injected subcutaneously and evenly into 6 injection sites, which were selected to drain lymph glands near the axilla, groin and pre-scapular. Each sheep was re-immunized every 28 days with 100 μ g of hTNF α and freund's incomplete adjuvant, according to strict state and national animal welfare ethide guidelines, and blood samples were collected at the processing facility of the Turretfield research center (Rosedale, south australia, australia) at approximately 4 weeks after 14 days. The animals were not finally bled. A total of 10 ml of blood per kg of body weight can be collected from the external jugular vein without damaging the animal.
The sheep antisera were then stored at-20 ℃.
Example 2
Purification of polyclonal sheep antibodies against human TNF alpha (intact anti-TNF alpha)
Sheep polyclonal antibody (PcAb) was purified using two different methods. The albumin can be precipitated and the IgG kept in solution using caprylic acid precipitation, or the IgG can be precipitated using sodium sulfate precipitation. The purified IgG was filtered and stored at-20 ℃ for inclusion in recommended formulations for rectal administration or for further characterization.
Example 3
Enzyme-linked immunosorbent assay for characterizing antibody binding
The specific antibodies generated bind to multiple epitopes on the surface of recombinant human TNF α (rhTNF α), but do not bind to recombinant rodent TNF α. The binding affinity is extremely high.
A direct ELISA assay was developed for the detection of anti-TNF α IgG (intact anti-TNF α) in sheep antisera and IgG purified fractions of the antisera (purified by caprylic acid precipitation). Immulon 4HBx microtiter plates were coated with 1. mu.g/mL hTNF α. Plates were washed 3 times with Phosphate Buffered Saline (PBS) containing 0.1% tween 20(PBST) and then blocked with blocking buffer (2.5% fetal bovine serum diluted in PBS) for 1 hour at 37 ℃. Plates were washed and washed at 37 ℃ with 1: 1000 initial dilutions were incubated with antiserum for 1 hour, followed by 1: 2 serial dilutions were incubated; washing with PBST; and incubated with donkey anti-goat IgG horseradish peroxidase conjugate for 1 hour at 37 ℃. After further washing, 3 ', 5, 5' -Tetramethylbenzidine (TMB) liquid substrate solution was added and after about 10 minutes the reaction was stopped by adding 1.0MHCl and then reading the optical density at 450 nm.
The developed detection method showed a very low background, which was tested with preimmune sheep serum (fig. 1).
Example 4
Immunocytotoxicity assay for characterizing antibody neutralization
L929 mouse fibrosarcoma cell line (purchased from Sigma-Aldrich, The Old Brickyard, New Road, Gillingham, Dorset, SP 84 XT, UK) was used to test The cytotoxic effects of TNF α and The neutralizing capacity of The antibody for TNF α. Thus, an assay was developed to test sheep PcAb in antiserum, which neutralizes the cytotoxic effects of rhTNF α by purified IgG (intact anti-TNF α) and fragments thereof (anti-TNF α fragments) in antiserum.
anti-TNF α fragments were prepared by papain digestion of a portion of the sheep antiserum stock of example 1. Fab is present at a concentration of 10g/L and about 10% of the total Fab is specific for TNF α. The affinity chromatography step is not included in its manufacture. In the presence of excess Fab, about 12 Fab molecules were attached to each TNF α trimer.
The IC90 hTNF α concentration determined according to the cytotoxicity assay (data not shown) was 13ng/ml, which was used as the priming dose. Briefly, L929 cells containing twice the requisite priming dose in DMEM were incubated with equal volumes of various dilutions of hTNF α antiserum or anti-TNF α fragments or intact anti-TNF α for 24 hours. As a positive control (maximum lethality), 2.5. mu.g/ml hTNF α was used. Antibody toxin neutralization titers were evaluated by a colorimetric assay based on neutral red blood cell staining.
Antibody toxin neutralization titers were evaluated by a colorimetric assay based on neutral red blood cell staining (representative curves are shown in figure 2). Advantageously, intact anti-TNF α exhibits significantly improved neutralizing capacity compared to anti-TNF α fragments.
Specific antibody concentrations were calculated as follows:
specific antibody concentration [ g/L]＝[CCD(μg/L–LC50(μg/L)]x[MW Ab/(MW Ag x BS)]x EC50 x 10-6
-CCD (μ g/L) -priming dose ═ 13 μ g/L (LC 90 determined according to the cytotoxicity of TNF α on L929 cells)
-LC50(μ g/L) ═ 0.3 μ g/L (determined by the cytotoxicity of L929 cells with TNF α)
-BS-binding site for whole IgG ═ 2
-MW Ab＝160 000Da
-MW Ag(TNFα)＝51 000Da
In view of the above, the specific PcAb concentration in the antiserum was calculated to be 2.9 g/L.
Comparative example 5
Comparative analysis of sheep serum (sheep) and hen egg specific antibody titers
A study was carried out to evaluate the concentration and affinity of specific IgY obtained from eggs compared to the concentration of specific antibodies obtained from sheep antisera.
A group of 10 chickens and 5 sheep were immunized with human interleukin-6 (hIL-6, a proinflammatory cytokine such as TNF α) and the titers and affinities of the resulting specific PcAbs were compared. Chicken IgY has an average affinity constant of 1.3X 1010L/mol, whereas the sheep antibody was 3.1X1010L/mol. However, the level of specific PcAb obtained in sheep (mean titer ≧ 1: 200,000) was more than ten times the titer in egg yolk (. ltoreq.1: 20,000). This ten-fold or greater difference in specific PcAb concentrations is also evident when sheep and hens are immunized with many other immunogens.
The above experiments show the advantages of antibodies derived from blood, in particular from sheep sources.
Example 6
Comparative analysis of specific antibody titres of sheep serum (sheep) and cow milk (cow) origin
A study was conducted to evaluate the potential of colostrum and milk from suitably immunized cows as a source of PcAb.
Cows were immunized with human TNF α and the titers of the resulting specific PcAb were determined first in colostrum and then in a series of cows. The maximum titer obtained in colostrum was 1: 275,000 (compared to 1: 800,000 in sheep antiserum) and this level rapidly drops to about 1: 27,500.
Thus, blood-derived sources have been shown to produce consistently higher concentrations of antibodies that bind to human TNF α than milk/colostrum-derived sources.
Example 7
Binding of sheep blood-derived polyclonal antibody to human TNF alpha
The titer of sheep blood-derived polyclonal anti-TNF α antibodies (intact anti-TNF α) was determined by indirect ELISA. ImmulonR4HBX Flat-bottomed microplates (Thermo Scientific) were coated with coating buffer (phosphate buffered saline, PBS, 8g/L NaCl, 0.2g/L KCl, 1.44g/L Na)2HPO4、0.24g/L KH2PO4pH 7.4) coating human recombinant TNF α at a concentration of 1. mu.g/mL (100. mu.l/well) and incubating at 4 ℃And (5) breeding over night. The plates were washed 3 times with PBS (PBST) containing 0.1% Tween 20 and blocked with 2.5% fetal bovine serum in PBS (150. mu.l/well) for 2 hours at 37 ℃. The plates were then washed 3 times with PBST and incubated with an anti-TNF α polyclonal antibody composition (serum that had been purified with 6% v/v caprylic acid and filtered with glass microfibers and 0.2um filters) (100 μ l/well) at appropriate dilution. PolyCAb B (binding to clostridium difficile toxin) was used as a negative control. The wells were washed three times with PBST, then 100 microliters of diluted (1: 10,000) donkey anti-sheep antibody conjugated to horseradish peroxidase (Sigma) was added and incubated for 1 hour at 37 ℃. The plate was then washed 3 times with PBST and 100ml of 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) substrate solution left for about 4 hours to reach room temperature was added. The plate was allowed to stand at room temperature for 5 minutes. The reaction was stopped by adding 50 ml/well of 1M HCl and plates were read at 450nm and 690nm in a Polarstar reader. All samples were tested in duplicate or triplicate. 50% binding titer was estimated using Graph Pad Prism 7.
The sheep blood-derived polyclonal antibody was found to have an EC of 0.77mg/L50Values bind to human TNF α.
Example 8
Cell survival assay (human TNF α)
The neutralizing capacity of blood-borne sheep polyclonal antibodies (whole anti-TNF α serum, which has been purified using 6% v/v caprylic acid and filtered using glass fibers and a 0.2 μm filter) and infliximab (Schering-Plough Ltd) was evaluated and quantified using a Neutral Red Uptake (NRU) based assay. L929 cells known to undergo cell death upon exposure to TNF α were used. The assay was performed using an indicator plate at 7.5x103Perwell (100. mu.L) was inoculated with L929 cells and allowed to incubate at 37 ℃ in 5% CO2Growth in humidified atmosphere for about 24 hours.
L929 cells were fed with Dulbecco's Modified Eagle Medium (DMEM) (Sigma) and supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 2mM L-glutamine (Sigma) and 5% penicillin/streptomycin (Sigma). Cells were stored at 75cm2In a flask at 2x10 weekly5Was inoculated in 30mL of medium. These cells are periodically fed.
For each antibody sample, two serial dilutions were performed in a 96-well dilution plate using DMEM, followed by addition of an equal amount of TNF α -containing medium. A fixed concentration of TNF α (12 ng/ml challenge dose) was used and was based on LC98 (lethal concentration resulting in 98% cell death). This concentration is sufficient to cause near 100% cell rounding. Each plate included a TNF- α cytotoxicity curve as a control for analysis of batch-to-batch variation, monitoring antigen stability and reproducibility.
Antibody in DMEM served as negative control, while TNF α challenge dose served as positive control. The dilution plates were incubated at room temperature for 1 hour. After incubation, 100 μ L of the sample on the dilution plate was transferred to the corresponding well in the indicator plate containing 100 μ L of medium, resulting in a total volume of 200 μ L. The indicator panel was then returned to 37 ℃ with 5% CO2In an incubator. After 48 hours, the plates were washed 3 times with phosphate buffered saline, then 100 μ l destaining solution (50% ethanol and 1% acetic acid) was added and placed on a flat bed shaker for 15 minutes. The plates were read in a Polarstar plate reader at 540nm (test measurement) and 690nm (background measurement) wavelengths. The absorbance of each well was calculated by subtracting the background measurement from the test measurement. The percent cell death was calculated as follows:
results were obtained from a total of 6 plates run in duplicate (n-2) over 3 days alone. The sheep polyclonal antibody reached 100% cell viability in the assay at the highest concentration used compared to infliximab (viability below 70%). Specifically, an average difference in the optimal survival rate of 26.36% (range: 15.45-39.5%) was observed between L929 cells treated with infliximab and sheep polyclonal antibody. This means that infliximab cannot completely neutralize hrTNF α.
The dilution of the sheep polyclonal antibody required to protect 50% of the cell monolayer was estimated using GraphPad Prism 7, resulting in an average EC50The value was 0.455 ug/ml. Surprisingly, similar preparations of polyclonal antibodies derived from bovine colostrum were reported to neutralize humansTNFα，EC50The value was 16 ug/ml. Therefore, the sheep blood-derived polyclonal antibody is greatly improved compared with the bovine polyclonal antibody.
Example 9
Comparison of blood-derived polyclonal antibodies with monoclonal antibodies
Comparative antigen binding assays were performed using a blood-derived sheep polyclonal antibody that binds to human TNF α and the monoclonal antibody infliximab (Schering-Plough Ltd) that binds to human TNF α.
Figure 3 shows that the monoclonal antibody infliximab does not bind to murine TNF α, whereas the ovine-derived polyclonal antibody binds to human TNF α.
The blood-borne polyclonal antibodies of the present invention are shown to bind and neutralize mouse TNF α, although at concentrations about 100-fold higher than those required to neutralize human TNF α. No neutralization of mouse TNF α was observed with infliximab, indicating an overall reduction in neutralizing capacity compared to the antibodies of the invention.
Example 10
Binding of sheep blood-borne polyclonal antibody and mouse TNF alpha
Titers of infliximab and sheep blood-derived polyclonal antibody (intact anti-TNF α) were determined by indirect ELISA. Immulon R4 HBX flat-bottomed microplates (Thermo Scientific) were plated with coating buffer (phosphate buffered saline, PBS, containing 8g/L NaCl, 0.2g/L KCl, 1.44g/L Na2HPO4、0.24g/L KH2PO4pH 7.4) was coated with mouse recombinant TNF α at a concentration of 1 μ g/mL (100 μ l/well) and incubated overnight at 4 ℃. The plates were washed 3 times with PBS (PBST) containing 0.1% Tween 20 and blocked with 2.5% fetal bovine serum in PBS (150. mu.l/well) for 2 hours at 37 ℃. The plates were then washed 3 times with PBST and incubated with an anti-TNF α polyclonal antibody composition (serum that had been purified with 6% v/v caprylic acid and filtered with glass microfibers and 0.2um filters) (100 μ l/well) at appropriate dilution. PolyCAb B (binding to clostridium difficile toxin) was used as a negative control. The wells were washed three times with PBST, then 100 microliters of diluted (1: 10,000) donkey anti-sheep antibody conjugated to horseradish peroxidase (Sigma) was added and incubated for 1 hour at 37 ℃. The plates were then washed 3 times with PBST and addedInto 100ml of a substrate solution 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) which was left to stand for about 4 hours to reach room temperature. The plate was allowed to stand at room temperature for 5 minutes. The reaction was stopped by adding 50 ml/well of 1M HCl and plates were read at 450nm and 690nm in a Polarstar reader. All samples were tested in duplicate or triplicate. 50% binding titer was estimated using Graph Pad Prism 7.
FIG. 4 shows that the monoclonal antibody infliximab does not bind to murine TNF α, whereas the sheep derived polyclonal antibody needle binds to human TNF α, with average EC5011.35mg/L (CV ═ 5.5%). Total 2 plates in triplicate (n-3).
Example 11
Cell survival assay (mouse TNF α)
The neutralizing capacity of blood-borne sheep polyclonal antibodies (whole anti-TNF α serum, which has been purified using 6% v/v caprylic acid and filtered using glass fibers and a 0.2 μm filter) and infliximab (Schering-Plough Ltd) was evaluated and quantified using a Neutral Red Uptake (NRU) based assay. L929 cells known to undergo cell death upon exposure to TNF α were used. The assay was performed using an indicator plate at 7.5x103Perwell (100. mu.L) was inoculated with L929 cells and allowed to incubate at 37 ℃ in 5% CO2Growth in humidified atmosphere for about 24 hours.
L929 cells were fed with Dulbecco's Modified Eagle Medium (DMEM) (Sigma) and supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 2mM L-glutamine (Sigma) and 5% penicillin/streptomycin (Sigma). Cells were stored at 75cm2In a flask at 2x10 weekly5Was inoculated in 30mL of medium. These cells are periodically fed.
For each antibody sample, two serial dilutions were performed in a 96-well dilution plate using DMEM, followed by addition of an equal amount of TNF α -containing medium. A fixed concentration of mouse TNF α (challenge dose 5ng/ml) was used and was based on LC98 (lethal concentration resulting in 98% cell death). This concentration is sufficient to cause near 100% cell rounding. Each plate included a TNF- α cytotoxicity curve as a control for analysis of batch-to-batch variation, monitoring antigen stability and reproducibility.
In DMEMThe antibody of (a) served as a negative control, while the TNF α challenge dose served as a positive control. The dilution plates were incubated at room temperature for 1 hour. After incubation, 100 μ L of the sample on the dilution plate was transferred to the corresponding well in the indicator plate containing 100 μ L of medium, resulting in a total volume of 200 μ L. The indicator panel was then returned to 37 ℃ with 5% CO2In an incubator. After 48 hours, the plates were washed 3 times with phosphate buffered saline, then 100 μ l destaining solution (50% ethanol and 1% acetic acid) was added and placed on a flat bed shaker for 15 minutes. The plates were read in a Polarstar plate reader at 540nm (test measurement) and 690nm (background measurement) wavelengths. The absorbance of each well was calculated by subtracting the background measurement from the test measurement. The percent cell death was calculated as follows:
the dilution required to protect 50% of the cell monolayer was estimated using GraphPad Prism 7.
Figure 5 shows that infliximab does not provide protection to cells exposed to mouse TNF α. In contrast, for the sheep polyclonal antibody, the mean EC was observed5095.15 μ g/ml (CV 25.6%), and-100% survival was observed at higher doses. A total of 5 plates were performed in duplicate (n-2) over 3 separate days.
Example 12
Composition preparation (suppository) for treating proctitis
The suppository prepared from the blood-borne polyclonal antibody comprises the following components:
example 13
Composition preparation (enema) for treating proctitis
The blood-borne polyclonal antibody is prepared into liquid enema as follows:
example 14
Case study
A50 year old male was diagnosed with proctitis. The subject rectally administers the suppository of example 12 twice daily, as instructed by their physician. Treatment is continued until proctitis (or its symptoms) no longer appears.
Example 15
Case study
A 33 year old female was diagnosed with proctitis. The subject rectally administered the suppository of example 12 three times a day according to the instructions of their physician. Once proctitis (or its symptoms) no longer appears, the frequency of administration is reduced to twice daily.
Example 16
Case study
A 67 year old female was diagnosed with proctitis. The subject was administered the enema of example 13(50ml) three times a day. Once proctitis (or symptoms thereof) is controlled, the enema is performed twice a day until proctitis (or symptoms thereof) no longer appears.
Sequence of
SEQ ID No.1
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL
SEQ ID No.2
MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL
SEQ ID No.3
MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCP
SEQ ID No.4
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCP
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 TNF. alpha. P84L)
MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTLSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGI IAL
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with certain 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.
Sequence listing
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Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe
35 40 45
Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro
50 55 60
Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser
65 70 75 80
Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro
85 90 95
Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu
100 105 110
Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser
115 120 125
Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly
130 135 140
Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala
145 150 155 160
Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro
165 170 175
Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu
180 185 190
Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu
195 200 205
Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly
210 215 220
Gln Val Tyr Phe Gly Ile Ile Ala Leu
225 230
<210> 3
<211> 146
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 3
Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala
1 5 10 15
Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe
20 25 30
Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe
35 40 45
Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro
50 55 60
Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser
65 70 75 80
Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro
85 90 95
Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu
100 105 110
Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser
115 120 125
Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly
130 135 140
Cys Pro
145
<210> 4
<211> 70
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 4
Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val
1 5 10 15
Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg
20 25 30
Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu
35 40 45
Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe
50 55 60
Lys Gly Gln Gly Cys Pro
65 70
<210> 5
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 5
Val Arg Ser Ser Ser Arg Thr Pro
1 5
<210> 6
<211> 18
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 6
His Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn
1 5 10 15
Arg Arg
<210> 7
<211> 6
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 7
Asn Gly Val Glu Leu Arg
1 5
<210> 8
<211> 5
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 8
Val Pro Ser Glu Gly
1 5
<210> 9
<211> 7
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 9
Cys Pro Ser Thr His Val Leu
1 5
<210> 10
<211> 11
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 10
Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys
1 5 10
<210> 11
<211> 13
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 11
Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys
1 5 10
<210> 12
<211> 16
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 12
Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala
1 5 10 15
<210> 13
<211> 233
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 13
Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala
1 5 10 15
Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe
20 25 30
Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe
35 40 45
Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro
50 55 60
Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser
65 70 75 80
Ser Arg Thr Leu Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro
85 90 95
Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu
100 105 110
Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser
115 120 125
Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly
130 135 140
Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala
145 150 155 160
Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro
165 170 175
Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu
180 185 190
Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu
195 200 205
Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly
210 215 220
Gln Val Tyr Phe Gly Ile Ile Ala Leu
225 230