METHOD FOR REDUCING THE IMMUNE RESPONSE TO A BIOLOGICALLY ACTIVE PROTEIN
20220169712 · 2022-06-02
Inventors
Cpc classification
C07K14/755
CHEMISTRY; METALLURGY
A61K47/64
HUMAN NECESSITIES
C07K14/535
CHEMISTRY; METALLURGY
International classification
A61K47/64
HUMAN NECESSITIES
C07K14/535
CHEMISTRY; METALLURGY
Abstract
A new use of a molecule comprising at least one moiety which is a biologically active protein and at least one moiety capable of binding to a serum albumin of a mammal is provided, for preparation of a medicament which elicits no or a reduced immune response upon administration to the mammal, as compared to the immune response elicited upon administration to the mammal of the biologically active protein per se. Also provided is a method of reducing or eliminating the immune response elicited upon administration of a biologically active protein to a human or non-human mammal, which comprises coupling the polypeptide to at least one moiety capable of binding to a serum albumin of the mammal.
Claims
1. A method of reducing the immune response elicited upon administration of a biologically active protein to a human subject, comprising: coupling a biologically active protein to at least one moiety capable of binding to human serum albumin to form a molecule which has a binding affinity for human serum albumin such that the K.sub.D of the interaction is less than or equal to 10.sup.−7 M, wherein the moiety capable of binding to human serum albumin comprises an albumin binding domain of a surface protein from a Gram, bacterium, or a fragment or derivative thereof which at least partially retains the capability of binding albumin; is capable of interacting with at least one of residues Phe-228, Ala-229, Ala-322, Val-325, Phe-326 and Met-329 in human serum albumin; and comprises amino acid residues that contribute to the hydrophobic core of the interaction surface between human serum albumin and the moiety or contribute to the surrounding hydrogen bond interactions, and correspond to residues Phe-27, Ala-31, Leu-44 and Ile-48 in the albumin binding GA module of protein PAB from Finegoldia magna, and wherein said molecule elicits a reduced immune response upon administration to said subject as compared to the immune response elicited upon administration to the subject of the biologically active protein that is not coupled to the moiety capable of binding to human serum albumin.
2. The method according to claim 1, wherein the molecule has a binding affinity for human serum albumin of less than or equal to 10.sup.−9 M.
3. The method according to claim 2, wherein the molecule has a binding affinity for human serum albumin of less than or equal to 10.sup.−12 M.
4. The method according to claim 1, wherein the immune response is a humoral immune response.
5. The method according to claim 1, wherein the biological activity of the biologically active protein comprises an ability to interact with a target molecule other than a serum albumin.
6. The method according to claim 5, wherein the biological activity of the biologically active protein comprises an ability to block the activity of the target molecule.
7. The method according to claim 5, wherein the target molecule is present on the surface of a cell.
8. The method according to claim 7, wherein the cell is a cancerous or precancerous cell.
9. The method according to claim 7, wherein the target molecule present on the surface of the cell is selected from HER2, CD4, CD20, CD22, CD74, CEA and EpCAM.
10. The method according to claim 5, wherein the target molecule is an enzyme.
11. The method according to claim 5, wherein the target molecule is selected from hormone receptors and cytokine receptors.
12. The method according to claim 1, wherein the biologically active protein is selected from antibodies, staphylococcal protein A, fibronectin, lipocalin, transferrin, and lectin.
13. The method according to claim 1, wherein the biological activity of the biologically active protein comprises an enzymatic activity.
14. The method according to claim 1, wherein the biological activity of the biologically active protein comprises a hormone activity.
15. The method according to claim 1, wherein the biological activity of the biologically active protein comprises a pharmaceutical activity.
16. The method according to claim 1, wherein the biologically active protein is selected from growth hormone, ciliary neurotrophic factor, granulocyte-macrophage colony stimulating factor, insulin, interferon β, factor VIII, erythropoietin, GLP1 and thrombopoietin.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLE 1
[0096] Humoral Immune Response in Mice Following Administration of Various Molecules
[0097] Molecules Studied
[0098] In this Example, the inventive concept was studied through a comparison of the antibody response in mice upon administration of different molecules. The molecules administered were the following:
[0099] His.sub.6Z.sub.Taq4:1—a variant of protein Z, in turn derived from the B domain of staphylococcal protein A. This Z variant was produced using recombinant DNA technology, through the expression of the DNA sequence encoding it, and simultaneously provided with a hexahistidyl tag according to known molecular biology procedures. Z variant Z.sub.Taq4:1 has previously been selected on the basis of its affinity for Taq DNA polymerase. A description of the Z.sub.Taq4:1 molecule, including its amino acid sequence and the procedure for selection thereof, is given in Gunneriusson E et al, Protein Eng 12: 10, 873-878 (1999) (see e.g.
[0100] Z.sub.Taq4:1-ABD—a fusion protein between the Z variant Z.sub.Taq4:1 and the 46 amino acid albumin binding domain (ABD) of streptococcal protein G strain G148 (Kraulis P J et al, FEBS Lett 378:190 (1996))). The fusion protein was prepared through expression of the corresponding DNA sequence in accordance with known molecular biology procedures. Used to illustrate the invention.
[0101] Streptokinase—a commercially available bacterial enzyme known to induce strong antibody responses. Purchased from Sigma (cat no S-8026, lot 092K1515) and used as a positive control.
[0102] Materials and Methods
[0103] Mice and administration schedule: Female NMRI mice (20 mice, plus 2 as a reserve) were used in this experiment. Body weight upon arrival was 20 g. At the start of the immunization experiments, the mice were from 8 to 12 weeks old. The mice were kept and fed in accordance with guidelines from the Swedish Ministry of Agriculture, Food and Fisheries. Food and water were given ad libitum. For the immunization experiment, the mice were split into five groups with four animals in each group. 20 μg of the molecules indicated in Table 1 were administered subcutaneously to each mouse in 0.1 ml NaCl, 0.9%.
TABLE-US-00001 TABLE 1 Mouse groups and administered molecules Group Mouse # Molecule 1 1-4 His.sub.6-Z.sub.Taq4:1 2 5-8 Z.sub.Taq4:1-ABD 3 17-20 Streptokinase
[0104] The solutions of test molecules were kept frozen at −20° C. and thawed before injection. Repeated injections were given at Day 0, 3, 6, 9, 12 and 21 of the study. Blood samples of 150 μl were taken from the orbital plexus of the mice at Day 0 (preplasma), 7, 14 and 21 of the study. At Day 34 of the study, the mice were sacrificed and the maximal amount of blood obtained. Blood was collected in K.sup.+EDTA tubes, and left to stand for one hour after sampling. Thereafter, samples were centrifuged at 6000 rpm for 6 min in order to separate plasma. Plasma was frozen at −20° C. for storage before analysis.
[0105] Analysis of plasma samples by specific ELISA: For analysis of plasma from mice receiving one of the molecules above, ELISA-plates (Costar, no 9018) were coated with the corresponding molecule diluted in coating buffer (15 mM Na.sub.2HCO.sub.3, 35 mM Na—HCO.sub.3, pH 9.6) to a final concentration of 1 μg/ml. 100 μl of the coating solution was added per well and plates were incubated for 1-3 nights at 4° C. The plates were then washed manually 3 times with deionized water and blocked with 200 μl/well, using PBS (2.68 mM KCl, 1.47 mM KH.sub.2PO.sub.4, 137 mM NaCl, 8.1 mM Na.sub.2HPO.sub.4, pH 7.4) with either 1% bovine serum albumin (BSA; from Sigma, cat no A-2153) or 2% dry milk (Semper A B, Stockholm, Sweden), for 1 to 2 hours. The blocking solution was then removed and 100 μl plasma was added to each well, diluted from 1:100 in blocking solution and then in a series of 2-fold dilutions. After 2 hours of incubation, plates were washed manually 3 times with PBS-T (PBS with 0.05% Tween 20; Tween 20 from Acros Organics, cat no 233362500). Thereafter, 100 μl of a secondary antibody, HRP-conjugated goat anti-mouse IgG (Southern Biotech no 1031-05), diluted 1:2000 in blocking solution, was added to each well. Plates were incubated for 1 hour. The three steps (blocking, addition of plasma sample, addition of secondary antibody) were performed on a shaker, and the last step in the dark. The plates were washed manually 5 times with PBS-T. Subsequently, 100 μl of a substrate solution (ImmunoPure® TMB; Pierce, cat no 34021) was added to each well and plates were subsequently incubated in the dark. Color development was stopped after 15 minutes by addition of 100 μl stop solution (2 M H.sub.2SO.sub.4; VWR, cat no 14374-1). Plates were read at 450 nm in an ELISA spectrophotometer (Basic Sunrise, Tecan).
[0106] Also included was a standard comprising a pool of mouse plasma previously obtained, containing 210 μg/ml of anti-His.sub.6-Z.sub.Taq4:1 IgG. This pool was used in 2-fold dilution series. The limit of detection in ELISA plates coated with His.sub.6-Z.sub.Taq4:1 was approximately 5 ng/ml.
[0107] Analysis of total IgG content in plasma samples: For determination of the total amount of IgG in the samples, quantitative ELISA analyses were performed using a Mouse IgG ELISA Quantitation kit (Bethyl, cat no E90-131), according to the manufacturer's instructions. In brief, ELISA plates were coated with anti-mouse IgG provided by the manufacturer. The plasma to be investigated and the standard plasma were added in 2-fold dilution series beginning with 2000 ng/ml. The detection antibody was diluted 1:100000 (anti-mouse IgG, HRP conjugate; provided by the manufacturer) in blocking buffer. The ELISA analyses were developed using ImmunoPure® TMB as described above.
[0108] Purification of IgG: IgG was purified from a pool of Day 34 plasma obtained from mice injected with Z.sub.Taq4:1-ABD. The pool was diluted 12.5 times with PBS-T before loading on a HiTrap column provided with an IgG-specific affinity ligand derived from staphylococcal protein A. The column had been previously equilibrated with PBS-T. The column was washed until absorbance values reached zero, and bound IgG was eluted using elution buffer (0.2 M glycine, 1 mM EGTA, pH 2.8). For neutralization, Tris base to a concentration of 50 mM and 1/10 volume of 10×PBS were added.
[0109] Data analysis: ELISA values were obtained using the Magellan software from Tecan. The values were exported to Microsoft Excel for analysis. IgG concentrations were calculated by comparison with a standard curve using the Xlfit.3 program (www.idbs.com). The values/curves obtained with the standard pool of anti-His.sub.6-Z.sub.Taq4:1 IgG and the Bethyl standard plasma were used for determination of specific and total IgG, respectively.
[0110] Results
[0111] His.sub.6Z.sub.Taq4:1: Mice #1-#4 were injected with His.sub.6-Z.sub.Taq4:1. Plasma samples obtained before immunization (preplasma), and after 7, 14, 21 and 34 days were analyzed for presence of His.sub.6-Z.sub.Taq4:1 specific antibodies, as described in the method section. ELISA plates were blocked with 1% BSA. There were no antibodies against the injected molecule in preplasma. All four mice responded by generating a moderate antibody response against the injected protein. The titre of specific antibodies rose steadily during the treatment, with the highest titre at Day 34.
[0112] Total IgG concentration was determined using the Bethyl-ELISA, and the concentration of IgG specific for His.sub.6-Z.sub.Taq4:1 was determined using the standard pool as described above. The results are presented in Table 2. The concentration of specific antibodies increased 50-500 times during the 34 days of treatment. Taking the total IgG concentration into account, the specific antibodies constituted about 1-4% of the total amount of IgG.
TABLE-US-00002 TABLE 2 Total and specific IgG in plasma following administration of His.sub.6-Z.sub.Taq4:1 Total IgG Specific IgG (μg/ml) (μg/ml) Day: Mouse # Molecule 0 34 0 7 14 21 34 1 His.sub.6-Z.sub.Taq4:1 2100 550 ND* ND* ND* 3.4 5.2 2 His.sub.6-Z.sub.Taq4:1 320 1200 ND* ND* 13 21 29 3 His.sub.6-Z.sub.Taq4:1 350 900 ND* ND* 1.8 13 37 4 His.sub.6-Z.sub.Taq4:1 810 740 ND* ND* 6.9 12 17 *ND—Not detectable (below detection limit)
[0113] Z.sub.Taq4:1-ABD: Mice #5-#8 were injected with Z.sub.Taq4:1-ABD. Plasma samples obtained before immunization (preplasma, data not shown), and after 7, 14, 21 and 34 days were analyzed for presence of antibodies specific for Z.sub.Taq4:1-ABD, as described in the method section. ELISA plates were blocked with 1% BSA.
[0114] The risk that mouse serum albumin interacted with the ABD moiety of the protein coated in the ELISA wells, and thus sterically hindered mouse antibodies from specific binding to the Z.sub.Taq4:1-ABD molecules, was tested. In the first approach, ABD was avoided by coating with His.sub.6-Z.sub.Taq4:1. Plasma from day 34 were tested, and results showed no specific interaction between mouse antibodies and His.sub.6-Z.sub.Taq4:1 (data not shown). Another approach was to remove albumin from the plasma. Total IgG was therefore purified from a pool of Day 34 plasma from mice #5-#8 using a HiTrap column as described in the “Materials and methods” section. The IgG fraction was tested on plates coated with ABD or His.sub.6-Z.sub.Taq4:1, and the results are shown in
[0115] Total IgG concentration was determined using the Bethyl-ELISA as described above, and the results are presented in Table 3.
TABLE-US-00003 TABLE 3 Total IgG in plasma following administration of Z.sub.Taq4:1-ABD Total IgG (μg/ml) Day: 0 34 Mouse # Molecule 5 Z.sub.Taq4:1-ABD 270 320 6 Z.sub.Taq4:1-ABD 280 370 7 Z.sub.Taq4:1-ABD 1000 950 8 Z.sub.Taq4:1-ABD 1400 1200
[0116] Streptokinase: Mice #17-#20 were injected with streptokinase, a bacterial enzyme that prevents clotting of blood and is known to induce strong antibody responses. Plasma samples obtained before injection (preplasma, not shown), and after 7, 14, 21 and 34 days were analyzed for the presence of streptokinase-specific antibodies, as described in the method-section. The ELISA plates were blocked with 2% dry milk.
[0117] Total IgG concentration was determined using the Bethyl-ELISA as described above, and the results are presented in Table 4. The total IgG concentration was in all but one case considerably higher at death bleeding than in preplasma, implying a normal maturation of the immune system and/or an ongoing immune response.
TABLE-US-00004 TABLE 4 Total IgG in plasma following administration of streptokinase Total IgG (μg/ml) Day: 0 34 Mouse # Molecule 17 Streptokinase 1500 1100 18 Streptokinase 1200 4800 19 Streptokinase 3000 6300 20 Streptokinase 280 4100
[0118] Endpoint titres: The concentration of antibodies specific for the administered molecule was determined only in plasma from mice injected with His.sub.6-Z.sub.Taq4:1, due to the lack of suitable standards for the other proteins. For this reason, endpoint titres were used for comparisons between different injected proteins. The endpoint titre was defined as the dilution where the OD value equaled two times the background, the background being the OD value obtained in preplasma from the same mouse. Table 5 shows the endpoint titres for each mouse. To summarize the results, the endpoint titres after immunization with streptokinase were considerably higher than those obtained with different Z variants. Of particular relevance to the present invention was that antibody titres after immunization with Z.sub.Taq4:1-ABD were not detectable.
TABLE-US-00005 TABLE 5 Endpoint titres of antibodies following administration of various molecules to mice His.sub.6-Z.sub.Taq4:1 Z.sub.Taq4:1-ABD Streptokinase Mouse Endpoint Mouse Endpoint Mouse Endpoint # titre # titre # titre 1 2,700 5 No value 17 78 000 2 14 000 6 No value 18 Value out of range 3 16 000 7 No value 19 110 000 4 8,000 8 No value 20 730 000
DISCUSSION
[0119] To study the effect of administration of different molecules on initiation of specific B-cell activation and antibody generation in an animal model, NMRI mice were injected subcutaneously with different molecules. In order to simulate a normal treatment cycle, six injections of adequate doses without adjuvant were administered to the mice (three days apart, with a booster injection at Day 21). Plasma from treated animals were analyzed for the presence of antibodies of IgG isotype with specificity for the injected molecules. In addition, levels of total IgG content of all animals were determined in preplasma and on the day of death bleeding (Day 34).
[0120] As shown in the “Results” section above, His.sub.6-Z.sub.Taq4:1 induced specific IgG responses in the studied animals. The antibody responses were low to moderate (5 to 37 μg/ml at Day 34), with the most apparent increase after booster injection at Day 21 (2-20 times increase). The responses peaked at Day 34.
[0121] Importantly, plasma from animals injected with Z.sub.Taq4:1-ABD did not show any specific binding to the injected molecules as determined by ELISA. This result was in great contrast to the specific IgG titres observed when analyzing His.sub.6-Z.sub.Taq4:1, i e the same Z sequence fused to His.sub.6 instead of ABD. All five time-points analyzed were negative for target-specific mouse IgG antibodies. The Limit of Detection (LOD) in the ELISA was about 5 ng/ml.
[0122] Streptokinase, a bacterial protein considered to be a strong immunogen, was used as positive control in the studies. As expected, high specific IgG responses were observed in animals injected with streptokinase.
[0123] In conclusion, the injected proteins can be ranked in the following manner, according to their ability to evoke specific IgG responses: streptokinase>>His.sub.6-Z.sub.Taq4:1>>Z.sub.Taq4:1-ABD. The most interesting and relevant result was that the fusion of ABD to Z.sub.Taq4:1 resulted in a specific IgG response that could not be detected.
EXAMPLE 2
[0124] Humoral Immune Response in Mice Following Administration of Different Molecules at Various Frequencies
[0125] Molecules Studied
[0126] In this Example, the inventive concept was again studied through a comparison of the antibody response in mice upon administration of different molecules. The molecules administered were the following:
[0127] His.sub.6-Z.sub.Taq4:5—a variant of protein Z, in turn derived from the B domain of staphylococcal protein A. The Z.sub.Taq4:5 variant was produced using recombinant DNA technology, through expression of the DNA sequence encoding it, and simultaneously provided with a hexahistidyl tag according to known molecular biology procedures. Z.sub.Taq4:5, including its selection and amino acid sequence, is described in Gunneriusson E et al, supra, where it is denoted Z.sub.TaqS1-1. Used for comparative purposes.
[0128] Z.sub.Taq4:1-ABD—as described in Example 1. Used to illustrate the invention.
[0129] ABD—the albumin binding domain (ABD) of streptococcal protein G strain G148 (see above for references). Prepared through expression of the corresponding DNA sequence in accordance with known molecular biology procedures. Used for comparative purposes.
[0130] Materials and Methods
[0131] Mice and administration schedule: Female NMRI mice (40 mice, plus 2 as a reserve) were used in this experiment. Body weight upon arrival was 20 g. At the start of the immunization experiments, the mice were from 8 to 12 weeks old. The mice were kept and fed in accordance with guidelines from the Swedish Ministry of Agriculture, Food and Fisheries. Food and water were given ad libitum. For the immunization experiment, the mice were split into seven groups according to Table 6. 20 μg of the molecules indicated in Table 6 were administered subcutaneously to each mouse in 0.1 ml NaCl, 0.9%.
TABLE-US-00006 TABLE 6 Mouse groups and administered molecules Group Mouse # Molecule 1 53-60 His.sub.6-Z.sub.Taq4:5 2 61-68 Z.sub.Taq4:1-ABD 3 69-76 ABD 4 82-85 His.sub.6-Z.sub.Taq4:5 5 86-89 Z.sub.Taq4:1-ABD 6 90-93 ABD
[0132] The solutions of test molecules were kept frozen at −20° C. and thawed before injection. The mice of groups 1-3 received subcutaneous injections at Day 0, 7 and 21 of the study (scheme 1, low frequency). The mice of groups 4-6 received subcutaneous injections at Day 0, 1 and 21 of the study (scheme 2, high frequency). Blood samples of 150 μl were taken from the orbital plexus of the mice at Day 0 (preplasma), 7, 14 and 21 of the study. At Day 34 of the study, the mice were sacrificed and the maximal amount of blood obtained. Blood was collected in K.sup.+EDTA tubes, and left to stand for one hour after sampling. Thereafter, samples were centrifuged at 6000 rpm for 6 min in order to separate plasma. Plasma was frozen at −20° C. for storage before analysis.
[0133] Analysis of plasma samples by specific ELISA: For analysis of plasma from mice receiving one of the molecules above, ELISA-plates (Costar, no 9018) were coated with the corresponding molecule diluted in coating buffer (15 mM Na.sub.2HCO.sub.3, 35 mM Na—HCO.sub.3, pH 9.6) to a final concentration of 1 μg/ml. 100 μl of the coating solution was added per well and plates were incubated for 1-3 nights at 4° C. The plates were then washed manually 3 times with deionized water and blocked with blocking buffer (200 μl/well; PBS (2.68 mM KCl, 1.47 mM KH.sub.2PO.sub.4, 137 mM NaCl, 8.1 mM Na.sub.2HPO.sub.4, pH 7.4) with 0.5% casein (Sigma, cat no C-8654), for 1 to 2 hours. The blocking buffer was then removed and 100 μl plasma was added to each well, diluted from 1:100 in blocking solution and then in a series of 2-fold dilutions. Also included was a standard comprising a pool of mouse plasma previously obtained, containing 210 μg/ml of anti-His.sub.6-Z.sub.Taq4:1 IgG. This pool was used in 2-fold dilution series. The limit of detection in ELISA plates coated with His.sub.6-Z.sub.Taq4:1 was approximately 5 ng/ml.
[0134] After 2 hours of incubation, plates were washed manually 3 times with PBS-T (PBS with 0.05% Tween 20). Thereafter, 100 μl of a secondary antibody, HRP-conjugated goat anti-mouse IgG (Southern Biotech no 1031-05), diluted 1:2000 in blocking buffer, was added to each well. Plates were incubated for 1 hour. All steps were performed on a shaker, and the last step in the dark. The plates were washed manually five times with PBS-T. Subsequently, 100 μl of a substrate solution (ImmunoPure® TMB; Pierce, cat no 34021) was added to each well and plates were subsequently incubated in the dark. Color development was stopped after 15 minutes by addition of 100 μl stop solution (2 M H.sub.2SO.sub.4; VWR, cat no 14374-1). Plates were read at 450 nm in an ELISA spectrophotometer (Basic Sunrise, Tecan) using Magellan software.
[0135] Analysis of total IgG content in plasma samples: For determination of the total amount of IgG in the samples, quantitative ELISA analyses were performed using a Mouse IgG ELISA Quantitation kit (Bethyl, cat no E90-131), according to the manufacturer's instructions. In brief, ELISA plates were coated with anti-mouse IgG (1 μg/ml) provided by the manufacturer. The plasma to be investigated and the standard plasma were added in 2-fold dilution series. The standard plasma was diluted from 2000 ng/ml. The detection antibody was used at a dilution of 1:100000 (anti-mouse IgG, HRP conjugate; provided by the manufacturer) in blocking buffer. The ELISA analyses were developed using ImmunoPure® TMB as described above.
[0136] Data analysis: ELISA values were obtained using the Magellan software from Tecan. The values were exported to Microsoft Excel for analysis. IgG concentrations were calculated by comparison with a standard curve using the Xlfit.3 program (www.idbs.com). The values/curves obtained with the standard pool of anti-His.sub.6-Z.sub.Taq4:1 IgG and the Bethyl standard plasma were used for determination of specific and total IgG, respectively.
[0137] Results
[0138] His.sub.6Z.sub.Taq4:5 Eight mice (#53-#60) were injected with His.sub.6-Z.sub.Taq4:5 at 20 μg/mouse and injection, according to scheme 1 (see above). Plasma samples obtained before (preplasma), during (Day 7, 14, 21) and after (Day 34) injections were analyzed for presence of His.sub.6-Z.sub.Taq4:5 specific antibodies as described above. The results are shown in
[0139] Four mice (#82-#85) were injected with His.sub.6-Z.sub.Taq4:5 according to scheme 2 (see above). Plasma samples obtained before (preplasma), during (Day 7, 14, 21) and after (Day 34) injection were analyzed for presence of His.sub.6-Z.sub.Taq4:5 specific antibodies as described above. The results are shown in
[0140] The concentration of specific IgG was determined using the standard pool of anti-His.sub.6-Z.sub.Taq4:1 IgG (see method section). This pool has previously been shown to contain 210 μg/ml of His.sub.6-Z.sub.Taq4:1 specific antibodies. Concentrations were calculated using the XLfit program and the one-site, dose-response formula. Both samples and standard were tested as singles, and the standard variation of the method is therefore unknown. In addition, the mathematical formula chosen for calculation also affects the values, and the variation depending on the formula has not been calculated. Thus, the concentrations given in the table below should be considered as relative, rather than absolute, values. Table 7 shows the concentration of His.sub.6-Z.sub.Taq4:5 specific antibodies in plasma from individual mice. As shown in the titration analysis above, the concentration of specific IgG at Day 34 varied considerably between individual mice in both groups. The seemingly higher concentration in the scheme 1 group was not statistically significant when tested with Student's T test (TTEST function, Microsoft Excel).
[0141] Total IgG concentrations were also determined in Day 34 plasma samples from the His.sub.6-Z.sub.Taq4:5 injected mice, using quantitative ELISA as described above. The results are presented in Table 7 (column 7).
TABLE-US-00007 TABLE 7 Total and specific IgG in plasma following administration of His.sub.6-Z.sub.Taq4:5 Total % specific IgG IgG of Specific IgG (μg/m1) (μg/m1) total IgG Day Day Day Day Day Day Scheme Mouse # 7 14 21 34 34 34 1 53 ND* 20 20 260 1100 26 54 ND ND ND 20 1500 1 55 ND ND 20 160 1500 9 56 ND 30 30 90 740 9 57 ND 50 50 10 1500 1 58 ND ND ND 1800 2000 80 59 ND ND ND 6 680 1 60 ND ND ND 60 510 12 82 ND ND ND 100 3430 3 2 83 ND ND ND ND 3430 0.01 84 ND ND ND 10 2483 0.4 85 ND ND ND 20 6400 0.3 *ND—Not detectable (below detection limit)
[0142] Z.sub.Taq4:1-ABD: Eight mice (#61-#68) were injected with Z.sub.Taq4:1-ABD using scheme 1. Plasma samples obtained before injection (preplasma, data not shown) and after 7, 14, 21 and 34 days were analyzed for presence of Z.sub.Taq4:1-ABD specific antibodies as described above.
[0143] There are high levels of mouse serum albumin (MSA) present in the analyzed samples. To circumvent the problem with MSA that might bind to the Z.sub.Taq4:1-ABD coated ELISA surface, plasma samples were also titrated on plates coated with His.sub.6-Z.sub.Taq4:1 (data not shown). The analyses were once again negative, confirming the observation that the Z.sub.Taq4:1-ABD molecule is non-immunogenic.
[0144] Four mice (#86-#89) were injected with Z.sub.Taq4:1-ABD according to scheme 2. Plasma samples obtained before injection (preplasma, data not shown) and after 7, 14, 21 and 34 days were analyzed for presence of Z.sub.Taq4:1-ABD specific antibodies as described above. The results are shown in
[0145] Total IgG concentration was determined using the Bethyl-ELISA as described above, and the results are presented in Table 8.
TABLE-US-00008 TABLE 8 Total IgG in plasma following administration of Z.sub.Taq4:1-ABD Scheme Mouse # Total IgG (μg/ml) at Day 34 1 61 500 62 1300 63 2600 64 1800 65 1700 66 1100 67 830 68 380 2 86 2500 87 870 88 2900 89 11100* *Unreliable, since value out of range
[0146] ABD: Eight mice (#69-#76) were injected with ABD according to scheme 1. Plasma obtained before injection (preplasma) and after 7, 14, 21 and 34 days was analyzed for presence of ABD-specific antibodies as described above. Very low titres of antibodies against the ABD molecule were detected, and only in Day 34 plasma (
[0147] Four mice (#90-#93) were injected with ABD according to scheme 2. Plasma obtained before injection (preplasma) and after 7, 14, 21 and 34 days was analyzed for presence of ABD-specific antibodies as described above. No antibody response could be measured (
[0148] Total IgG concentration was determined using the Bethyl-ELISA as described above, and the results are presented in Table 9.
TABLE-US-00009 TABLE 9 Total IgG in plasma following administration of ABD Total IgG (μg/ml) Scheme Mouse # at Day 34 1 69 750 70 400 71 940 72 410 73 390 74 100 75 1100 76 304 2 90 3200 91 5100 92 6500 93 5800
DISCUSSION
[0149] In accordance with the results of Example 1, Z.sub.Taq4:1-ABD was unable to induce specific IgG responses in the mice. Likewise, the administration of ABD itself, devoid of fusion partner, did not elicit any measurable specific IgG response. The animals in these groups were administered the same amount of protein as in Example 1. Neither low frequency or high frequency injections generated detectable IgG responses. Since it is believed that the Z protein and its derivatives are T-dependent antigens, they would be expected to generate specific antibodies predominantly of the IgG isotype. Therefore, since these results show that no IgG antibodies are formed, they imply that no antibodies of other isotypes are formed either, in response to the Z.sub.Taq4:1-ABD fusion protein.
EXAMPLE 3
[0150] Humoral Immune Response in Mice Following Administration of Multimers of a Z Variant Provided with an Albumin Binding Moiety
[0151] Molecules Studied
[0152] As in Examples 1 and 2, the inventive concept was again studied through a comparison of the antibody response in mice upon administration of different molecules. The molecules administered were the following:
[0153] His.sub.6-Z.sub.Taq4:5—as described in Example 2. Used for comparative purposes.
[0154] ABD-Z.sub.her2:4—a fusion protein between the albumin binding domain (ABD) of streptococcal protein G strain G148 (see above) and the Z variant Z.sub.her2:4. Z.sub.her2:4 was selected from a library of combinatorial variants of Z and characterized. In the selection procedure, purified protein corresponding to the cancer antigen HER2 (also described in the literature as neu, HER2/neu or c-erbB-2) was used as the target molecule. Z.sub.her2:4 was found to interact with HER2 with a K.sub.D value of approximately 50 nM. The amino acid sequence of Z.sub.her2:4, in standard one-letter code, is:
TABLE-US-00010 (SEQ ID NO: 4) VDNKFNKELR QAYWEIQALP NLNWTQSRAF IRSLYDDPSQ SANLLAEAKK LNDAQAPK
[0155] The fusion protein was prepared through expression of the corresponding DNA sequence together with the DNA sequence encoding the ABD moiety, in accordance with known molecular biology procedures. The ABD-Z.sub.her2:4 fusion protein is used to illustrate the invention.
[0156] ABD-(Z.sub.her2:4).sub.2—a fusion protein between the albumin binding domain (ABD) and a dimer of the Z variant Z.sub.her2:4, prepared in accordance with known molecular biology procedures with the added knowledge of Z.sub.her2:4 sequence information. Used to illustrate the invention.
[0157] ABD-(Z.sub.her2:4).sub.3—a fusion protein between the albumin binding domain (ABD) and a trimer of the Z variant Z.sub.her2:4, prepared in accordance with known molecular biology procedures with the added knowledge of Z.sub.her2:4 sequence information. Used to illustrate the invention.
[0158] ABD-(Z.sub.her2:4).sub.4—a fusion protein between the albumin binding domain (ABD) and a tetramer of the Z variant Z.sub.her2:4, prepared in accordance with known molecular biology procedures with the added knowledge of Z.sub.her2:4 sequence information. Used to illustrate the invention.
[0159] Materials and Methods
[0160] Mice and administration schedule: Female NMRI mice (30 mice, plus 2 as a reserve) were used in this experiment. Body weight upon arrival was 20 g. At the start of the immunization experiments, the mice were from 8 to 12 weeks old. The mice were kept and fed in accordance with guidelines from the Swedish Ministry of Agriculture, Food and Fisheries. Food and water were given ad libitum. For the immunization experiment, the mice were split into five groups according to Table 10. 20 μg of the molecules indicated in Table 10 were administered subcutaneously to each mouse in 0.1 ml NaCl, 0.9%.
TABLE-US-00011 TABLE 10 Mouse groups and administered molecules Group Mouse # Molecule 1 114-119 His.sub.6-Z.sub.Taq4:5 2 120-125 ABD-Z.sub.her2:4 3 126-131 ABD-(Z.sub.her2:4).sub.2 4 132-137 ABD-(Z.sub.her2:4).sub.3 5 138-143 ABD-(Z.sub.her2:4).sub.4
[0161] The solutions of test molecules were kept frozen at −20° C. and thawed before injection. The mice of group 1 received subcutaneous injections at Day 0, 3, 6, 9, 12 and 63 of the study (scheme 1). Blood samples of 150 μl were taken from the orbital plexus of the mice of group 1 at Day 0 (preplasma), 7, 14, 21, 34, 49 and 63 of the study. At Day 73 of the study, these mice were sacrificed and the maximal amount of blood obtained. The mice of groups 2-5 received subcutaneous injections at Day 0, 3, 6, 9, 12 and 21 of the study (scheme 2). Blood samples of 150 μl were taken from the orbital plexus of the mice of groups 2-5 at Day 0 (preplasma), 7, 14 and 21 of the study. At Day 34 of the study, these mice were sacrificed and the maximal amount of blood obtained. Blood was collected in K.sup.+EDTA tubes, and left to stand for one hour after sampling. Thereafter, samples were centrifuged at 6000 rpm for 6 min in order to separate plasma. Plasma was frozen at −20° C. for storage before analysis.
[0162] Analysis of plasma samples by specific ELISA: In general, a volume of 100 μl per well was used for all incubation steps except for blocking, where 200 μl were used. ELISA plates (Costar, no 9018) were incubated 1-3 days for coating, 1-2 hours for blocking and plasma, 1 hour for secondary antibody and 15 min for substrate solution. The incubations were performed on a shaker at room temperature, except coating, which was incubated at 4° C. Washing was done between all steps, unless otherwise stated, using the ELISA SkanWasher 300 (Skatron) with 4×350 μl washing buffer (PBS-T, see Example 1) per well. Plates were read at 450 nm in a Tecan ELISA reader using the Magellan v3.11 software. Blocking buffer was used for all dilutions except coating, where coating buffer (15 mM Na.sub.2HCO.sub.3, 35 mM NaHCO.sub.3, pH 9.6) was used instead.
[0163] ELISA-plates were coated with His.sub.6-Z.sub.Taq4:5 or ABD-(Z.sub.her2:4).sub.2, diluted to a concentration of 5 μg/□ml. After coating, plates were blocked with PBS+0,5% casein (PBS (2.68 mM KCl, 1.47 mM KH.sub.2PO.sub.4, 137 mM NaCl, 8.1 mM Na.sub.2HPO.sub.4, pH 7.4) with 0.5% casein (Sigma, cat no C-8654)). Blocking was removed and plasma was added, diluted from 1:100 and then in 3-fold dilution series. Plasma from mice injected with His.sub.6-Z.sub.Taq4:5 was analyzed on ELISA plates coated with His.sub.6-Z.sub.Taq4:5, whereas plasma from mice injected with the four different Z.sub.her2:4 constructs was analyzed on ELISA plates coated with ABD-(Z.sub.her2:4).sub.2. Also included was a standard comprising a pool of mouse antibodies previously obtained. The pool included IgG directed against Z.sub.Taq4:5 and Z.sub.her2:4. HRP conjugated goat anti-mouse IgG (Southern Biotech, cat no 1031-05), diluted 1:2000, was used as the secondary reagent, and the reaction was developed using ImmunoPure® TMB substrate solution (Pierce, cat no 34021). This incubation was performed in the dark. The colour development was stopped after 15 minutes by the addition of stop solution (2 M H.sub.2SO.sub.4; VWR, cat no 14374-1).
[0164] Results
[0165] Plasma samples obtained before immunization (preplasma), and after 7, 14, 21 and 34 days were analyzed for presence of specific antibodies, as described in the “Materials and methods” section. There were no antibodies specific for the injected molecules in any preplasma (data not shown). The diagrams presented in
[0166] His.sub.6Z.sub.Taq4:5. The results are shown in
[0167] ABD-Z.sub.her2:4: The results are shown in
[0168] ABD-Z.sub.her2:4).sub.2: The results are shown in
[0169] ABD-Z.sub.her2:4).sub.3: The results are shown in
[0170] ABD-Z.sub.her2:4).sub.4: The results are shown in
[0171] Also tested was the ability of the four different ABD-(Z.sub.her2:4).sub.n constructs to elicit a specific IgM response. No such responses were detected (data not shown).
DISCUSSION
[0172] The results of this experiment confirm the finding from Examples 1 and 2 that the provision of an albumin-binding moiety reduces or eliminates the immune response to a biologically active protein. Importantly, the effect was shown to be valid for proteins of increasing size. The tetramer of Z.sub.her2:4 comprises more than 230 amino acid residues, but despite its size does not elicit a substantial antibody response upon administration in mice.
EXAMPLE 4
[0173] Reduction of Immune Response Tested with Dimers of Further Biologically Active Molecules
[0174] Molecules Studied
[0175] The aim of this study was also to assess if the antibodies, generated after immunization, are able to inhibit the binding between specific AFFIBODY® molecules and their target protein. The previous observation of a reduction in immune response when a biologically active protein is coupled to an albumin binding domain was confirmed using two other AFFIBODY® molecules: (Z.sub.Aβ3).sub.2 and ABD-(Z.sub.Aβ3).sub.2.
[0176] (Z.sub.Aβ3) 2—a dimer of a variant of protein Z, in turn derived from the B domain of staphylococcal protein A. The Z.sub.Aβ3 variant was produced using recombinant DNA technology, through expression of the DNA sequence encoding it, according to known molecular biology procedures. Used for comparative purposes.
[0177] ABD-(Z.sub.Aβ3).sub.2— a fusion protein between the albumin binding domain (ABD) and a dimer of the Z variant Z.sub.Aβ3 prepared in accordance with known molecular biology procedures.
[0178] Used to illustrate the invention.
[0179] His.sub.6-Z.sub.Taq4:1—the his-tagged variant of protein Z described in Example 1. Used for comparative purposes.
[0180] His.sub.6-Z.sub.Taq4:5—the his-tagged variant of protein Z described in Example 2. Used for comparative purposes.
[0181] Materials and Methods
[0182] Mice were injected with two different dimeric AFFIBODY® molecules, (Z.sub.Aβ3).sub.2 and ABD-(Z.sub.Aβ3).sub.2. Plasma from mice was obtained as before, four times during the treatment scheme and after booster and was analyzed for presence of AFFIBODY®-specific IgG. The results showed that the mice injected with (Z.sub.Aβ3).sub.2, generated a strong IgG response. The antibody production started around day 14 and peaked at day 34. The average concentration of specific IgG at death bleeding was 278 μg/ml (Z.sub.Aβ3).sub.2. No specific antibodies were detected in plasmas from mice treated with ABD-(Z.sub.Aβ3).sub.2.
[0183] Total IgG-concentration was determined in plasma at day 0 and day 34 from both groups of mice. The concentration of total IgG increased approximately 2 times in both groups over the 34 day period.
[0184] An inhibition-ELISA was set up to test if anti-(Z.sub.Aβ3).sub.2-antibodies were able to neutralize the interaction between the AFFIBODY® molecule and the target protein. The results showed that (Z.sub.Aβ3).sub.2-specific IgG did not neutralize the interaction between (Z.sub.Aβ3).sub.2 and □-Amyloid 40.
[0185] Mice and Administration Schedule
[0186] The mice were treated with (Z.sub.Aβ3).sub.2 and (Z.sub.Aβ3).sub.2-ABD according to the scheme (Table 11). The injection and bleeding scheme is shown in Table 12. Mouse 5 in group 1 became sick and was removed from the study after bleeding day 22.
TABLE-US-00012 TABLE 11 μg/ ml/ Mouse animal/ animal/ Animal Group Treatment strain Admin. injection injection number 1 (Z.sub.Aβ3).sub.2 NMRI s.c. 20 0.1 1-6 2 ABD-(Z.sub.Aβ3).sub.2 NMRI s.c. 20 0.1 7-12
TABLE-US-00013 TABLE 12 Day Treatment 0 Preserum (= Sample 1) and Injection 1 3 Injection 2 6 Injection 3 7 Sample 2 (day 7) 9 Injection 4 12 Injection 5 14 Sample 3 (day 14) 21 Sample 4 (day 21), and Injection 6 34 Sample 5 (= death bleeding, day 34)
[0187] Purification of IgG from Mouse Plasma
[0188] Total IgG was purified from pooled plasma day 34 (mouse no 5 day 21) from the mice immunized with (Z.sub.Aβ3).sub.2. The plasma pool, 2400 μl, was diluted 5 times with PBS-T to a total amount of 12 ml before loading on a ZWt-coupled High-Trap column (L0091-98) previously equilibrated with PBS-T. The column was washed until absorbance values reached zero and bound IgG was eluted using an acidic elution buffer (0.2 M Glycine, 1 mM EGTA, pH 2.8). For neutralization, 1M Tris base was added to a final concentration of 50 mM. The buffering capacity was restored by adding 1/10 of the elution volume of 10×PBS (2.68 mM KCl, 1.47 mM KH.sub.2PO.sub.4, 137 mM, NaCl, 8.1 mM Na.sub.2HPO.sub.4, pH 7.4, PBS-Tween (PBS-T), 1×PBS with 0.05% Tween). [0189] ELISA-plates (96 well, flat bottom, high binding Costar No. 9018) were coated with the appropriate AFFIBODY® molecule diluted in coating buffer to a final concentration of 2 μg/ml. 100 μl of the coating solution was added per well and plates were incubated for 1-3 nights at 4° C. The plates were then washed manually four times with deionized water and blocked with blocking buffer (0.5% Casein (Sigma) in 1×PBS; 200 μl/well) for 1 to 2 hours. Blocking buffer was removed, and 100 μl of serum was added to each well in dilution series. After 1 hour incubation, the plates were washed with the automated ELISA-washer or manually four times with PBS-T, and 100 μl of the second step antibody, HRP-conjugated goat anti-mouse IgG diluted 1:2000 in blocking buffer, was added to each well. The plates were subsequently incubated for 1 hour. Plates were washed four times with PBS-T and 100 μl of substrate solution (IMMUNOPURE® TMB) was added to each well followed by incubation in the dark. The colour development was stopped after 15 minutes by the addition of 100 l of stop solution 2M H.sub.2SO.sub.4. Plates were read at 450 nm in an ELISA-reader with the use of the Magellan software.
[0190] Mouse Anti Z.sub.Aβ3-Specific IgG ELISA
[0191] Plates were coated with (Z.sub.Aβ3).sub.2 2 μg/ml in coating buffer and incubated overnight at 4° C. After washing, plates were blocked as described above. Plasma from mice immunized with (Z.sub.Aβ3).sub.2 or with ADB-(Z.sub.Aβ3).sub.2 was added in 3-fold dilution series starting from 1/100. After incubation, plates were treated as described above. To measure the concentration of AFFIBODY®-specific IgG, a standard plasma pool was used, the Scheele 8 pool. The concentration of anti-Z IgG in this serum pool had been determined on His.sub.6-Z.sub.Taq4:1-coated plates to 97 μg/ml. The concentration of anti-His.sub.6-Z.sub.Taq4:5 and anti-(Z.sub.Aβ3).sub.2 IgG in Scheele 8 pool was determined to be 291 and 97 μg/ml respectively (L0242-14/16). As a positive control we used plasma from mouse #117 (Scheele 7; day 73, in a dilution of 1:10.000, which should give an OD value close to the inflection point of the standard curve. Negative control was blocking buffer. The limit of detection was 3 ng/ml.
[0192] Total IgG ELISA
[0193] Plasmas from day 0 and day 34 (mouse no 5 day 21) from both groups of mice were analyzed regarding the total amount of IgG. In this assay, ELISA-plates were coated with (Fab).sub.2 fragments of goat anti-mouse IgG-Fc antibodies (Southern Biotech no. 1031-05 0.5 μg/ml). Plasma or standard (mouse IgG) in 3-fold dilution series starting from 1:10000 and 100 ng/ml respectively was added to coated wells. The reaction was developed with HRP-conjugated (Fab).sub.2 fragments of goat anti-mouse IgG-fab antibodies (0.2 μg/ml). Substrate solution (ImmunoPure® TMB Pierce no. 34021) was added and the colour development was stopped after 10 minutes incubation in dark by the addition of stop solution.
[0194] Inhibition ELISA Analysis
[0195] Plates were coated with (Z.sub.Aβ3).sub.2, 2 μg/ml in coating buffer and incubated overnight in 4° C. After washing plates were blocked as described above. Purified anti-(Z.sub.Aβ3).sub.2 IgG was added in 3-fold dilution series starting from 1p g/ml. After 1 h incubation the protein β-Amyloid 40, biotinylated, was added to the wells without washing in between. The final concentration of β-Amyloid 40 BioSite A2275-74D was 10 μg/ml. The plates were washed after one additional hour of incubation and depending on what reaction to analyze, either goat-anti-mouse IgG HRP (Dako P0397 diluted 1/2000) or streptavidin-HRP (diluted 1/5000) was added. After the final incubation, plates were washed and developed as described above.
[0196] Data Analysis
[0197] Magellan (Tecan) was used as ELISA reader software. The results were exported to Excel for data analysis and presentation. For concentration determinations, the XLfit program, formula “Dose response-one site”, equation 205, was used. Processed data from each day and type of measurement i.e. IgG, concentration etc, were represented by an Excel file.
[0198] Results
[0199] AFFIBODY®-Specific IgG ELISA Group 1
[0200] Plasmas from six mice injected with (Z.sub.AB3).sub.2 was titrated on (Z.sub.Aβ3).sub.2-coated plates. All mice responded by day 14, although mouse 5 showed a very low response. The maximal anti-AFFIBODY®-IgG level was observed at day 34 (
TABLE-US-00014 TABLE 14 Concentration of specific IgG Concentration of specific IgG in μg/ml Group 1 day 7 day 14 day 21 day 34 mouse 1 ND 3.7 9.8 121.3 mouse 2 ND 13.5 51.3 216.3 mouse 3 ND 19.8 78.2 530.8 mouse 4 ND 3.4 17.2 113.7 mouse 5 ND ND 7.6 — * mouse 6 ND 8.0 71.1 407.9 Average ND 9 39 278 St dev 6 29 165 (ND = Not Detectable) ND: Not detected, values were below the detection limit 3 ng/ml. * Mouse 5 became sick and was removed from the study day 22.
[0201] AFFIBODY®-Specific IgG ELISA Group 2
[0202] Plasma from six mice injected with ADB-(Z.sub.Aβ3).sub.2 was titrated on (Z.sub.Aβ3).sub.2-coated plates. No specific IgG concentration could be detected in any of the bleeding samples. (
[0203] Total IgG ELISA
[0204] Plasma from both groups of mice day 0 and day 34 (mouse no 5 day 21), was titrated on (Z.sub.Aβ:3).sub.2 coated plates. As shown in Table 15 the amount of total IgG increased over the 34 day period in both groups. However, considering that day 0 levels are surprisingly low, the increase may reflect the normal expansion in a mouse over that particular period in life. Plasma from vehicle mice would have been the appropriate control.
TABLE-US-00015 TABLE 15 Total IgG concentrations group 1 Group 1 (Z.sub.Aβ:3).sub.2 Group 2 ABD-(Z.sub.Aβ:3).sub.2 Mouse Day 0/ Day 34/death Day 0/ Day 34/death no preplasma bleeding preplasma bleeding 1 828.9 1724.4 812.9 1850.6 2 984.6 1948.5 931 2108.9 3 1142.1 2673 437.4 1091.7 4 2074.5 3418.2 900.3 1180.1 5 1789.2 3936.6 778.6 1839.3 6 829.8 1263.6 1322.9 2573.1
[0205] Inhibition ELISA: Target Protein
The likelihood that AFFIBODY©-specific IgG antibodies neutralize/inhibit the interaction between the target protein and the AFFIBODY® molecule was explored by an inhibition ELISA. The ELISA-plate was coated with (Z.sub.Aβ3).sub.2 as described above. Total IgG, purified from a pool of plasma from group 1 day 34, was added. The IgG antibodies were added in 3-fold dilutions from 1p g/ml. The antibodies were allowed to bind for one hour to the coated AFFIBODY® molecule before the target protein β-Amyloid 40 was added to a final concentration of 10 μg/ml. The reaction was developed with streptavidin-HRP to visualize the interaction between (Z.sub.Aβ3).sub.2 and the target protein (blue line. The interaction between the IgG-antibodies and the coated AFFIBODY® molecule was visualized with anti-mouse IgG HRP (
[0206] As control the titrated IgG-antibody was allowed to react with the coated AFFIBODY® molecule without adding any target protein.
EXAMPLE 5
[0207] Reduction of Immune Response Tested in Additional Mouse Strain
[0208] As shown in Example 4 we have previously observed that His.sub.6-Z.sub.Taq4:1 but not Z.sub.Taq4:1-ABD induces an antibody response in out bred NMRI mice. In this study, an additional out bred mouse strain (CD1) was tested and the results showed that CD1-mice responded as NMRI mice by producing specific IgG when administered with His.sub.6-Z.sub.Taq4:1 but not with Z.sub.Taq4:1-ABD. Thus, the immune unresponsiveness observed upon injection of ABD-fused AFFIBODY® molecule (Z.sub.Taq4:1) seems to be a general phenomenon in mice.
[0209] The nature of ABD-induced unresponsiveness was further analyzed in NMRI mice. Four groups of mice received ten (10) AFFIBODY® injections with a change of injected molecule after the fifth injection (according to treatment scheme 2). The results showed that mice, primed with Z.sub.Taq4:1-ABD, produced AFFIBODY specific-IgG after antigen-switch to His.sub.6-Z.sub.Taq4:1 (group 4). The antibody production started approximately 14 days after the switch to His.sub.6-Z.sub.Taq4:1, which is the number of days usually necessary for naïve mice to produce antibodies. Mice that were primed with Z.sub.Taq4:1-ABD before receiving a mixture of Z.sub.Taq4:1-ABD and His.sub.6-Z.sub.Taq4:1 (group 5) also started to produce specific IgG against the AFFIBODY® molecule. The observed specific response in group 5 was smaller than seen in group 4 which is most likely due to fact that group 5 received half of the injected dose of His.sub.6-Z.sub.Taq4:1 as compared to group 4. Mice in group 6 were primed with His.sub.6-Z.sub.Taq4:1 before receiving Z.sub.Taq4:1-ABD and this treatment resulted in a continued AFFIBODY®-specific IgG production after the antigen switch although the titre decreased by time.
[0210] This Scheele 9 study was performed mainly for two reasons: 1) does a second out bred mouse strain (CD1) respond to the administration of ABD-fused and unfused AFFIBODY® molecules in the same way as NMRI mice (BT1-PAR07, BT11-PAR03), and 2) when Z.sub.Taq4:1-ABD is injected and followed by His.sub.6-Z.sub.Taq4:1, will the mice generate an AFFIBODY®-specific IgG response or not? The answer to the latter question helped us clarify whether the observed unresponsiveness towards ABD-fused AFFIBODY® molecules is an active or passive suppression process. The molecules used in each group of animals are listed in Table 16 and the two treatment schemes are presented in Tables 17 and 18. The setup of this study is illustrated in
TABLE-US-00016 TABLE 16 Animal number scheme for the Scheele 9 study; AFFIBODY® 1: His.sub.6-Z.sub.Taq4:1; AFFIBODY® 3: Z.sub.Taq4:1-ABD μg/ mL/ Mouse animal/ animal/ Animal Group Treatment strain Admin. injection injection number 1 Affibody 1 CD1 s.c. 20 0.1 144-148 2 Affibody 3 CD1 s.c. 20 0.1 149-153 3 Affibody 1 NMRI s.c. 20 0.1 154-158 4 Affibody 3 + 1 NMRI s.c. 20 0.1 159-163 5 Affibody 3 + 1/3 NMRI s.c. 20 0.1 164-168 6 Affibody 1 + 3 NMRI s.c. 20 0.1 169-173
TABLE-US-00017 TABLE 17 Treatment scheme 1 for group 1 and 2 Day Treatment 0 Preplasma (= Sample 1) 0 Injection 1 3 Injection 2 6 Injection 3 7 Sample 2 9 Injection 4 12 Injection 5 14 Sample 3 21 Sample 4 21 Injection 6 34 Death-bleeding (= Sample 5)
TABLE-US-00018 TABLE 18 Treatment scheme 2 for group 3, 4, 5, and 6 Day Treatment 0 Preplasma (= sample 1) 0 Injection 1 3 Injection 2 6 Injection 3 7 Sample 2 9 Injection 4 12 Injection 5 14 Sample 3 21 Sample 4 From now on In group 4, 5, and 6: change of antigen 21 Injection 6 24 Injection 7 27 Injection 8 28 Sample 5 30 Injection 9 33 Injection 10 35 Sample 6 45 Death-bleeding (= sample 7)
[0211] Materials and Methods
[0212] Affibody® ELISA
[0213] 96 well, flat bottom, high binding Costar ELISA plates were coated with His.sub.6-Z.sub.Taq4:1 diluted in coating buffer (10× coating buffer, 150 mM Na.sub.2CO.sub.3, 350 mM NaHCO.sub.3, pH9.6) to a final concentration of 5 μg/ml. 100 μl of the coating solution was added per well, and plates were incubated for 1-4 nights at 4° C. The plates were then washed manually four times with deionized water and blocked with blocking buffer (200 μl/well) for 1 to 2 hours. Blocking buffer was removed and 100 μl of plasma was added to each well, diluted from 1:100 in blocking buffer, and then in 3-fold dilution series. To measure the concentration of AFFIBODY®-specific IgG, a standard plasma pool was used, the Scheele 6B pool. The concentration of anti His.sub.6-Z.sub.Taq4:1 IgG in this plasma pool had been determined to 120 μg/ml. As a positive control we used plasma from mouse #118 of the Scheele 7 study, day 73, in a dilution of 1:9200, which should give an OD value near the inflection point of the standard curve. Negative control was blocking buffer.
[0214] After 2 hours of incubation, the plates were washed four times with PBS-T (Phosphate buffered saline (10×PBS) 2.68 mM KCl, 1.47 mM KH.sub.2PO.sub.4, 137 mM NaCl, 8.1 mM Na.sub.2HPO.sub.4, pH 7.4; PBS-Tween (PBS-T), 1×PBS with 0.05% Tween) and 100 μl of a second step antibody, HRP-conjugated goat anti-mouse IgG diluted 1:2000 in blocking buffer (PBS-Tween (PBS-T), 1×PBS with 0.05% Tween) was added in each well. The plates were thereafter incubated for 1 hour. Plates were washed four times with PBS-T and 100 μl of the substrate solution (ImmunoPure® TMB) was added to each well. Plates were incubated in the dark and the color development was stopped after 15 minutes by the addition of 100 l of stop solution (2M H.sub.2SO.sub.4). Plates were read at 450 nm in an ELISA-reader with the use of the Magellan software.
[0215] Total IgG ELISA
[0216] For determination of total IgG in plasma, a quantitative ELISA was performed. The procedure was the same as for the AFFIBODY® ELISA, with the following differences: ELISA-plates were coated with AffiniPure (Fab′).sub.2 fragment goat anti-mouse IgG Jackson 115-006-008 (Fcγ-fragment specific), in a concentration of 0.5 μg/ml. Dilutions of the first and second step were done in PBS-T without casein, and the dilution series of plasma began at 1:10.000. Standard IgG was ChromPure mouse IgG Jackson 015-000-003, whole molecule, in a dilution series beginning with 100 ng/ml. Peroxidase-conjugated AffiniPure F(ab′).sub.2 fragment goat anti-mouse IgG (F(ab′).sub.2-fragment specific) Jackson 115-036-006 diluted 1:2000 was used as second step antibody.
[0217] Data Analysis
[0218] Magellan (Tecan) was used as ELISA reader software. The results were exported to Excel for data analysis and presentation. For concentration determinations, the XLfit program, formula “Dose response-one site”, equation 205, was used.
[0219] Results
[0220] Treatment Scheme 1
[0221] Group 1 and 2 consisted of CD1 mice. The animals were injected with either His.sub.6-Z.sub.Taq4:1 (group 1) or Z.sub.Taq4:1-ABD (group 2), according to treatment scheme 1 (Table 17). Blood samples obtained before, during, and after immunization were analyzed on His.sub.6-Z.sub.Taq4:1 coated plates for detection of IgG-antibodies specifically recognizing His.sub.6-Z.sub.Taq4:1.
[0222] Group 1
[0223] The five mice that were injected with His.sub.6-Z.sub.Taq4:1 developed IgG antibodies as expected. All five mice responded by day 14 showing a maximal anti AFFIBODY®-IgG response at day 34 (
TABLE-US-00019 TABLE 19 Concentration of specific and total IgG in group 1 Concentration of IgG in ug/ml Specific IgG Total IgG Day 7 Day 14 Day 21 Day 34 Day 34 m. 144 ND 7.8 14.2 115.3 3706 m. 145 ND 42.9 70.3 107.9 3065 m. 146 ND 7.7 16.0 63.5 4306 m. 147 0.3 16.7 49.6 128.9 3612 m. 148 ND 8.3 8.0 10.2 2472 average 0.3 8.0 31.6 85.2 3432 (ND = not detectable)
[0224] Group 2
[0225] Group 2 consisted of five CD1 mice that were injected with Z.sub.Taq4:1-ABD. As shown in
TABLE-US-00020 TABLE 20 Concentration of total IgG in group 2 Concentration of total IgG in μg/ml Day 34 m. 149 1435 m. 150 794 m. 151 1638 m. 152 1050 m. 153 2971 Average 1578
[0226] Treatment Scheme 2
[0227] Group 3 to 6 consisted of NMRI mice that were injected 10 times. Group 3 received His.sub.6-Z.sub.Taq4:1 at all injection times while the other groups had an injection scheme with a switch of antigen after the first 5 injections (treatment scheme 2, Table 3.3). Blood samples obtained before, during, and after immunization were analyzed on His.sub.6-Z.sub.Taq4:1 coated plates for IgG-antibodies directed against His.sub.6-Z.sub.Taq4:1. No antibody reactivity was found in any of the pre-plasma samples of group 3 to 6 (data not shown).
[0228] Group 3
[0229] Group 3 was injected with His.sub.6-Z.sub.Taq4:1 according to treatment scheme 2 (Table 18). Specific antibody production could be observed from day 14. The concentration of specific IgG increased over-time to reach a maximum in the death-bleedings (
TABLE-US-00021 TABLE 21 Concentration of specific and total IgG in group 3 Concentration of IgG in μg/ml Specific IgG Total IgG Day 7 Day 14 Day 21 Day 28 Day 35 Day 45 Day 45 m. 154 ND 1.0 4.8 8.3 18.7 32.6 2992 m. 155 ND 1.6 4.7 10.3 32.7 65.5 2576 m. 156 ND 4.0 6.2 22.1 68.5 167.5 5703 m. 157 ND 2.9 3.1 9.5 13.7 35.0 4088 m. 158 ND 3.3 2.9 8.7 34.1 56.1 2285 average ND 2.5 4.3 11.8 33.5 71.3 3529 (ND = not detectable)
[0230] Group 4
[0231] Group 4 received 5 injections of Z.sub.Taq4:1-ABD during the first 3 weeks, and then 5 additional injections of His.sub.6-Z.sub.Taq4:1. The animals did not react to the first antigen, Z.sub.Taq4:1-ABD, but generated His.sub.6-Z.sub.Taq4:1-specific antibodies from day 35 and on. Day 35 corresponds to 14 days after the switch of injected AFFIBODY® molecule (
TABLE-US-00022 TABLE 22 Concentration of specific and total IgG in group 4 Concentration of IgG in μg/ml Specific IgG Total IgG Day 7 Day 14 Day 21 Day 28 Day 35 Day 45 Day 45 m. 159 ND ND ND ND 0.8 9.6 9497 m. 160 ND ND ND ND 0.7 1.0 3172 m. 161 ND ND ND ND 0.7 1.2 2554 m. 162 ND ND ND ND 2.5 5.6 3719 m. 163 ND ND ND ND 1.0 no no plasma plasma average ND ND ND ND 1.1 4.4 4736 (ND = not detectable)
[0232] Group 5
[0233] Group 5 received five injections of Z.sub.Taq4:1-ABD during the first 3 weeks and then another five injections of a mixture of Z.sub.Taq4:1-ABD and His.sub.6-Z.sub.Taq4:1 (10 μg/protein). There was no detectable immune response in the animals upon injection of the first antigen i.e. Z.sub.Taq4:1-ABD. After switching to His.sub.6-Z.sub.Taq4:1 injections low levels of specific antibodies could be detected from day 35 (
TABLE-US-00023 TABLE 23 Concentration of specific and total IgG in group 5 Concentration of IgG in μg/ml Specific IgG Total IgG Day 7 Day 14 Day 21 Day 28 Day 35 Day 45 Day 45 m. 164 ND ND ND ND 0.4 1.5 2565 m. 165 ND ND ND ND 0.6 0.8 1617 m. 166 ND ND ND ND ND 0.5 1284 m. 167 ND ND ND ND 0.3 1.1 1988 m. 168 ND ND ND ND ND 0.3 2347 Average ND ND ND ND 0.4 0.8 1864 (ND = not detectable)
[0234] Group 6
[0235] Group 6 received five His.sub.6-Z.sub.Taq4:1 injections during the first 3 weeks followed by 5 injections of Z.sub.Taq4:1-ABD. The results are illustrated in
TABLE-US-00024 TABLE 24 Concentration of specific and total IgG in group 6 Concentration of IgG in μg/ml Specific IgG Total IgG Day 7 Day 14 Day 21 Day 28 Day 35 Day 45 Day 45 m. 169 ND 2.3 5.8 14.4 11.6 13.6 3295 m. 170 ND 2.2 12.6 82.2 90.7 63.3 2659 m. 171 ND 3.8 18.4 39.4 27.2 25.5 2737 m. 172 ND 3.5 9.6 115.0 60.3 56.0 5431 m. 173 ND 8.0 18.7 58.3 32.4 53.3 2941 Average ND 4.0 13.0 61.9 44.4 42.3 3530 (ND = not detectable)
[0236] The results are illustrated in
DISCUSSION
[0237] There were two main purposes to this study, more precisely a) to investigate the immunogenicity of His.sub.6-Z.sub.Taq4:1 and Z.sub.Taq4:1-ABD in an additional out bred mouse strain and b) to determine whether the observed unresponsiveness of ABD-fused AFFIBODY® molecules is due to an active suppression of the animals' immune system or is merely a passive ignorance of the same.
[0238] The results showed that the out bred CD1 mice responded similar to earlier studied NMRI mice by generating specific antibodies recognizing and binding His.sub.6-Z.sub.Taq4:1 but not Z.sub.Taq4:1-ABD. Thus the ABD-induced unresponsiveness seems to be a general phenomenon rather than a response pattern only connected to the NMRI strain.
[0239] As mentioned above, the mice in group 3 to 6 were injected with different combinations of ABD-fused and unfused Z.sub.Taq4:1 to determine whether the ABD-mediated unresponsiveness is an active or passive process. Mice in group 4 and 5 were injected with five doses of Z.sub.Taq4:1-ABD with the main purpose of inducing unresponsiveness and then followed by five injections of His.sub.6-Z.sub.Taq4:1 (group 4) or a mixture of His.sub.6-Z.sub.Taq4:1 and Z.sub.Taq4:1-ABD (group 5). Both groups responded by producing a His.sub.6-Z.sub.Taq4:1 specific IgG response suggesting that injections with ABD-fused AFFIBODY® molecules do not result in an active suppression of the protein by the immune system i.e. anergy. The results of this study strongly indicate that ABD-fused AFFIBODY® molecules, by binding to serum albumin, are ignored by the mice' immune system rather than actively suppressed by the same. Mice that first received His.sub.6-Z.sub.Taq4:1 continued to produce AFFIBODY®-specific IgG after the switch to Z.sub.Taq4:1-ABD although the levels decreased slightly.
EXAMPLE 6
[0240] Immunogenicity of Chronically Administered Biologically Active Molecules in Rat
[0241] In this study, the immune responses generated in rats injected with different AFFIBODY® molecules were analyzed over an extended period. This report covers data up to 96 days after immunization. The molecules used were (Z.sub.Aβ3).sub.2 and ABD-(Z.sub.Aβ3).sub.2 as described above in Example 4. The aim of this study was to 1) analyze the ability of (Z.sub.Aβ3).sub.2 to induce specific immune responses in rats and 2) investigate if ABD-(Z.sub.Aβ3).sub.2 gives a lower immune response compared to an AFFIBODY® molecule without ABD. Blood samples obtained before, nine times during the immunization scheme and after the last injection were analyzed for reactivity. On plates coated with (Z.sub.Aβ3).sub.2 the result showed that administration with (Z.sub.Aβ3).sub.2 generated an IgG response that increased over time with a large individual variability. In contrast, there were no detectable or very low specific IgG in serum from all rats injected with ABD-fused (Z.sub.Aβ3).sub.2-molecules. In addition, no adverse effects were seen in the rats.
[0242] Methods
[0243] General ELISA Method
[0244] In general, a volume of 100 □l per well was used for all incubation steps except for blocking where a volume of 200 □l was used. Plates were incubated 1 day for coating, 1-2 hours for blocking and plasma, 1 hour for second step antibody and 10 min for substrate solution. Incubations were done at room temperature except coating that was incubated at 4° C. Washing was done between all steps unless otherwise stated, using the ELISA SkanWasher 300, with 4×350 □l washing buffer (PBS-T) per well. Plates were read at 450 nm in a Tecan ELISA reader with use of the Magellan software. PBS-T buffer was used for all dilutions except coating, where coating buffer was used instead.
[0245] Rat Anti (Z.sub.Aβ3).sub.2-Specific IgG ELISA
[0246] Plates were coated with 2 μg/ml of (Z.sub.Aβ3).sub.2, in coating buffer and incubated overnight at 4° C. After washing, plates were blocked as described above. Serum from rabbits injected with (Z.sub.Aβ3).sub.2 or ABD-(Z.sub.Aβ3).sub.2 was added in 3-fold dilution series starting from 1/10. After incubation, plates were washed and HRP-conjugated goat anti-rat IgG, Southern Biotechnology 3050-05 diluted 1:6000, was added. After the final incubation, plates were washed and developed as described above.
[0247] Data Analysis
[0248] Magellan2 (Tecan) was used as ELISA reader software. The results were exported to Excel for data analysis and presentation. For concentration determinations the Xlfit3.0 program, formula “Dose response-one site”, equation 205 was used. Processed data from each day and type of measurement i.e. IgG, concentration etc, are represented by an Excel file.
[0249] Results
[0250] Treatment and Injection Scheme
[0251] Two groups of rats, ten per group, were injected with the same dose of AFFIBODY® molecules (200 μg/ml), about every 28 days. Serum was drawn before (day 0), according to the schedule during the injection scheme and two weeks after the last injection (death-bleeding).
TABLE-US-00025 TABLE 25 List of AFFIBODY® molecules and the corresponding group-number in the study. μg/ ml/ animal/ animal/ Test Rat Group Treatment Admin. injection injection tube no. 1 (Zaβ3)2 s.c. 100 0.25 A 1-10 2 ABD-(Zaβ3)2 s.c. 100 0.25 B 11-20
TABLE-US-00026 TABLE 26 Injection and bleeding scheme for group 1 and 2. Day Treatment 0 Preserum/sample 1, Injection 1 (100 μg/animal; tube A-B) 3 Injection 2 (100 μg/animal; tube A-B) 6 Injection 3 (100 μg/animal; tube A-B) 7 Sample 2 14 Sample 3 21 Sample 4 21 Injection 4 (100 μg/animal; tube A-B) 36 Sample 5 51 Injection 5 (100 μg/animal; tube A-B) 66 Sample 6 81 Injection 6 (100 μg/animal; tube A-B) 96 Sample 7 111 Injection 7 (100 μg/animal; tube A-B) 141 Injection 8 (100 μg/animal; tube A-B) 156 Sample 8 171 Injection 9 (100 μg/animal; tube A-B) 201 Injection 10 (100 μg/animal; tube A-B) 216 Sample 9 231 Injection 11 (100 μg/animal; tube A-B) 261 Injection 12 (100 μg/animal; tube A-B) 276 Sample 10 291 Injection 13 (100 μg/animal; tube A-B) 306 Sample 11
[0252] Injection with (Zaβ3).sub.2 and ABD-(Zaβ3).sub.2
[0253] Serum samples from individual rats were titrated in 3-fold dilution series on (ZAβ3).sub.2 coated ELISA-plates and analyzed for the presence of specific antibodies, as described in the Methods section.
[0254] Titration Curves, (ZAβ3).sub.2
[0255] As shown in
[0256] Titration Curves, ABD-(ZAβ3).sub.2
[0257] As shown in