TACI-FC FUSION PROTEINS AND USES THEREOF

Abstract

The present invention relates to an optimized TACI-Fc fusion protein and use of the same in the manufacture of a medicament for treating systemic lupus erythematosus, and a dosage regimen or method for treating systemic lupus erythematosus using the TACI-Fc fusion protein.

Claims

1. A method for treating systemic lupus erythematosus (SLE), comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical preparation of a recombinant TACI-Fc fusion protein in a dose greater than or equal to 50 mg per administration, wherein the recombinant TACI-Fc fusion protein comprises amino acids 13-118 of extracellular domain of TACI and the Fc region of human immunoglobulin.

2. The method of claim 1, wherein the dose is greater than or equal to 60 mg per administration.

3. The method of claim 1, wherein the dose is about 50-240 mg per administration.

4. The method of claim 1, comprising administering the subject in need thereof once every week, once every two weeks, once every three weeks, or once every four weeks.

5. The method of claim 4, comprising administering the subject in need thereof once every week.

6. The method of claim 3, wherein the pharmaceutical preparation is a lyophilized preparation or a liquid preparation.

7. The method of claim 6, wherein the pharmaceutical preparation is a lyophilized preparation.

8. The method of claim 6, wherein the pharmaceutical preparation is administered by subcutaneous injection, intraperitoneal injection, intramuscular injection, or intravenous injection.

9. The method of claim 8, wherein the pharmaceutical preparation is administered by subcutaneous injection.

10. The method of claim 3, wherein the pharmaceutical preparation is administered to the subject in need thereof for consecutive 12 weeks, 24 weeks, 36 weeks, 48 weeks, 60 weeks, 72 weeks or more.

11. The method of claim 10, wherein the pharmaceutical preparation is administered to the subject in need thereof for consecutive 48 weeks or longer.

12. The method of claim 1, wherein two of the recombinant TACI-Fc fusion proteins form a double-stranded structure through interchain disulfide bond formed in Fc hinge region in a liquid pharmaceutical preparation.

13. The method of claim 12, wherein the amino acids 13-118 of the extracellular domain of TACI has a sequence as shown in amino acids 1-106 of SEQ ID NO: 1, or has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with amino acids 1-106 of SEQ ID NO: 1.

14. The method of claim 13, wherein the Fc region of human immunoglobulin has a sequence as shown in amino acids 107-333 of SEQ ID NO: 1, or has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with amino acids 107-333 of SEQ ID NO: 1.

15. The method of claim 14, wherein the recombinant TACI-Fc fusion protein has a sequence as shown in SEQ ID NO: 1.

16. The method of claim 1, wherein the systemic lupus erythematosus is mild to moderate systemic lupus erythematosus or moderate to severe systemic lupus erythematosus.

17. A recombinant TACI-Fc fusion protein, comprising an amino acid sequence as shown in SEQ ID NO: 1.

18. A nucleotide sequence, encoding the amino acid sequence of recombinant TACI-Fc fusion protein as shown in SEQ ID NO: 1.

19. A vector, comprising the nucleotide sequence of claim 18.

20. A pharmaceutical composition, comprising the recombinant TACI-Fc fusion protein of claim 17 and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition is a liquid preparation.

22. The pharmaceutical composition of claim 21, wherein two of the TACI-Fc fusion proteins form a double-stranded structure through interchain disulfide bond formed in the Fc hinge region in the pharmaceutical composition.

23. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition is manufactured into a preparation containing 80-240 mg of the recombinant TACI-Fc fusion protein per unit.

24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition is a lyophilized preparation or a liquid preparation, and is administered by subcutaneous route in a dose of about 80-240 mg per administration.

25. (canceled)

26. A method of treating an autoimmune disease, comprising administering recombinant TACI-Fc fusion protein of claim 17 to a subject in need thereof.

27. The method of claim 26, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, neuromyelitis optica, and a neuromyelitis optica spectrum disorder.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0030] FIG. 1: The overlay of the ADCC effect curve (effector-target ratio (E:T)=10:1) of TACI-Fc fusion protein (SEQ ID NO.1) (green, solid)/rituximab (red, hollow).

[0031] FIG. 2: The overlay of the ADCC effect curve (effector-target ratio (E:T)=20:1) of TACI-Fc fusion protein (SEQ ID NO.1) (green, solid)/rituximab (red, hollow).

DETAILED DESCRIPTION

Example 1. Comparison Test of the ADCC Effect of TACI-Fc Fusion Protein (SEQ ID NO. 1)

[0032] 1.1 Experimental Materials

TABLE-US-00002 TABLE 1 Materials Manufacturer Batch No RPMI 1640 medium Gibco A10491 Rituximab Roche 10001031283948 96-well cell plate Costar 3599 PBMC cells Hemacare PB009C-1 LDH detection kit Roche 1164793001 FBS Excellbio FND500 TritonX-100 Solarbio T8200

[0033] 1.2 Experimental Principle

[0034] Raji cells express APRIL on the surface, and TACI is the co-receptor of APRIL and BLyS. Therefore, APRIL on the surface of Raji cells can bind to the TACI terminal of TACI-Fc fusion protein (SEQ ID NO: 1). If the Fc terminal of the TACI-Fc fusion protein (SEQ ID NO: 1) has a binding site of crystallizable fragment γ receptor (Fc γ receptor, FcyR), it can bind to the FcyR of effector cell and trigger the ADCC effect. Rituximab was used as a positive control in the test because rituximab is a Cluster of Differentiation 20 (CD20) monoclonal antibody capable of inducing the ADCC effect. Raji cell expresses CD20 on the surface, and therefore, rituximab can bind to CD20 on the surface of Raji cells to trigger ADCC effects. The experimental results are shown in FIG. 1 and FIG. 2: The control rituximab induces ADCC effect, and the inhibition rate is concentration-dependent. Compared with the control, TACI-Fc fusion protein does not induce ADCC effect at each concentration.

Example 2: Comparison Test of the CDC Effect of TACI-Fc Fusion Protein

[0035] 2.1 Experimental Materials

TABLE-US-00003 TABLE 2 Materials Manufacturer Batch No RPMI 1640 medium Gibco A10491 Rituximab Roche 10001031283948 Human serum complement Donor Cell titer-gloluminescent Promega G7572 cell viability assay Transparent 96-well plate Costar 3610 with white bottom

[0036] 2.2 Experimental Principle

[0037] APRIL on the surface of Raji cells can bind to the TACI terminal of TACI-Fc fusion protein (SEQ ID NO: 1). If the Fc terminal of the TACI-Fc fusion protein (SEQ ID NO: 1) has a complement binding site, it can trigger the CDC effect. Rituximab was used as a positive control in the test because rituximab can induce the CDC effect. Raji cell expresses CD20 on the surface, and therefore, rituximab can bind to CD20 on the surface of Raji cells to trigger CDC effects. Monoclonal antibody that does not bind to Raji cells was used as a negative control.

[0038] 2.3 Experimental Procedure

[0039] After adjusting the concentration of Raji cells to 10,000 cells/well, the cells were added a 96-well plate and incubated in a CO.sub.2 incubator for 20 minutes. Then, diluted series of samples of different concentration gradients were added and then the cells were incubated in the CO.sub.2 incubator for 20 minutes. Then, 10% normal human serum complement was added and then the cells were incubated in the CO.sub.2 incubator for 2 hours. After 10 minutes of color development using the ATP method, a microplate reader was used for reading. After subtracting the serum control value, GraphPad Prism 7.00 was used to make a 4-PL curve with the concentration (ng/ml) of Ig on the abscissa and the inhibition rate (%) on the ordinate. The experimental results are as follows:

TABLE-US-00004 TABLE 3 Results of the he CDC effect Inhibition rate % TACI-Fc fusion Final concentration protein (SEQ ID Negative (ng/ml) Rituximab NO: 1) control 30000 38.17 4.88 5.60 3000 24.65 2.01 9.63 300 11.85 9.12 −0.14 30 4.83 1.85 −0.76 3 −0.68 2.27 −0.66

[0040] Experimental Results:

[0041] 1. Compared with the cell control group, each serum complement and drug control group has no obvious killing effect.

[0042] 2. The positive control group (10% serum complement-rituximab) has a highest inhibition rate of 40%, and has a significant concentration-dependent effect.

[0043] 3. The administration group (10% serum complement-TACI-Fc fusion protein (SEQ ID NO.1)) has a highest inhibition rate less than 10% and has no concentration-dependent effect.

[0044] 4. The negative control group (10% serum complement-RC48) has a highest inhibition rate less than 10% and has no concentration-dependent effect.

[0045] It can be seen that rituximab can induce a CDC effect, and the inhibition rate is obviously concentration-dependent, while the TACI-Fc fusion protein (SEQ ID NO.1) basically induce no CDC effect.

Example 3: The Clinical Effect Trials of the Treatment for Patients with Systemic Lupus Erythematosus

[0046] 1. The experimental purpose is to preliminarily evaluate the safety and effectiveness of TACI-Fc fusion protein (with amino acid sequence shown in SEQ ID NO: 1) in standard combination therapy compared with placebo in standard combination therapy in the treatment of moderate to severe SLE patients, and analyze the dose-effect relationship to provide dose basis for follow-up clinical trials. The effective count of subjects in this experiment is 249 and the administrations in those showing no effect are stopped according to the dosing schedule.

[0047] 2. Inclusion Criteria

[0048] 1. Patients diagnosed with active SLE who meet at least 4 of the 11 SLE standards revised by the American College of Rheumatology in 1997;

[0049] 2. Subject with age from 18 to 65 years old, both male and female, no gender ratio limit;

[0050] 3. Inpatients or outpatients with good compliance, and providing an informed consent form before the trial.

[0051] 4. Subject with SELENA-SLEDAI score ≥8 points during the screening period. For those with low complement and/or anti-ds-DNA antibody score, the score for the clinical symptoms of SELENA-SLEDAI (except for low complement and/or anti-ds-DNA antibody) should be ≥6 points;

[0052] 5. Subjects with positive in autoantibody serological test. Positive is defined as ANA positive and/or anti-double-stranded DNA antibody positive results defined by the clear reference value range of the research center laboratory, and critical values are not accepted;

[0053] 6. Subjects with stable standard treatment regimen for at least 30 days before the first day (ie, the day when the test drug is administered fist). The standard treatment regimen refers to the stable use of any one of the following (alone or in combination): corticosteroids, antimalarials, non-steroidal anti-inflammatory drugs (NSAIDs), other immunosuppressants or immunomodulators including azathioprine, mycophenolate (including mycophenolate mofetil, sodium mycophenolate), cyclophosphamide, methotrexate, leflunomide, tacrolimus, cyclosporine.

[0054] 3. Exclusion Criteria

[0055] 1. Subjects with severe lupus nephritis (defined as urine protein>6 g/24 hours or serum creatinine>2.5 mg/dL or 221 umol/L) or required hemodialysis or received high-dose corticosteroids (prednisone>100 mg/day or equivalent)≥14 days in the last 2 months;

[0056] 2. Subjects with central nervous system disease caused by SLE or not caused by SLE (including epilepsy, psychosis, organic brain syndrome, cerebrovascular accident, encephalitis, central nervous system vasculitis) in the last 2 months;

[0057] 3. Subjects with severe diseases and disease history in the heart, liver, kidney and other important organs, blood, endocrine system; evaluation criteria for severity includes: a. ALT or AST≥2 times the upper limit of normal range; b. endogenous creatinine clearance rate <30 mL/min; c. white blood cell count<2.5×10.sup.9/L; d. hemoglobin<85 g/L; e. platelet count<50×10.sup.9/L.

[0058] 4. Subjects currently suffering from active hepatitis or having severe liver disease or disease history. Hepatitis B: patients having positive HBsAg will be excluded. For subject having negative HBsAg and positive anti-HBc antibodies, they will be classified according to the HBV-DNA test, in which patients having positive HBV-DNA will be excluded and not allowed to participate in the study, and patients having negative HBV-DNA may be allowed to participate in the study. Hepatitis C: patients with positive hepatitis C antibodies will be excluded.

[0059] 5. Patients with immunodeficiency, uncontrolled severe infection, and active or recurrent peptic ulcer;

[0060] 6. Pregnant women, breastfeeding women, and men or women who have planned fertility in the next 12 months;

[0061] 7. Allergic reactions: subject with a history of allergies to human-derived biological products;

[0062] 8. Those who received live vaccines in the last month;

[0063] 9. Those who have participated in any clinical trial 28 days before the initial screening and/or within 5 times the half-life of the study compound (whichever is longer);

[0064] 10. Those who received B-cell targeted therapies, such as Rituxan or Epalizumab within one year;

[0065] 11. Those who received tumor necrosis factor inhibitors and interleukin receptor blockers within one year;

[0066] 12. Those who have received intravenous gamma globulin (IVIG), prednisone ≥100 mg/d for more than 14 days or undergo plasma exchange within one month;

[0067] 13. Those having active infections (such as herpes zoster, HIV infection, active tuberculosis, etc.) during the screening period;

[0068] 14. Mental patients with depression or suicidal thoughts;

[0069] 15. Subjects who are considered unsuitable to participate in this trial by the researcher.

[0070] IV. Grouping

[0071] Treatment group: recombinant TACI-Fc fusion protein (with amino acid sequence shown in SEQ ID NO: 1) freeze-dried powder preparation; strength: 80 mg per injection; subcutaneous injection, dosage: 80 mg, 160 mg or 240 mg per administration (the indication of 80 mg, 160 mg, or 240 mg refers to the mass of the active ingredient TACI-Fc fusion protein in the lyophilized powder preparation), administered once a week for consecutive 48 weeks.

[0072] Control group: simulant of the recombinant TACI-Fc fusion protein lyophilized powder, administered by subcutaneous once a week for consecutive 48 weeks.

[0073] The specific grouping is as follows:

TABLE-US-00005 TABLE 4 Distribution of cases in each center (randomized cases) RC18 240 mg RC18 160 mg RC18 80 mg Placebo Total (N = 62) (N = 63) (N = 62) (N = 62) (N = 249) Summary Screened case 356 Enrolled case 62 63 62 62 249 Completed case 46 44 45 36 171 FAS 62 63 62 62 249 PPS 48 46 48 50 192 SS 62 63 62 62 249

[0074] FAS: Full Analysis Set, refers to the set of eligible cases and dropped cases, but does not include excluded cases

[0075] PPS: cases that meet the test protocol, have good compliance, and complete the connects filled according the CRF requirements

[0076] SS: cases that receives at least one treatment and have actual data recorded by safety indicators

[0077] V. Endpoint Index

[0078] The evaluation methods of clinical trial effect are as follows:

TABLE-US-00006 TABLE 5 Evaluation Selection of the No Indicator time endpoint indicator Primary 1 SLE response index at 12 months after administration. Week 48 Effectiveness endpoint SLE response is defined as follows: index indicators a. SELENA-SLEDAI score drop ≥4 points; and b. no new organs reach A grade in the BILAG score, or evaluation new organs do not exceed one B grade time c. the physician's global assessment (PGA) does not increase by more than 0.3 points from the baseline. The physician's global assessment uses a 100 mm visual analog scale (VAS) to assess the comprehensive disease status before and after medication Secondary 1 (1) the proportion of subjects with SELENA-SLEDAI Week 48 Effectiveness endpoint score decreased by ≥4 points after administration. (2) index indicators The change in the physician's global assessment score and from baseline after administration. (3) The proportion of evaluation subjects with prednisone dose ≤7.5 mg/d or a decrease time of dose ≥25% from baseline at 44-48 weeks after administration. (4) The change in seroiogical test index (IgG, IgA, IgM, total B cell (CD19+), anti-dsDNA antibodies, complement (C3, C4) from baseline

[0079] VI. Results of Clinical Trials

[0080] The clinical trial data analyzed by FAS (full analysis set) shows that the treatment group (TACI-Fc fusion protein: SEQ ID NO: 1) has a significantly higher response. The specific data are as follows:

TABLE-US-00007 TABLE 6 Therapeutic doses Efficacy at week 48 (SRI4) 240 mg group 75.8%, p <0.0001 160 mg group 68.3%, p = 0.0001 80 mg group 71.0%, p <0.0001 Control group 33.9%, p <0.0001

[0081] The SELENA-SLEDAI score at week 48 shows that more patients in the treatment group (TACI-Fc fusion protein: SEQ ID NO.1) has a reduction of 4 points or more (SELENA-SLEDAI score). The details are as follows: 80 mg dose group (75.8%, p=0.003), 160 mg dose group (77.8%, p=0.001), 240 mg dose group (79.0%, p<0.001), and placebo group (50.0%).

[0082] No more patients are found to have worsened PGA scores, as follows: 80 mg dose group (96.8%, p<0.001), 160 mg dose group (92.1%, p=0.013), 240 mg dose group (96.8%,p<0.001)), and placebo group (75.8%).

[0083] The analysis results from PPS (Per protocol set) and FAS (full analysis set) are consistent.

[0084] The data on clinical adverse events (AEs) and serious adverse events (SAEs) are as follows:

TABLE-US-00008 TABLE 7 Incidence of AEs and SAEs Group Incidence of AEs Incidence of SAEs 240 mg group 93.5% 12.9% 160 mg group 92.1% 15.9% 80 mg group 90.3% 12.9% Placebo group 82.3% 16.1%

[0085] In the 240 mg group, there is one death unrelated to the test drug. The most common adverse events are upper respiratory tract infections and injection site reactions.

[0086] In addition, during clinical trials, there are 11 unexpected pregnancies in the TACI-Fc (SEQ ID NO.1) treatment group (4 cases in the 240 mg dose group, 3 cases in the 160 mg dose group, and 4 cases in the 240 mg dose group), while no pregnancy event in the placebo group. Of the 11 pregnancies, 10 cases chose selective termination and 1 case chose pregnancy and live birth.

[0087] Therefore, the three dose groups of recombinant TACI-Fc fusion protein (SEQ ID NO.1) achieve very significant therapeutic effects and are safe and effective as proved by clinical data. The administrations for patients in the treatment group are well tolerated, and the ability to conceive of some patients may be unexpectedly restored by treating the patients with systemic lupus erythematosus.